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Methods in Cell Biology

VOLUME 101
The Zebrafish: Cellular and Developmental Biology, Part B
Third Edition
Series Editors
Leslie Wilson
Department of Molecular, Cellular and Developmental Biology
University of California
Santa Barbara, California

Paul Matsudaira
Department of Biological Sciences
National University of Singapore
Singapore
Methods in Cell Biology

VOLUME 101
The Zebrafish: Cellular and Developmental Biology, Part B
Third Edition

Edited by
H. William Detrich III
Department of Biology, Northeastern University, Boston, MA, USA

Monte Westerfield
Institute of Neuroscience, University of Oregon, Eugene, OR, USA

Leonard I. Zon
Division of Hematology/Oncology, Children’s Hospital of Boson,
Department of Pediatrics and Howard Hughes Medical Institute,
Harvard Medical School, Boston, MA, USA

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DEDICATION

We dedicate the Third Edition of Methods in Cell Biology: The Zebrafish to


Wolfgang Driever, Mark C. Fishman, Charles Kimmel, and Christiane N€ usslein-
Volhard. Through their foresighted embrace of the zebrafish as a model vertebrate
and their pursuit of genetic screens to illuminate vertebrate development, they
fostered the emergence of the vibrant zebrafish research community.

v
CONTRIBUTORS

Numbers in parentheses indicate the pages on which the author’s contributions begin.

R. Craig Albertson (225), Department of Biology, Syracuse University, Syracuse NY,


Tufts Univeristy, Boston, Massachusetts
James F. Amatruda (19), Department of Pediatrics; Molecular Biology; Internal
Medicine, UT Southwestern Medical Center, Dallas, Texas, USA
Jennifer L. Anderson (111), Carnegie Institution for Science, Department of
Embryology, Baltimore, Maryland, USA
Viktoria Andreeva (225), Department of Oral and Maxillofacial Pathology, Tufts
Univeristy, Boston, Massachusetts
Andrei Avanesov (39), Division of Craniofacial and Molecular Genetics, Tufts
University, Massachusetts, USA
Maira Carrillo (197), Department of Biological Sciences, University of North Texas,
Denton, Texas, USA
Juliana D. Carten (111), Carnegie Institution for Science, Department of
Embryology, Baltimore, Maryland, USA
Joanne Chan (181), Vascular Biology Program and Department of Surgery,
Children’s Hospital Boston, Harvard Medical School, Boston, Massachusetts, USA
W. James Cooper (225), Department of Biology, Syracuse University, Syracuse NY,
Tufts Univeristy, Boston, Massachusetts
H. William Detrich III (1), Department of Biology, Northeastern University,
Boston, Massachusetts 02115, USA
Judith Eisen (143), Institute of Neuroscience, 1254 University of Oregon, Eugene,
Oregon
Steven A. Farber (111), Carnegie Institution for Science, Department of
Embryology, Baltimore, Maryland, USA
Robert Gerlai (249), Department of Psychology, University of Toronto, Mississauga,
Ontario, Canada
Wolfram Goessling (205), Genetics Division, Brigham and Women’s Hospital,
Harvard Medical School, Boston, Massachusetts, USA; Harvard Stem Cell
Institute, Cambridge, Massachusetts, USA
Sean Hasso (181), Vascular Biology Program and Department of Surgery, Children’s
Hospital Boston, Harvard Medical School, Boston, Massachusetts, USA
Pudur Jagadeeswaran (197), Department of Biological Sciences, University of
North Texas, Denton, Texas, USA
Seongcheol Kim (197), Department of Biological Sciences, University of North
Texas, Denton, Texas, USA

xi
xii Contributors

Jade Li (39), Departments of Genetics, Yale University School of Medicine, New


Haven, Connecticut, USA
Jarema Malicki (39), Division of Craniofacial and Molecular Genetics, Tufts
University, Massachusetts, USA
Sean G. Megason (1), Department of Systems Biology, Harvard Medical School,
Boston, Massachusetts 02115, USA
Timothy J. Mitchison (1), Department of Systems Biology, Harvard Medical School,
Boston, Massachusetts 02115, USA
Grant I. Miura (161), Division of Biological Sciences, University of California, San
Diego, La Jolla, California
Trista E. North (205), Department of Pathology, Beth Israel Deaconess Medical
Center, Harvard Medical School, Boston, Massachusetts, USA; Harvard Stem
Cell Institute, Cambridge, Massachusetts, USA
Nikolaus D. Obholzer (1), Department of Systems Biology, Harvard Medical
School, Boston, Massachusetts 02115, USA
Kevin J. Parsons (225), Department of Biology, Syracuse University, Syracuse NY,
Tufts Univeristy, Boston, Massachusetts
Uvaraj P. Radhakrishnan (197), Department of Biological Sciences, University of
North Texas, Denton, Texas, USA
Surendra K. Rajpurohit (197), Department of Biological Sciences, University of
North Texas, Denton, Texas, USA
Iain Shepherd (143), Department of Biology, Emory University Rollins Research
Building, Atlanta Georgia
David L. Stachura (75), Division of Biological Sciences, Section of Cell and
Developmental Biology, University of California San Diego; Department of
Cellular and Molecular Medicine, University of California San Diego School of
Medicine, La Jolfla, California, USA
Zhaoxia Sun (39), Departments of Genetics, Yale University School of Medicine,
New Haven, Connecticut, USA
David Traver (75), Division of Biological Sciences, Section of Cell and
Developmental Biology, University of California San Diego; Department of
Cellular and Molecular Medicine, University of California San Diego School of
Medicine, La Jolfla, California, USA
Daniel Verduzco (19), Department of Pediatrics; Molecular Biology, UT
Southwestern Medical Center, Dallas, Texas, USA
Martin W€ uhr (1), Department of Systems Biology, Harvard Medical School, Boston,
Massachusetts 02115, USA
Pamela C. Yelick (225), Department of Oral and Maxillofacial Pathology, Tufts
Univeristy, Boston, Massachusetts
Deborah Yelon (161), Division of Biological Sciences, University of California, San
Diego, La Jolla, California
Shiaulou Yuan (39), Departments of Genetics, Yale University School of Medicine,
New Haven, Connecticut, USA
PREFACE

Building on the foundation of our first (1999) and second (2004) editions of
Methods in Cell Biology: The Zebrafish, Monte, Len, and I are pleased to continue
this Third Edition with Methods in Cell Biology Volume 101, Cellular and
Developmental Biology, Part B. In this volume (and its previously released com-
panion, Volume 100, Part A), our contributors present the latest technical advances
in the Cell, Developmental and Neural Biology of the zebrafish that have appeared
since the second edition. One theme that clearly emerges from these chapters is that
the zebrafish is the preeminent vertebrate model for mechanistic cellular studies of
developmental processes in vivo.
Subsequently, Genetics, Genomics, and Informatics will cover new technologies
in Forward and Reverse Genetics, Transgenesis, The Zebrafish Genome and
Mapping Technologies, Informatics and Comparative Genomics, and
Infrastructure. The Third Edition will also introduce Disease Models and
Chemical Screens, two rapidly emerging and compelling applications of the zebra-
fish. We trust that the Third Edition will prove valuable to both seasoned zebrafish
investigators and those who are newly adopting the zebrafish model as part of their
research armamentarium.
We thank the Series Editors, Leslie Wilson and Paul Matsudaira, and the staff of
Elsevier/Academic Press, especially Zoe Kruze, for their enthusiastic support of our
Third Edition of Methods in Cell Biology: The Zebrafish. Their help, patience, and
encouragement are profoundly appreciated.
H. William Detrich, III
Monte Westerfield
Leonard I. Zon

xiii
CHAPTER 1

Live Imaging of the Cytoskeleton in Early


Cleavage-Stage Zebrafish Embryos
M. W€uhr,* N.D. Obholzer,* S.G. Megason,* H.W. Detrich IIIy
and T.J. Mitchison*
*
Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115, USA
y
Department of Biology, Northeastern University, Boston, Massachusetts 02115, USA

Abstract
I. Introduction
II. Maintaining the Breeding Competence of Zebrafish throughout the Day
III. Mounting Zebrafish Embryos for Live Imaging
A. Rationale
B. Methods
IV. Live Imaging of Microtubules in Cleaving Zebrafish Embryos
A. Rationale
B. Methods
C. Results
V. Live Imaging of Microfilaments in Cleaving Zebrafish Embryos
A. Rationale
B. Method
C. Results
VI. Comparison of Microscopic Techniques for Imaging the Cytoskeleton of Cleaving
Zebrafish Embryos
VII. Discussion and Future Directions
Acknowledgments
Appendix A Supplementary Movies
References

Abstract
The large and transparent cells of cleavage-stage zebrafish embryos provide
unique opportunities to study cell division and cytoskeletal dynamics in very large
animal cells. Here, we summarize recent progress, from our laboratories and others,

METHODS IN CELL BIOLOGY, VOL 101 0091-679X/10 $35.00


Copyright 2011. Elsevier Inc. All rights reserved. 1 DOI 10.1016/B978-0-12-387036-0.00001-3
2 uhr et al.
M. W€

on live imaging of the microtubule and actin cytoskeletons during zebrafish embry-
onic cleavage. First, we present simple protocols for extending the breeding com-
petence of zebrafish mating ensembles throughout the day, which ensures a steady
supply of embryos in early cleavage, and for mounting these embryos for imaging.
Second, we describe a transgenic zebrafish line [Tg(bactin2:HsENSCONSIN17-
282-3xEGFP)hm1] that expresses the green fluorescent protein (GFP)-labeled
microtubule-binding part of ensconsin (EMTB-3GFP). We demonstrate that the
microtubule-based structures of the early cell cycles can be imaged live, with single
microtubule resolution and with high contrast, in this line. Microtubules are much
more easily visualized using this tagged binding protein rather than directly labeled
tubulin (injected Alexa-647-labeled tubulin), presumably due to lower background
from probe molecules not attached to microtubules. Third, we illustrate live imaging
of the actin cytoskeleton by injection of the actin-binding fragment of utrophin fused
to GFP. Fourth, we compare epifluorescence-, spinning-disc-, laser-scanning-, and
two-photon-microscopic modalities for live imaging of the microtubule cytoskele-
ton in early embryos of our EMTB-3GFP-expressing transgenic line. Finally, we
discuss future applications and extensions of our methods.

I. Introduction
The zebrafish embryo has long been recognized as an excellent model system for
molecular–genetic analysis of vertebrate embryonic development (Detrich et al.,
1999), one whose advantages complement, and perhaps exceed, those of the mouse
(Orkin and Zon, 1997). Forward genetic screens using large-scale zygotic (Driever
et al., 1996; Haffter et al., 1996), maternal (Pelegri and Mullins, 2004), and numer-
ous targeted strategies have generated thousands of mutations in the zebrafish that
affect all levels of development. Systematic identification and cloning of the
mutated genes, whether by candidate (Skromne and Prince, 2008), positional
(Bahary et al., 2004), or insertional (Amsterdam and Hopkins, 2004) approaches,
has greatly enhanced our understanding of the signaling pathways that regulate
expression of the vertebrate body plan. Modern deep sequencing methods will make
gene identification even faster.
The advantages of the zebrafish for mechanistic studies of developmental pro-
cesses in vivo at the cellular level have been less well appreciated although the tide is
clearly turning (Beis and Stainier, 2006). The remarkable optical clarity of the large
blastomeres of the pre-pigmentation embryo facilitates the microscopic examination
of cellular processes that underlie morphogenesis. The reduced pigmentation mutant
lines nacre (Lister et al., 1999) and casper (White et al., 2008) extend tissue and
organ transparency to juvenile and adult animals. As researchers apply transgenic
approaches to tag proteins of interest with a fluorescent protein (FP), we foresee a
major shift of cellular research to the context of the living fish. Zebrafish excel over
amphibian models for live imaging of early development because their meroblastic
cleavage separates the transparent blastodisc from the opaque yolk, whereas the
1. Live Imaging of the Cytoskeleton in Early Cleavage-Stage Zebrafish Embryos 3

holoblastic cleavage of amphibian embryos renders cells nontransparent at early


stages due to distributed yolk particles. The high fecundity of the zebrafish and its
low maintenance costs are also major advantages, particularly in comparison to the
mouse.
Characterization of the cytoskeleton of zebrafish eggs and embryos and its role in
morphogenesis of the zygote began in the early 1990s. These studies, which had been
stimulated by the pioneering work of J. P. Trinkaus on epiboly and gastrulation in
embryos of Fundulus heteroclitus (Betchaku and Trinkaus, 1978; Trinkaus, 1949,
1951), focused initially on microtubules and microfilaments. Using ultraviolet irra-
diation and antimitotic drugs, Str€ ahle and Jesuthasan (1993) and Solnica-Krezel and
Driever (1994) demonstrated that microtubules participate either directly or indi-
rectly in epibolic cell movements, and Jesuthasan and Str€ ahle (1997) concluded that
specification of the zebrafish dorsoventral axis required the microtubule-dependent
transport of dorsal determinants from the vegetal pole to marginal blastomeres. In
recent years, numerous studies have shown that maternal products of the zebrafish
oocyte and early embryo are organized, and reorganized, by microtubules and
microfilaments during oogenesis and embryogenesis (Dekens et al., 2003; Knaut
et al., 2000; Strasser et al., 2008; Theusch et al., 2006; Yabe et al., 2009; reviewed by
Lindeman and Pelegri, 2010).
To date, the cytoskeletal components of zebrafish oocytes and embryos have
generally been analyzed by the application of immunofluorescence light microscopy
and/or electron microscopy to fixed preparations. Although methods of fixation to
optimize cytoskeletal preservation in embryos have been developed (reviewed by
Topczewski and Solnica-Krezel, 2009) and their use has led to important discoveries
(reviewed by Lindeman and Pelegri, 2010), research on the function of the cytoskel-
eton in zebrafish development would benefit enormously from live-cell imaging of
fluorescent cytoskeletal proteins. Such studies have revolutionized our understand-
ing of cytoskeleton organization and dynamics in somatic cells, where essentially all
cutting-edge cytoskeletal work is now performed using live imaging. Various labo-
ratories have embarked on live-imaging strategies to study cytoskeletal dynamics in
zebrafish; examples include microtubule imaging by injection of rhodamine-labeled
tubulin into zebrafish zygotes (Li et al., 2006, 2008), the labeling of microfilaments
by injection of plasmids that drive the transient expression of the F-actin-binding
domain of utrophin fused to mCherry (Andersen et al., 2010), and the creation of a
transgenic zebrafish line that expresses a GFP-tagged a-tubulin (Asakawa and
Kawakami, 2010).
For any experiment aimed at live visualization of the cytoskeleton, the key question
is, ‘‘What probe to use?’’’ Useful probes must fulfill multiple criteria: they must not
perturb the biology, they must report faithfully on the organization and dynamics of
the filament system, they must emit as many photons as possible for as long as
possible, and they must provide optimal contrast in the face of background signal
from the cytoplasm. The last consideration is often under-appreciated. For all cyto-
skeleton filaments and their associated binding proteins, there exist at least two
protein pools: (1) molecules that are in filaments or binding to filaments and
4 uhr et al.
M. W€

(2) molecules that are free in the cytoplasm and often exchange rapidly with the
filament-associated pool. In the thick cells of an early embryo, the majority of signal
may come from the soluble pool, which lowers the contrast for imaging the filament.
For this reason, the best probes for filament visualization in embryos are often not
tagged versions of the primary polymer subunits themselves (e.g. tubulin, actin), but
rather probes that bind selectively to the polymeric form of the subunit and thus have
a lower pool of free proteins. Such polymer-binding probes must be critically eval-
uated for unwanted interactions; they may tend to stabilize or bundle the polymer if
their levels are too high, and they may also bind selectively to certain subsets of the
filaments. Despite these caveats, this strategy has been quite successful, and here we
discuss its application to microtubule and actin visualization in zebrafish.
In this chapter, we describe methods for live imaging of microtubules and micro-
filaments in cleaving zebrafish embryos, the former by use of a transgenic zebrafish
line (W€ uhr et al., 2010) that expresses the GFP-tagged microtubule-binding domain
of ensconsin (Faire et al., 1999) and the latter by injection of the actin-binding
domain of utrophin bearing a GFP tag (Burkel et al., 2007), respectively. We also
compare the quality of images obtained by various optical platforms.

II. Maintaining the Breeding Competence of Zebrafish


throughout the Day

In the wild, zebrafish spawn at the onset of light in the morning (Detrich et al.,
1999). In the lab, this behavior potentially limits the time frame for experimentation
on cleavage-stage embryos. Several procedures exist for circumventing this restric-
tion: (1) use of isolation cabinets on light cycles that shift ‘‘morning’’’ for zebrafish
mating ensembles to suit the investigator or (2) use of in vitro fertilization, in which
females are squeezed and their eggs collected in defined medium or salmon ovarian
fluid to prevent activation (Corley-Smith et al., 1999; Sakai et al., 1997). The former
technique requires considerable cabinetry, whereas the latter can delay egg activa-
tion by at most 6 h (Siripattarapravat et al., 2009) and females require significant
time to recover from egg donation.
We have found that it is possible to obtain newly fertilized embryos over a large
portion of the day (6–8 h) with a simpler routine. We maintain males and females in
separate tanks in our zebrafish facility. One day before the experiment, two females
and one male are placed together in a crossing cage and allowed to acclimate, which
appears to predispose them to mate. The following morning (as defined by the light
cycle), males and females are separated immediately after they have spawned
sufficient embryos for initiation of experimentation. When more embryos are
required, the connubial trio is reunited. We are careful not to separate a threesome
for more than 2 h, because they become refractory to further mating that day.
Using this method, we typically obtain sufficient embryos to conduct three to four
experiments at 2-h intervals in a single day, provided that the fish are at optimal
age (4–12 months), well fed, and husbanded.
1. Live Imaging of the Cytoskeleton in Early Cleavage-Stage Zebrafish Embryos 5

III. Mounting Zebrafish Embryos for Live Imaging


A. Rationale
Proper mounting of cleaving embryos is one of the most important steps for live
imaging. To obtain images of high quality, one must immobilize the embryos and
place them within the working distance of moderate to high numerical aperture (NA)
objectives. In this context, upright and inverted microscopes have different experi-
mental advantages and disadvantages. Mounting of dechorionated embryos on an
upright microscope with a water-immersion or air objective is comparatively easy,
but one is limited to using objectives of modest NA. This restriction will be reduced as
vendors build immersion lenses with increasingly high numerical NA and long work-
ing distance. For example, Nikon has introduced a 25  1.1 NA water objective with a
2-mm working distance (Nikon Inc.), which delivers an extraordinary increase in
performance at low magnification. Unfortunately, such lenses are extremely expensive
and may require special adapters. In contrast, mounting embryos for inverted micros-
copy is more difficult but permits the use of oil-immersion, high NA objectives.
In our experience, the best method to immobilize a dechorionated embryo is to
place it in the pocket of an agarose specimen chamber cast on a Petri dish. The
lateral dimensions of the pockets are slightly smaller than the diameter of an
embryo (Fig. 1A) so that friction between the gently compressed embryo and the
walls of the pocket resists embryo movement. To cast a chamber with pockets that
can accommodate embryos of differing size, we use a plastic mold that creates
squares with sides of 550–700 mm; the depth of each pocket is 400 mm. Mounting
of the embryo is performed on a dissecting microscope after which the Petri dish is
transferred to the microscope used for imaging.
The increased difficulty of mounting an embryo for inverted microscopy arises
from its natural tendency to rotate so that the heavy yolk faces downward, that is,
opposite to the desired yolk-up orientation in the mounting pocket. In addition, the
specimen chamber must be cast on the glass cover slip of a suitable culture dish (Fig.
1B). The key, in our experience, is to use an agarose pocket of optimal dimensions, so
the embryo is prevented from rotating but not overly compressed.

B. Methods
1. Machine the embryo mounting mold from a suitable plastic or metal (e.g. plex-
iglass, aluminum) to the dimensions shown in Fig. 1A.
2. Pour melted 2% agarose in egg water (Westerfield, 2007) into a Petri dish. Insert
the mold into the agarose solution and put a weight on top. Allow the agarose to
set at 4 C. If mounting for inverted microscopy, use a coverslip-bottom culture
dish (e.g. MatTek Corp., Ashland, MA, USA).
3. Remove the mold and add egg water and embryos into the dish.
4. Using the dissecting microscope for visualization, dechorionate embryos with
two sharp No. 5 forceps.
6 uhr et al.
M. W€

[(Fig._1)TD$IG]

Fig. 1 Mounting of cleavage-stage zebrafish embryos. (A) Mold used to prepare agarose mounting pockets for cleavage-stage
embryos. Pockets of differing dimensions provide flexibility in mounting of embryos of heterogeneous size. (B) Configuration of
embryos for observation using an upright microscope equipped with a water-immersion (dipping) objective (left panel) or for
imaging via inverted microscopy and an oil-immersion objective (right panel).

5. With a blunt glass rod, maneuver an embryo into a square chamber whose
dimensions are slightly smaller than the embryo’s diameter. Orient the embryo
as desired and, if necessary, add low-melting-point agarose (0.8%) to fix the
embryo in position. Position additional embryos in remaining pockets as desired.
6. To reduce swaying when transferring, remove most of the egg water from the dish,
leaving just enough to cover the embryo. Transfer the dish to the microscope stage
and add egg water to a level sufficient for immersing the objective (Fig. 1B).
7. For inverted microscopy, carefully transfer the culture dish to the microscope stage
and bring the objective into oil contact with the dish’s coverslip (Fig. 1B). Add egg
water to cover the embryos so that they do not dehydrate during imaging.

IV. Live Imaging of Microtubules in Cleaving


Zebrafish Embryos

A. Rationale
Li et al. (2006, 2008) demonstrated real-time imaging of microtubules in cleaving
zebrafish embryos by injection of rhodamine-labeled tubulin at the one-cell stage.
Due to the thickness of early zebrafish blastomeres and the large proportion of
1. Live Imaging of the Cytoskeleton in Early Cleavage-Stage Zebrafish Embryos 7

rhodamine–tubulin that remains monomeric, the signal-to-noise ratio of fluorescent


microtubule polymer relative to the fluorescent subunit pool is substantially lower
than that achieved by comparable injection of thin, adherent tissue culture cells
(Zhai et al., 1996). Fig. 2A (right panel) shows an image from our laboratory of a
cleaving zebrafish embryo whose microtubules are labeled by incorporation of
injected Alexa-647-labeled tubulin. Although the contrast is unusually high, astral
microtubules are barely visible over the background from soluble tubulin. To obtain
higher contrast images of microtubule dynamics and organization in cleaving zebra-
fish blastomeres and to circumvent the injection step, we (W€ uhr et al., 2010)
generated a transgenic fish line, Tg(bactin2:HsENSCONSIN17-282-3xEGFP)hm1,
that expresses the microtubule-binding domain of ensconsin fused to three sequen-
tial GFP moieties (EMTB-3GFP). This probe was first tested in tissue culture cells,
where it was shown to associate tightly but dynamically with microtubules without

[(Fig._2)TD$IG]

Fig. 2 Microtubule imaging in cleaving embryos from the Tg(bactin2:HsENSCONSIN17-282-3xEGFP)hm1 zebrafish line.
(A) Transgenic, EMTB-3GFP-expressing embryos were injected with Alexa-647-labeled tubulin. The GFP and Alexa-647
signals were imaged simultaneously as described in Section IV.B.3. (B) Time lapse images of microtubules labeled by EMTB-
3GFP (enlargements of the boxed region of panel A, left side). The dynamic instability of the ends of individual microtubules can
be followed; the green arrows delineate a growing microtubule end, whereas the red arrows show a shortening microtubule.
(C) Ratiometric image generated as the composite of the two images shown in panel A. The image has been pseudocolored
to differentiate regions labeled preferentially by EMTB-3GFP (red) or by Alexa-647-labeled tubulin (blue). See Section IV.C for
further details. (D) Simultaneous labeling of microtubules and chromatin in embryos expressing EMTB-3GFP and H2B-
mCherry. See Section IV.C for details. (See Plate no. 1 in the Color Plate Section.)
8 uhr et al.
M. W€

perturbing either microtubule organization or dynamics when expressed at levels


appropriate for imaging (Faire et al., 1999). Later, EMTB-3GFP was adapted for
imaging microtubules in echinoderm embryos, where its increased contrast relative
to directly labeled tubulin was a compelling advantage (von Dassow et al., 2009). In
that work, the probe was introduced by mRNA injection at the one-cell stage, which
precluded live imaging of the first division. We introduced this probe into zebrafish
by making a transgenic line. In addition to permitting visualization immediately
after egg spawning and fertilization due to maternal expression of the transgene, the
transgenic approach has the advantage that we know the probe has not perturbed
embryonic development since the fish line is fully fertile. Below we compare the two
methods directly by injection of Alexa-647-labeled tubulin into the transgenic
zebrafish line. We have also used EMTB fused to a single GFP and expressed in
bacteria to visualize microtubules live in Xenopus egg extracts, demonstrating the
versatility of ensconsin-based probes.

B. Methods
1. Generation of Transgenic Zebrafish Lines
The transgenic line, Tg(bactin2:HsENSCONSIN17-282-3xEGFP)hm1, was created
in an unspecified wild-type strain by use of the Tol2Kit (Kawakami, 2004; Kwan
et al., 2007; Urasaki et al., 2006; W€
uhr et al., 2010). EMTB-3GFP expression is
driven by the beta actin promoter, chosen for its high expression levels. Beginning
with eight founders, we selected progeny that gave the highest expression levels
without detectable developmental toxicity. The line is now in its third generation, the
transgene is mostly stably transmitted, and expression of EMTB-3GFP remains
robust. EMTB-3GFP expression levels do tend to decline with generational passage
of the transgene, and we compensate for this by selecting adult females that express
the brightest eggs for propagation. Using identical methods, we have created a
transgenic zebrafish line in wild-type strain AB that expresses human histone
H2B fused in frame to mCherry2 for visualization of chromatin dynamics.

2. Preparation of Alexa-647-Labeled Tubulin and Embryo Microinjection


Tubulin was purified from calf brain and labeled with Alexa647-succinimide-
ester (Invitrogen) as described by Hyman et al. (1991). The ratio of fluorophore to
tubulin dimer was 0.7. Tg(bactin2:HsENSCONSIN17-282-3xEGFP)hm1 zebrafish
were mated, and, shortly after fertilization, 5 nL of labeled tubulin (11 mg/ml)
were injected through the yolk into the blastodiscs of embryos.

3. Laser-Scanning Confocal Microscopy


Images were recorded using a Zeiss LSM 710 inverted microscope equipped with
a 63 plan-apochromat objective (NA = 1.4). The pinhole was set at 63 mm, the
1. Live Imaging of the Cytoskeleton in Early Cleavage-Stage Zebrafish Embryos 9

pixel size was 0.11 mm, and pixel dwell time was 0.79 ms. Specimens were illumi-
nated simultaneously by argon (488 nm, 25 mW) and helium–neon (633 nm, 5 mW)
lasers. The emission spectra of GFP and Alexa 647 were recorded from 492 to
598 nm and from 637 to 755 nm, respectively. Images of a single focal plane were
collected at 7.7-s intervals.

C. Results
Fig. 2A compares the labeling of spindle microtubules by EMTB-3GFP and by
Alexa-647-tubulin in early anaphase of the second mitosis in an injected, transgenic
embryo: the left panel shows the EMTB-3GFP signal, whereas the right panel shows
the Alexa-647 signal. Movie S1 shows the same embryo in both imaging modalities
as anaphase commences. Spindle microtubules were brightly labeled in the green
channel, and their contrast with respect to the background was high. Neither spindle
morphology nor function appears to be perturbed by binding of EMTB-3GFP to
spindle microtubules (see Movie S1), as expected since the transgenic line is fertile.
Furthermore, the dynamic instability (Mitchison and Kirschner, 1984) of individual
microtubules at the front of asters as they expanded in telophase was readily detected
(see time-lapse imagery of Fig. 2B, which shows enlargements of the boxed region of
2A). In contrast, the same spindle observed in the Alexa-647 channel was less clearly
visualized; the central spindle microtubules were bright, but the background fluo-
rescence of the cytoplasm was high and individual microtubule ends in the asters
could not be identified with confidence.
The clarity of microtubule labeling by the transgenic zebrafish line is striking, but
one may ask whether EMTB-3GFP, which interacts with microtubules noncova-
lently, faithfully delineates all of the microtubules throughout the spindle.1 The
ensconsin (GFP) and tubulin (Alexa-647) signals correlate well, but subtle differ-
ences are apparent that cannot be explained by lower background fluorescence. To
compare microtubule labeling by the two approaches, we generated a composite,
ratiometric image from the two images of Fig. 2A. Fig. 2C shows that EMTB-3GFP
preferentially labels certain microtubule populations, which are shown in red in the
pseudocolored ratio image. These include the distal ends (distal with respect to the
centrosome) of astral microtubules and microtubules of the furrow microtubule
array [microtubules to the left of the spindle (Danilchik et al., 2003)]. Astral
microtubules proximal to the centrosomes label equivalently with the two probes
(green pseudocolor). EMTB-3GFP staining of the aster interaction zone (W€ uhr
et al., 2010), where the two asters meet, is very low (blue pseudocolor) compared
to the signal in the tubulin channel, suggesting the probe is selectively excluded from
these microtubules. Possible non-mutually exclusive explanations for the differential
labeling of microtubules by EMTB-3GFP could be: (1) EMTB-3GFP’s local

1
In this discussion we make the explicit assumption that labeling of microtubules by Alexa-647-tubulin is
uniform throughout the spindle.
10 uhr et al.
M. W€

concentration within the asters and the aster interaction zones might not be suffi-
ciently high to saturate its binding sites on the polymer, whereas this condition is met
for the dispersed microtubule ends at the astral peripheries; (2) EMTB-3GFP may
compete with other microtubule-associated proteins for binding to specific subsets
or subregions of spindle microtubules; and/or (3) the affinity of EMTB-3GFP may
be altered by regional regulation of posttranslational modification. We are currently
working on evaluating these hypotheses, with the aim of engineering a modified
probe with less differential binding. However, for most purposes the current probe
provides excellent microtubule imaging and is clearly superior to directly labeled
tubulin. For visualization of the advancing front of astral microtubules, selective
binding of the probe is even advantageous.
To observe microtubule and chromatin dynamics simultaneously during embry-
onic cleavage, we crossed the EMTB-3GFP line (hm1) with a beta-actin:H2B-
mCherry2 line [Tg(ba:h2b-mCherry2)hm13] (Fig. 2D, Movie S2). Fig. 2D shows
that the mCherry2-tagged histone labels the chromosomes at the metaphase plate (red
signal), and Movie S2 shows a blastomere undergoing a complete mitotic cycle of
chromosome condensation, spindle assembly, and chromosome partition. The high
signal-to-noise ratios of the EMTB-3GFP-labeled microtubules and of the
mCherry2-H2B-labeled chromosomes in these movies should facilitate quantitative
analysis of cleavage in the large blastomeres of the meroblastic zebrafish embryo.

V. Live Imaging of Microfilaments in Cleaving


Zebrafish Embryos
A. Rationale
Live imaging of microfilaments in the large blastomeres of the zebrafish embryo
is even more problematic than live imaging of microtubules, most likely because the
concentration of soluble, unpolymerized actin is very high compared to polymerized
actin in fibers. In a comparable embryo (Xenopus laevis), actin is present at
20 mM, and most is bound to sequestering proteins (Rosenblatt et al., 1995).
Sequestered monomer probably contributes to very high background staining if
actin is imaged via immunofluorescence of labeled actin monomers. Rhodamine-
labeled phalloidin, which binds only to F-actin with extremely high selectivity, has
been used to study microfilaments during cleavage of zebrafish embryos (Li et al.,
2008; Theusch et al., 2006), but this approach typically requires fixation and restricts
fixation methods to those that preserve filament structure (aldehyde fixation works,
organic solvent fixation does not). When labeled phalloidin has been used to image
microfilaments in living zebrafish embryos (Li et al., 2008), the probe must be
restricted to very low concentrations and thus low signal since phalloidin is in fact a
toxin derived from the death cap mushroom. Bement and co-workers developed
several FP-tagged probes for microfilaments based on the actin-binding calponin
homology domain of utrophin (Utr-CH)(Burkel et al., 2007). They showed that
1. Live Imaging of the Cytoskeleton in Early Cleavage-Stage Zebrafish Embryos 11

transient expression of GFP-Utr-CH in Xenopus oocytes by injection of synthetic


mRNA or plasmid constructs reports on the distribution of F-actin without perturb-
ing actin dynamics and is less toxic than phalloidin. It is possible, even likely, that
this probe binds selectively to a certain population of filaments, but this is difficult to
determine when we lack alternative methods for filament visualization. We
attempted to generate a transgenic fish line that would express Utr-CH-GFP, but
we were unable to establish founder fish, most likely due to probe toxicity during
development. As an alternative, we developed a protocol for injection of bacterially
expressed Utr-CH-GFP (kindly provided by David Burgess, Boston College) into the
one-cell stage of the zebrafish embryo. With this probe we could visualize cortical
microfilaments in living embryos with excellent contrast.

B. Method
His-tagged Utr-CH-GFP was expressed in Escherichia coli, purified via chroma-
tography on nickel columns, and flash frozen in 150 mM aspartic acid and 10 mM
HEPES solution (pH 7.2–7.3) in the laboratory of David Burgess. Shortly after
fertilization, zebrafish embryos were injected through the yolk into the blastodisc
with 2 nL of utrophin–GFP (1 mg/ml) and then mounted for upright microscopy as
described in Section III.B. Images were recorded using a Zeiss LSM 710 upright
microscope equipped with a 20 water-immersion objective (plan-apochromat DIC,
NA = 1.0). The pinhole was set at 32 mm, the pixel size was 0.59 mm, and pixel dwell
time was 1.58 ms. Specimens were illuminated with a 25-mWArgon laser at 488 nm.
Emission spectra were recorded from 492 to 598 nm. Images of a single focal plane
were collected at 41-s intervals.

C. Results
Figure 3 shows the lateral views of a one-cell zebrafish embryo undergoing
cytokinesis; the boxed regions are enlarged and shown at higher contrast below.
The cortical actin filaments are brightly labeled by Utr-CH-GFP as the cleavage
furrow develops. Cleavage appears to be unaffected by the utrophin probe (Movie
S3, the same embryo). At t = 0 min, the cell is in late telophase, and Utr-CH-GFP
fluorescence marks the aster–aster interaction zone (W€ uhr et al., 2010), where
cytokinesis will cleave the cell. By t = 26 min, the daughter cells have re-entered
mitosis, and Utr-CH-GFP stains comet tails behind rapidly moving vesicles (see
Movie S3 beginning at t = 18 min). These comet tails in mitotic cells presumably
represent Arp2/3 nucleated assemblies akin to Listeria comet tails, which have
been seen before in live embryos (Taunton et al., 2000; Velarde et al., 2007).
Comet tail assembly and vesicle movement were abolished by treatment of
embryos with the actin-depolymerizing agent cytochalasin B but were insensitive
to the anti-microtubule drug nocodazole (data not shown). Although further
validation of our utrophin-based labeling strategy is required, we consider it likely
12 uhr et al.
M. W€

[(Fig._3)TD$IG]

Fig. 3 F-actin imaging in an embryo injected with Utr-CH-GFP at the one-cell stage. Viewed from the side, the cortex is
brightly labeled. The enlargements (shown at higher contrast below the top panels) illustrate the dynamics of the microfilaments.
At t = 0 min, the cell is in late telophase, and Utr-CH-GFP staining marks the aster-aster interaction zone (yellow arrows). As the
daughter cells from the first division re-enter mitosis (t = 26 min), Utr-CH-GFP-labeled comets propel vesicles (e.g. red arrow)
whose movement is actin-dependent (see Section V.C for details). (See Plate no. 2 in the Color Plate Section.)

to be a useful, non-perturbing method for live imaging of the structure and


function of the actin cytoskeleton in zebrafish embryos. One important question
for future work is to what extent this probe reports on localization of all actin
filaments versus a subset with particular structure or biochemistry.

VI. Comparison of Microscopic Techniques for Imaging the


Cytoskeleton of Cleaving Zebrafish Embryos

Table I summarizes the advantages and disadvantages of four fluorescence-imag-


ing modalities we tested and provides representative micrographs obtained by each.
We note that these comments apply to the instrument we used and may not represent
fundamental limitations. For example, new gallium arsenide and avalanche photo-
diode detectors for scanning microscopes may increase sensitivity and lower noise
and photobleaching, albeit at additional cost. In the two-cell embryo imaged by
1. Live Imaging of the Cytoskeleton in Early Cleavage-Stage Zebrafish Embryos 13

Table I
Imaging the zebrafish microtubule cytoskeleton during cleavage: Advantages and disadvantages of four fluorescence-
imaging modalities

Type Advantages Disadvantages Example

Epifluorescence - Least expensive - Blurry, especially in thick


microscopy - Fast specimens at high
- Efficient collection of photons magnification (low
(low bleaching and contrast)
phototoxicity) - Low depth penetration
- CCD camera (low noise, high - Low contrast of
sensitivity) microtubules

Movie S4
Spinning disc confocal - Fast - Fixed pinhole size, only
microscopy - Less expensive than LSCM or optimal for certain
(SDCM) 2PM objectives
- Faster than LSCM and 2PM - Cross-talk between
- Fairly efficient collection of pinholes
photons (low bleaching and - Higher background than
phototoxicity) LSM especially in a thick
- Reasonable background specimen, leading to
suppression lower contrast images
- CCD camera
- Can generate optical sections Movie S5
Laser-scanning confocal - Can generate optical sections - Slow scan rate
microscopy - Good depth penetration - PMT is noisy (8-bit)
(LSCM) - Good background suppression - Expensive
- Very few photons collected
(bright signal required,
fast bleaching,
phototoxicity is likely
without care to limit
exposure)

Movie S6
Two-photon microscopy - Can generate optical sections - Slow scan rate
(2PM) - Excellent depth penetration - PMT is noisy (8-bit)
- Very good background - Expensive
suppression - Low brightness, fast
bleaching, phototoxicity
is likely (probably
because common
fluorophores are
optimized for one-photon
excitation)
Movie S6
14 uhr et al.
M. W€

conventional epifluorescence, the spindles of each blastomere are quite blurry due to
high background signal from out-of-focus fluorescence. Although basic measure-
ments of mitosis, such as rate of spindle assembly, spindle size, and spindle orien-
tation, could be made (see Movie S4), the dynamics of single microtubules, or even
bundles, cannot be resolved. Spinning disc confocal microscopy provides greater
clarity and single microtubule resolution of early mitotic spindles (Table I, Movie
S5) provided that they are relatively close to the surface of the embryo. Spinning disc
confocal often seems to provide an advantage over scanning laser confocal for live
imaging of the cytoskeleton in tissue culture cells due to lower photobleaching and in
some cases superior signal to noise. However, the lack of depth of penetration is a
problem for application of current Yokogawa spinning discs to zebrafish embryos.
We look forward to development of new spinning disc units with smaller pinholes
that are optimized for lower magnification work at depth. For the large, cleaving
blastomeres of the zebrafish embryo, laser-scanning confocal microscopy and two-
photon microscopy were clearly superior because of their greater depth of penetra-
tion. With care to limit exposure, photobleaching and phototoxicity were not a
problem with the one-photon modality. Both produced images of mitotic spindles
with very high spatial resolution and contrast (Table I, Movie S6). The dynamics of
individual microtubules were easily observed. Our current method of choice is laser-
scanning microscopy with one-photon excitation. This is partly due to the faster
bleaching of GFP caused by two-photon excitation, but of greater importance is the
higher signal-to-noise that was obtained with one-photon excitation (Movie S6). We
do not understand to what extent these factors represent fundamental advantages of
one-photon excitation versus limitations of the particular instruments we used.

VII. Discussion and Future Directions


In this chapter, we describe methods to image microtubules and actin filaments
in the thick cells of living cleavage-stage zebrafish embryos. Our methods make
use of FP-tagged filament-binding proteins, EMTB-3GFP for microtubules and Utr-
CH-GFP for microfilaments, that appear not to affect the dynamics or organization of
the respective polymers. These probes yield superior contrast during live imaging
when compared to filament labeling by fluorescently derivatized polymer subunits
themselves (i.e. tubulin, actin), presumably because the free pools of the binding
proteins are much lower than those of the filament subunits. Furthermore, we have
successfully developed a transgenic zebrafish line that expresses EMTB-3GFP and
shown that it yields valuable information about microtubule organization and function
in cleavage-stage zebrafish embryos (W€ uhr et al., 2010). For analysis of microtubule
function at later stages of development, we suggest that the EMTB-3GFP probe be
introduced into strains lacking pigmentation [e.g. nacre (Lister et al., 1999) and casper
(White et al., 2008)]. A disadvantage of EMTB-3GFP is that the probe turns over
rapidly on microtubules (Bulinski et al., 2001), which prevents its use to measure
microtubule turnover or sliding by photoactivation experiments (Mitchison, 1989). To
1. Live Imaging of the Cytoskeleton in Early Cleavage-Stage Zebrafish Embryos 15

enable such measurements, we suggest the creation of a zebrafish line that expresses
tubulin linked to a photoconvertible FP (McKinney et al., 2009). Although we have not
been able to generate a transgenic line that constitutively expresses Utr-CH-GFP,
suggesting subtle toxic effects of this probe expressed at high levels, we consider it
probable that such a line can be created, perhaps by using a weaker, or inducible,
promoter. Indeed, we envision a bright future for live analysis of cellular dynamics of
all kinds by use of transgenic zebrafish that express appropriate FP-tagged probes.
We chose to study the zebrafish embryo not only for its potential in understanding
cytoskeletal function during development, but also because the blastomeres created
during cleavage are among the largest of vertebrate cells. One of our goals is to
understand how the cytoskeleton scales with cell size to solve the physical challenges
of organizing large cells (W€uhr et al., 2008). To this end, a combination of the unique
experimental advantages of Xenopus egg extracts and transgenic zebrafish embryos
is likely to yield important experimental synergisms. Xenopus egg extracts provide
the opportunity to observe cytoskeletal function in vitro with single molecule reso-
lution (Needleman et al., 2010), and the system can be easily titrated with proteins
and drugs. Conversely, the zebrafish embryo provides experimental read out from a
truly in vivo system. The two systems, used in combination, are likely to lead to rapid
advances in our knowledge of cytoskeletal function and other cellular processes.

Acknowledgments
We thank David Burgess for generous gift of purified Utr-CH-GFP. H.W.D. was supported by NSF
grant ANT-0635470. N.D.O and S.G.M. were supported by NHGRI P50 HG004071 and NIDCD R01
DC010791. This work was supported by the National Institutes of Health (NIH) grant GM39565.

Appendix A. Supplementary Movies


Supplementary data associated with this chapter can be found, in the online
version, at http://www.elsevierdirect.com/companions/9780123870360.

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CHAPTER 2

Analysis of Cell Proliferation, Senescence,


and Cell Death in Zebrafish Embryos
Daniel Verduzco*,y and James F. Amatruda*,y,z
*
Departments of Pediatrics, UT Southwestern Medical Center, Dallas, Texas, USA
y
Molecular Biology, UT Southwestern Medical Center, Dallas, Texas, USA
z
Internal Medicine, UT Southwestern Medical Center, Dallas, Texas, USA

Abstract
I. Introduction: The Cell Cycle in Zebrafish
A. Forward-Genetic Screens
II. Zebrafish Embryo Cell-Cycle Protocols
A. Analysis of Cell Proliferation and Mitosis
B. Analysis of DNA Damage, Senescence, and Apoptosis
C. In Situ Hybridization
III. Screening for Chemical Suppressors of Zebrafish Cell-Cycle Mutants
IV. Conclusions
V. Reagents and Supplies
Acknowledgments
References

Abstract
Proper control of cell proliferation is critical for normal development, growth,
differentiation, and tissue homeostasis. Dysregulation of cell division and cell
death underlies almost all cancers, and contributes to the pathology of birth
defects and degenerative diseases. The zebrafish has proved to be an excellent
system for elucidating the roles of the cell cycle in normal development, and
ways in which dysregulation of cell proliferation contributes to disease. This
chapter describes the methods for studying the cell cycle in zebrafish embryos,
including protocols to examine cell proliferation, DNA damage, senescence, and
cell death.

METHODS IN CELL BIOLOGY, VOL 101 0091-679X/10 $35.00


Copyright 2011. Elsevier Inc. All rights reserved. 19 DOI 10.1016/B978-0-12-387036-0.00002-5
20 Daniel Verduzco and James F. Amatruda

I. Introduction: The Cell Cycle in Zebrafish


In multicellular organisms, the cell cycle is a fundamental feature of cellular
physiology that is critical for normal development, organogenesis, and tissue
homeostasis. Reflecting this central role, the molecular pathways that regulate cell
division in eukaryotes are evolutionarily conserved. Aberrations in the control of the
cell cycle are common in degenerative diseases and cancer. Therefore, analysis of
the cell cycle in nonmammalian organisms can illuminate the processes underlying
human development and disease. Forward genetic screens in yeast and Drosophila
have been invaluable for gene discovery and have made important contributions to
understanding pathways regulating cell proliferation. Importantly, it has been found
that the human orthologs of some genes identified in these organisms are misex-
pressed in human tumors (Hariharan and Haber, 2003). Zebrafish have proven to be
an excellent model of early vertebrate development (Driever et al., 1996; Haffter
et al., 1996) and also of a wide variety of human diseases such as cancer, anemia,
cardiovascular defects, neuromuscular conditions, kidney disease, and host–patho-
gen interaction, to name a few examples (Ackermann and Paw, 2003; Bassett and
Currie, 2003; Drummond, 2005; Goessling et al., 2007; Hsu et al., 2007; Lambrechts
and Carmeliet, 2004; Miller and Neely, 2004).
The particular advantages that make zebrafish ideal for developmental embryol-
ogy – including external fertilization of oocytes, transparent embryos, and rapid
embryonic development – also provide the opportunity to study early cell divisions,
tissue-specific cellular proliferation, and, more broadly, the role of cell-cycle genes
in development and disease. A number of methods and markers have been success-
fully applied to investigate the cell cycle in zebrafish embryos, including video
microscopy (Kane, 1999; Kane et al., 1992), histone-green fluorescent protein
fusions (Pauls et al., 2001), 5-bromo-2-deoxyuridine (BrdU) labeling (Baye and
Link, 2007; Link et al., 2000), proliferating cell nuclear antigen (PCNA) RNA and
protein expression (Koudijs et al., 2005; Wullimann and Knipp, 2000), phosphohis-
tone H3 (pH3) immunohistochemistry (Shepard et al., 2005), and minichromosome
manintenance protein expression (Ryu and Driever, 2006).
Studies of the developing zebrafish embryo have revealed similarities to the
early cell divisions of other vertebrates such as Xenopus. In the zebrafish, the
first seven cell divisions are synchronous and cycle rapidly between DNA rep-
lication (S phase) and mitosis (M phase) without the intervening gap phases, G1
and G2 (Kimmel et al., 1995). The midblastula transition (MBT) ensues during
the 10th cell division, which is approximately 3 h postfertilization. MBT is
accompanied by loss of division synchrony, increased cell-cycle duration, acti-
vation of zygotic transcription, and the onset of cellular motility (Kane and
Kimmel, 1993). Embryonic cells first exhibit a G1 gap phase between the M
and S phases during MBT. Recently, Dalle Nogare et al. demonstrated that during
cycles 11–13, embryonic cells acquire a G2 phase in a transcription-independent
fashion, through inhibition of Cdk1 and its activating phosphatase, Cdc25a
(Dalle Nogare et al., 2008).
2. Analysis of Cell Proliferation, Senescence, and Cell Death in Zebrafish Embryos 21

Further understanding of cell-cycle regulation in zebrafish embryos was obtained


by studying their responses to various cell-cycle inhibitors, including aphidocolin,
hydroxyurea, etoposide, camptothecin, and nocodazole (Ikegami et al., 1997a,
1997b, 1999). Exposure to these agents after MBT induces cell-cycle arrest, some-
times accompanied by initiation of an apoptotic program. However, prior to MBT,
the embryonic cells continue to divide, often with deleterious effects, after exposure
to cell-cycle inhibitors. These studies indicate that zebrafish embryos do possess
cell-cycle checkpoints, but they are not functional until after MBT.
Later developmental stages of zebrafish embryogenesis provide the opportunity to
study the cell cycle in distinct tissue types. Studies of cell-cycle regulation in older
embryos (10–36 h postfertilization) have focused on the developing eyes and central
nervous system. Lineage analysis of CNS progenitor cells revealed a correlation
between morphogenesis and cell-cycle number, implying that the nervous system
development may be at least partially regulated by the cell cycle (Kimmel et al.,
1994). Although most developing vertebrate embryos exhibit a constant lengthening
of the cell-cycle duration throughout the development, meticulous analysis of cell
number in the developing zebrafish retina revealed a surprising mechanism of
modulated cell-cycle control. Li et al. (2000) reported that the retinal cell-cycle
duration temporarily slows between 16 and 24 h postfertilization (hpf), followed by
an abrupt change to more rapid cell divisions.
Several studies have elucidated a role for the zebrafish cell-cycle machinery in
tissue differentiation during development and in the regenerative response to injury.
Bessa et al. (2008) found that Meis1, a marker of the eye primordium, promotes
G1-S progression and a block of differentiation in the zebrafish eye through regu-
lation of cyclin D1 and c-myc expression. Fischer and coworkers showed that loss of
caf1b in zebrafish (by mutation or MO injection) leads to an S-phase arrest and
eventual apoptosis that can be rescued by p53 deficiency. However, loss of caf1b also
leads to a block in differentiation in tissues that express caf1b, implicating caf1b in
the switch from proliferation to differentiation (Fischer et al., 2007). The effect of
loss of early mitotic inhibitor 1 (emi1) on somite formation was evaluated by Zhang
et al. These authors found that cell-cycle progression was required for proper somite
morphogenesis, but not for the formation of the segmentation clock (Zhang et al.,
2008). The role of the cell cycle in regeneration has also been assessed. Certain
traumas result in loss of hair cell precursors, which results in deafness in vertebrates.
Hernandez et al. (2007) used BrdU labeling and transgenic green fluorescent protein
reporter lines to study hair cell regeneration, identifying proliferation-dependent and
-independent mechanisms of hair cell renewal.

A. Forward-Genetic Screens
Several groups have carried out forward-genetic screens to identify mutations that
alter cell proliferation in embryos. Shepard et al. used pH3 as a marker of cell
proliferation in a two-generation haploid genetic screen. They identified seven
mutant lines with different alterations in pH3 immunoreactivity. At least two of
22 Daniel Verduzco and James F. Amatruda

these lines demonstrate aneuploidy and increased cancer susceptibility as hetero-


zygotes (Shepard et al., 2005, 2007). Using a similar screening strategy, Pfaff et al.
identified a further set of genes required for cell proliferation mutants, among which
was Scl-interrupting locus, which was identified as a novel, vertebrate-specific
regulator of mitotic spindle assembly (Pfaff et al., 2007). Koudijs et al. (2005) used
PCNA expression in the CNS as a readout to identify new mutations in repressors of
the hedgehog signaling pathway. Finally, another screen for genes that control eye
growth uncovered two zebrafish lines mutant for the anaphase-promoting complex/
cyclosome (APC/C) (Wehman et al., 2006). Loss of APC/C results in a loss of
mitotic progression and apoptosis; in this study, co-labeling with BrdU and pH3
revealed cells undergoing mitotic catastrophe.
In this chapter, we provide protocols to characterize the various phases of cell
division in zebrafish embryos, and protocols to detect DNA damage, senescence,
and cell death. Assays discussed in this chapter include DNA content analysis by
flow cytometry, whole-mount embryonic antibody staining, mitotic spindle anal-
ysis, BrdU incorporation, cell death analysis, and in situ hybridization with cell-
cycle regulatory genes. Each assay targets different phases of the cell cycle and in
total create a detailed picture of zebrafish embryo cell proliferation. Although our
studies have focused on embryonic assays for cell-cycle characterization, it is
likely that these protocols can be modified to study adult tissues. These proto-
cols can be applied to a variety of experiments such as characterization of the
cell-cycle phenotypes of mutants or the analysis of RNA overexpression and
morpholino knockdown of cell-cycle regulatory genes. Furthermore, the genetic
tractability of the zebrafish system (Patton and Zon, 2001) makes it an excellent
organism in which to pursue forward genetic screens for mutations or chemical
screens for novel compounds that alter cell division using one or more of these
cell-cycle assays.

II. Zebrafish Embryo Cell-Cycle Protocols1

A. Analysis of Cell Proliferation and Mitosis


1. DNA Content Analysis
A profile of the cell cycle in disaggregated zebrafish embryos or adult tissue can
be obtained through DNA content analysis. In this technique, cells are stained with a
dye that fluoresces upon DNA binding, such as Hoechst 33342 or propidium iodide.
The intensity of fluorescence is proportional to the amount of DNA in each cell
(Krishan, 1975). Analysis by fluorescence-activated cell sorting (FACS) generates a
histogram showing the proportion of cells that have an unreplicated complement of
DNA (G1 phase), those that have a fully replicated complement of DNA (G2 or M
phase), and those that have an intermediate amount of DNA (S phase).

1
Items in boldface indicate reagents and supplies listed in Section V.
2. Analysis of Cell Proliferation, Senescence, and Cell Death in Zebrafish Embryos 23

Protocol:
All steps are performed on ice except for the dechorionation (step 1) and RNAse
incubation (step 9).
1. Dechorionate embryos and wash with E3. Analysis of single embryos is possi-
ble, though in practice, we typically pool approximately 40 embryos/tube.
2. Disaggregate embryos (using small pellet pestle) in 500 ml of DMEM (or other
tissue culture medium) + 10% fetal calf serum in a matching homogenizing tube.
3. Bring volume to 1 mL with DMEM/serum and remove aggregates by passing
cell suspension sequentially through 105 and 40 mm mesh.
4. Count a sample using a hemocytometer.
5. Place volume containing at least 2  106 cells in a 15 mL conical tube, and bring
volume to 5 mL with 1 phosphate-buffered saline (PBS).
6. Spin at 1200 rpm for 10 min at 4  C.
7. Carefully aspirate off liquid and gently resuspend cell pellet in 2 mL propidium
iodide solution.
8. Add 2 mg of DNAse-free RNAse (Roche). This step is necessary to remove
double-stranded RNA, which binds propidium iodide.
9. Incubate in the dark at room temperature for 30 min.
10. Place samples on ice and analyze on FACS machine.
Note: Samples can also be fixed in Ethanol, allowing multiple samples or time
points to be collected for subsequent analysis.
1. Harvest cells and prepare single cell suspension in DMEM/serum as above, steps
1–4.
2. Wash cells in PBS and resuspend at 1–2  106 cells/mL.
3. To 1 mL cells in a 15 mL polypropylene, V-bottom tube, add 3 mL ice-cold
absolute EtOH. To avoid clumping, add the ethanol dropwise while vortexing
the sample.
4. Fix cells for at least 1 h at 4  C. Cells may be stored for several weeks at 20  C
before undergoing PI staining.
5. Wash cells twice in 1 PBS. We typically increase the speed of centrifugation to
2500 rpm because the cells do not pellet as readily after EtOH fixation.
6. Resuspend the pellet in 1 mL propidium iodide solution. Add 2 mg of DNAse-
free RNAse (Roche) and incubate for 3 h at 4  C.
7. Place samples on ice and analyze on FACS machine.

2. Whole Mount Immunohistochemistry with Mitotic Marker pH3


Histone H3 phosphorylation is considered to be a crucial event for the onset of
mitosis and this antibody has been widely used in Drosophila and mammalian cell
lines as a mitotic marker (Hendzel et al., 1997). Two members of the Aurora/AIK
kinase family, Aurora A and Aurora B, phosphorylate histone H3 at the serine 10
residue (Chadee et al., 1999; Crosio et al., 2002). Increased serine 10 phosphorylation
24 Daniel Verduzco and James F. Amatruda

of histone H3 has been seen in transformed fibroblasts (Chadee et al., 1999), suggest-
ing that this antibody could make an excellent marker for cell proliferation in the
zebrafish as well as detecting cell-cycle mutations that may result in transformed
phenotypes. In zebrafish, the pH3 antibody stains mitotic cells throughout the embryo
(Fig. 1A). pH3 staining in developing organs like the nervous system increases as they
undergo proliferation during distinct developmental stages.

Protocol:
1. Fix embryos overnight at 4  C in 4% paraformaldehyde (PFA).
2. Permeabilize embryos for 7 min in 20  C acetone.

[(Fig._1)TD$IG]

Fig. 1 Useful techniques for the study of the cell cycle, proliferation, or apoptosis as shown in zebrafish embryos. (A)
Antibody staining against phosphorylated histone H3 in wild-type 24 hpf embryos. (B) BrdU incorporation to mark cells in S
phase in the tail of a 28 hpf wild-type embryo. (C and D) Apoptotic cells can be visualized by TUNEL (C, wild-type 24 hpf
embryo) or acridine orange (D, 24 hpf crash&burn mutant embryo). (E) Anti-a tubulin can be used to examine mitotic spindle
formation. (F) DNA content analysis shows the population of embryonic cells present in all phases of the cell cycle.
2. Analysis of Cell Proliferation, Senescence, and Cell Death in Zebrafish Embryos 25

3. Wash embryos in H2O followed by two 5-min washes in PBS plus Tween-20
(PBST).
4. Incubate for 30 min at room temperature in block.
5. Incubate overnight at 4  C in rabbit anti-pH3 at a concentration of 1.33 mg/mL
in block. Two different sources of antibody have been used: Santa Cruz
Biotechnology and an antiphosphopeptide polyclonal antibody to the sequence
(ARKS[PO4]TGGKAPRKQLC) made and affinity purified by Genemed
synthesis.
6. Wash 4  15 min in PBST.
7. Incubate for 2 h at room temperature in horseradish peroxidase-conjugated
secondary goat anti-rabbit IgG (Jackson Immunoresearch) at a concentration
of 3 mg/mL in block.
8. Wash 4  15 min in PBST.
9. Develop in the dark for 3–5 min at room temperature in diaminobenzidine
(DAB) solution (0.67 mg/mL DAB in 15 mL of PBST to which 12 ml of 30%
H2O2 has been added).
10. Wash in PBST and store embryos at 4  C in PFA.

3. Mitotic Spindle/Centrosome Detection


Study of the mitotic spindle and centrosomes is an important step in understanding
mutants with cell-cycle defects, particularly those whose phenotypes appear to be
related to problems in mitosis. Genomic instability is one of the main alterations seen
in human cancers and such unequal segregation of chromosomes can be caused by
problems in mitotic spindle formation or centrosome number (Kramer et al., 2002).
In this protocol, anti-a-tubulin labels the mitotic spindle, anti-b-tubulin the centro-
some, and diamidino-2-phenylindole (DAPI) the DNA.

Protocol:
1. Fix embryos in PFA for 4 h at room temperature.
2. Dehydrate in methanol at 20  C for at least 30 min.
3. Rehydrate embryos in graded methanol:PBST series (3:1, 1:1, 1:3) for 5 min each.
4. Wash 1  5 min in PBST.
5. Place in 20  C acetone for 7 min.
6. Wash 3  5 min in PBST.
7. Incubate for 1 h at room temperature in block.
8. Incubate in monoclonal mouse a-tubulin antibody (Sigma) at a concentration of
1:500 and in polyclonal rabbit g-tubulin antibody (Sigma) at a concentration of
1:1000 (both diluted in block) at 4  C overnight.
9. Wash 4  15 min in PBST.
10. Incubate in rhodamine-conjugated goat anti-mouse secondary (Molecular
Probes) at 1:600 dilution and fluorescein-conjugated goat anti-rabbit secondary
(Jackson Immunoresearch) at 1:600 dilution for 2 h room temperature.
26 Daniel Verduzco and James F. Amatruda

11. Wash 2  15 min in PBST.


12. Include a 1:500 dilution of 100 mM DAPI during the third wash to stain DNA.
13. Wash 2  15 min in PBST.
14. For observation by epifluorescence microscopy, embryos are mounted on glass
slides with VectaShield mounting media (Vector Labs) and coverslipped. To
permit the specimen to lie flat, it is helpful to remove the yolk using forceps or a
tungsten needle. Alternatively, for embryos >18 hpf, the tail can be cut off from
the embryo and mounted on the slide.

4. BrdU Incorporation
BrdU is a nucleoside analog that is specifically incorporated into DNA during S
phase (Meyn et al., 1973) and can subsequently be detected with an anti-BrdU-specific
antibody. This technique has been used to label replicating cells in zebrafish embryos
(Larison and Bremiller, 1990) and adults (Rowlerson et al., 1997). The following
protocol is designed to label a fraction of proliferating cells in zebrafish embryos, to
allow comparison of the replication fraction of different embryos (Fig. 1 B). If the
embryos are chased for varying amounts of time after the BrdU pulse, then fixed and
stained for both BrdU and pH3 (section B), the transit of cells from S phase into G2/M
can be assessed. This is useful in analyzing mutants with mitotic phenotypes.

Protocol:
1. Dechorionate embryos and chill 15 min on ice in E3.
2. Prepare cold 10 mM BrdU/15% dimethylsulfoxide in E3 and chill on ice. Place
embryos in BrdU solution and incubate for 20 min on ice to allow uptake of BrdU.
3. Change into warm E3 and incubate exactly for 5 min at 28.5  C. Note: longer
incubation times will result in more cells being labeled.
4. Fix for 2 h at room temperature in PFA. Longer fixation may decrease the staining.
5. Transfer to methanol at 20  C overnight. All subsequent steps are performed
at room temperature unless otherwise noted.
6. Rehydrate in graded methanol:PBST series (3:1, 1:1, 1:3) for 5 min each.
7. Wash 2 in PBST, 5 min.
8. Digest embryos in 10 mg/mL proteinase K, 10 min.
9. Wash in PBST. Refix in PFA for not more than 20 min.
10. Wash quickly 3 in H2O, then 2 in 2N HCl.
11. Incubate for 1 h in 2N HCl. This step denatures the labeled DNA to expose the
BrdU epitope.
12. Remove the 2N HCl solution from the embryos and neutralize in 0.1 M borate
buffer, pH 8.5, 20 min, room temparature.
13. Rinse several times in PBST. Block for 30 min in BrdU blocking solution.
14. Incubate in monoclonal anti-BrdU antibody at a dilution of 1:100 in BrdU block
for 2 h at room temperature or overnight at 4  C. (If carrying out simultaneous
BrdU/pH3 staining, add the primary anti-pH3 antibody as described in Section
B, except that BrdU block is used.)
2. Analysis of Cell Proliferation, Senescence, and Cell Death in Zebrafish Embryos 27

15. Wash 5  10 min in PBST.


16. Incubate for 2 h room temperature with horseradish peroxidase or fluorophore-
conjugated anti-mouse secondary antibody. (For simultaneous BrdU/pH3 stain,
add a fluorescent anti-rabbit antibody as well.)
17. Wash 5  10 min in PBST. If using fluorescent secondary, mount embryos as
described in Section C, step 14.
18. If using HRP-conjugated secondary antibody, develop in the dark for 3–5 min at
room temperature in DAB solution (0.67 mg/mL DAB in 15 mL of PBST to
which 12 mL of 30% H2O2 has been added). When staining is complete, wash
3  5 min in PBST, then fix in PFA.

B. Analysis of DNA Damage, Senescence, and Apoptosis


1. COMET Assay
The Comet Assay, also known as the single cell microgel electrophoresis assay, is a
highly sensitive technique that is used to detect DNA damage at the single-cell level
(Singh et al., 1988). Cells are embedded into a thin agarose gel, through which a
current is run allowing for migration of DNA. Smaller fragments of DNA, resulting
from DNA damage, will travel more quickly and appear as a tail to the nucleus
‘‘comet head.’’ The comets can be visualized using a nuclear stain, such as SYBR
green, and visualized under a fluorescent microscope. The following protocol is
designed to isolate cells from zebrafish embryos and detect any kind of DNA
damage. Variations of this technique allows for specific detection of double-stranded
break.

Protocol:
1. Dechorionate embryos and wash with E3. Typically, about 25–50 embryos are
used.
2. Disaggregate embryos (using small pellet pestle) in 500 mL of DMEM (or other
tissue culture medium) + 10% fetal calf serum or lamb serum in a matching
homogenizing tube.
3. Bring volume to 1 mL with DMEM/serum.
4. Count the samples using a hemocytometer.
5. Spin down cells at 3000 RPM and resuspend in PBS to a concentration of
1  105 cells/mL.
6. Combine 10 mL of cells with 90 mL of molten LMAgarose (Trevigen) pre-
warmed to 37  C. Pipette 75 mL of the cell agarose mixture onto a
CometSlide (Trevigen) prewarmed to 37  C.
7. Incubate the slide flat at 4  C for 30 min in the dark to allow the gel to
solidify.
8. Immerse the slide in Lysis solution (Trevigen) containing 9% DMSO. After this
point, it is very important to maintain the slide in low-light conditions.
9. Dry off the slide, and immerse it in alkaline solution for 30 min.
28 Daniel Verduzco and James F. Amatruda

[(Fig._2)TD$IG]

Fig. 2 The COMET assay reveals double-strand DNA breaks. Images of zebrafish embryo cells from a
single-cell microgel electrophoresis experiment. (A) Unirradiated control cell. (B) A cell after exposure to
2000 R (20 Gy) g-irradiation. The arrow indicates the comet tail, composed of fragmented DNA
(Verduzco and Amatruda, unpublished data).

10. Prepare a large horizontal electrophoresis apparatus by filling the chamber with
fresh alkaline electrophoresis buffer and adjusting the volume of the alkaline
electrophoresis buffer such that the current is 300 mA when the voltage is set to
25 V. Additionally, the chamber should be prepared and used in a 4  C room.
11. Place the slide in the electrophoresis apparatus. Run for 30 min at 4  C in the
dark.
12. Dry off the slide. Rinse by dipping in ddH2O.
13. Incubate the slide in 70% EtOH for 5 min at room temperature in the dark.
14. Air-dry the slide for 1 h.
15. Pipette 50 mL of SYBR green staining solution (Trevigen) onto the microgel on
the slide.
16. View the slide using fluorescence microscopy under a fluorescein filter (Fig. 2).
17. Comets can be analyzed using CometScore by Tritek Corp or another similar
software program.

2. Detection of Senescence-Associated b-Galactosidase


The study of cellular senescence was initiated by Hayflick and Moorhead (1961).
Cellular senescence pertains to the cessation of cell replication and certain morpho-
logical and transcriptional changes that occur when cells permanently cease divid-
ing. Although it is unclear whether the events that occur during in vitro cellular
senescence also occur during organismal aging (Hayflick, 2007; Masoro, 2006),
studies have revealed strong connections between cellular senescence, cancer, and
age-related diseases (Campisi, 2005). Cellular senescence most likely arose evolu-
tionarily as a mechanism to defend against tumorigenesis (Shay and Roninson,
2004). When a cell is afflicted by stress that may result in transformation (such as
oxidative stress, DNA damage, or overepxpression of oncogenes), tumor-suppressor
genes such as p53 may force the cell to undergo senescence-induced arrest. Arrested
cells are functional but are not a risk for tumor initiation. Senescence also occurs as
the ends of chromosomes, the telomeres, shorten. During reach replication cycle, if
2. Analysis of Cell Proliferation, Senescence, and Cell Death in Zebrafish Embryos 29

no active telomerase is present (Bodnar et al., 1998), the telomeres shorten, leading
eventually to critically short telomeres that may interfere with gene expression and
genomic stability (Shay and Wright, 2006). Normal cells senesce before telomeres
shorten to the point of causing genomic instability, therefore instilling a counting
mechanism that confirms Hayflick’s observation in 1961 (Shay and Wright, 2006).
Senescent cells lose sensitivity to mitogens or growth factors, repress cell-cycle
genes, such as cdk2, and become insensitive to apoptotic signals. Morphological
changes occur resulting in an enlarged shape and flattened body (Ben-Porath and
Weinberg, 2005), as well as expression of unique markers, and many of unknown
function such as b-galactosidase activity at pH 6.0 (Dimri et al., 1995). Kishi and
coworkers have used senescence-associated b-galactosidase staining in several stud-
ies to characterize senescence in normal and mutant zebrafish embryos and during
aging of zebrafish adults (Kishi, 2004; Kishi et al., 2003, 2008; Tsai et al., 2007).

Protocol:
We have used the Senescence-Associated b-Galacotsidase Detection Kit from
Sigma (CS 0030). The following protocol adapts the manufacturer’s instructions
specifically for use with zebrafish embryos, and is kindly provided by Jenny
Richardson and Dr. Elizabeth Patton, Edinburgh Cancer Research Center:
1. Dechorionate embryos and add 1.5 mL of 1 fixation buffer (prepared from
10 Sigma Senescence Fixation Buffer). Incubate overnight at 4  C.
2. Wash embryos 4 times in 1 PBS, 1 h each wash.
3. Make up the senescence staining mixture as per the manufacturer’s protocol. Add
1 mL to embryos and incubate for 24 h at 37  C.
4. Wash embryos 3 times in 1 PBS, 10 min each wash.
5. Embryos can be stored at 4  C in 1 PBS and 0.1% NaN3 or in 70% glycerol at
4  C.
An alternative protocol was described by Kishi et al. (2008) in a recent paper
describing a senescence-based genetic screen. The following protocol is adapted
from Kishi et al., ‘‘The identification of zebrafish mutants showing alterations in
senescence-associated biomarkers,’’ PLoS Genet. 2008 Aug 15;4(8):e1000152:
1. Fix embryos or adult zebrafish in 4% PFA in 1 PBS at 4  C (for 3 days in adults
and overnight in embryos).
2. Wash three times for 1 h in PBS with pH 7.4 and for a further 1 h in PBS with pH
6.0 at 4  C.
3. Stain the samples overnight at 37  C in 5 mM potassium ferrocyanide, 5 mM
potassium ferricyanide, 2 mM MgCl2, and 1 mg/ml X-gal in PBS adjusted to pH
6.0.

3. Apoptosis Detection by TUNEL Staining


Apoptosis is a form of programmed cell death that eliminates damaged or
unneeded cells. It is controlled by multiple signaling pathways that mediate
30 Daniel Verduzco and James F. Amatruda

responses to growth, survival, or death signals. Cell-cycle checkpoint controls are


linked to apoptotic cascades and these connections can be compromised in diseases
including cancer. The defining characteristics of apoptosis are membrane blebbing,
cell shrinkage, nuclear condensation, segmentation, and division into apoptotic
bodies that are phagocytosed (Wyllie, 1987). The DNA strand breaks that occur
during apoptosis can be detected by enzymatically labeling the free ends with
modified nucleotides, which can then be detected with antibodies (Gavrieli et al.,
1992).
Protocol:
1. Embryos are fixed overnight at 4  C in PFA.
2. Wash in PBS and transfer to methanol for 30 min at 20  C.
3. Rehydrate embryos in a graded methanol:PBST series (3:1, 1:1, 1:3) for 5 min
each.
4. Wash 1 5 min in PBST.
5. Digest embryos in proteinase K (10 mg/mL) at room temperature (1 min for
embryos younger than 16 hpf, 2 min for embryos older than 16 hpf).
6. Wash twice in PBST.
7. Postfix in PFA for 20 min at room temperature.
8. Wash 5 5 min in PBST.
9. Postfix for 10 min at 20  C with prechilled ethanol:acetic acid 2:1.
10. Wash 3 5 min in PBST at room temperature.
11. Incubate for 1 h at room temperature in 75 mL equilibration buffer (TdT –
ApopTag Peroxidase In Situ Apoptosis Detection Kit from Serologics
Corporation).
12. Add small volume of working strength TdT (reaction buffer and TdT at a ratio of
2:1 plus 0.3% Triton) (Serologics Corporation).
13. Incubate overnight at 37  C.
14. Stop reaction by washing in working strength stop/wash buffer (1 mL concen-
trated buffer from Serologics Kit with 34 mL water) for 3–4 h at 37 C.
15. Wash 3  5 min in PBST.
16. Block with 2 mg/mL BSA, 5% sheep serum in PBST for 1 h at room
temperature.
17. Incubate in anti-digoxigenin peroxidase antibody included in kit (full strength).
18. Wash 4  30 min PBST at room temperature.
19. Develop in the dark for 5 min at room temperature in DAB solution (0.67 mg/
mL in 15 mL of PBST) and 12 ml 30% H2O2.
20. Wash in PBST and store embryos at 4  C in PFA.

4. Apoptosis Detection by Acridine Orange


Another method of apoptotic cell detection that can be performed on living
embryos is acridine orange staining. The basis of this method is that the ATP-
dependent lysosomal proton pump is preserved in apoptotic but not necrotic cells;
2. Analysis of Cell Proliferation, Senescence, and Cell Death in Zebrafish Embryos 31

therefore, apoptotic cells will take up the acridine orange dye whereas living or
necrotic cells will not (Darzynkiewicz et al., 1992). This method is useful for
identifying mutants based on an apoptotic phenotype in order to further characterize
them in living assays.

Protocol:
1. Live dechorionated embryos are incubated in a 2 mg/mL solution of acridine
orange (Sigma) in 1 E3 for 30 min at room temperature.
2. Embryos are washed 5 quickly in E3 and then visualized on a stereo dissecting
microscope equipped for FITC epifluorescence.

C. In Situ Hybridization
RNA expression analysis by in situ hybridization of antisense probes in whole-
mount zebrafish embryos is a commonly used technique to localize expression of
developmental regulatory genes. While the technique is not exceptionally quantita-
tive, it can reveal stark differences in gene expression. More quantitative analysis of
gene expression, such as Northern blotting, and real-time polymerase chain reaction
do not permit the examination of alterations in tissue-specific expression or an
expression pattern.
Cell division is a highly controlled process that involves regulation at both the
transcriptional and posttranslational stages. Cyclins are a class of proteins that play
critical roles in guiding cells through the G1, S, G2, and M phases of the cell cycle by
regulating the activity of the cyclin-dependent kinases. The name cyclin alludes to the
fact that their expression levels oscillate between peaks and nadirs that are coordi-
nated with particular phases of the cell cycle (reviewed in Murray, 2004). The tightly
regulated expression of these important cell-cycle genes incorporates transcriptional,
translational, and posttranslational controls. Many genes involved in cell-cycle reg-
ulation are specifically expressed during the cell-cycle phase in which they act.
Zebrafish orthologs of cell-cycle regulatory genes such as PCNA and cyclins have
been found to possess similar expression patterns throughout the proliferative tissues
of developing zebrafish embryos (C. Thisse, B. Thisse, unpublished data and www.
zfin.org). In situ hybridization for cell-cycle regulatory genes can be performed
using previously published in situ hybridization protocols (Jowett, 1999; Thisse
et al., 1993, 1994).

III. Screening for Chemical Suppressors of Zebrafish Cell-Cycle


Mutants

Another way to probe the cell cycle is via chemical agents. Chemical screens
could identify novel compounds that are useful tools for studying the cell cycle.
Furthermore, mutations in cell-cycle genes are commonly found in human cancer.
32 Daniel Verduzco and James F. Amatruda

Given the need to improve upon current cancer therapy, one approach is to identify
small molecule suppressors that bypass the consequences of specific cell-cycle gene
mutations. Akin to the use of genetic modifier screens to identify secondary muta-
tions that enhance or suppress a primary defect (St Johnston, 2002), chemical
suppressor screens would directly identify small molecules that rescue a genetic
phenotype. If the phenotype is disease-related, such compounds might represent lead
therapeutic agents.
Zebrafish have recently been utilized in chemical screens to identify compounds
that perturb specific aspects of development (Anderson et al., 2007; Bayliss et al.,
2006; den Hertog, 2005; Khersonsky et al., 2003; Peterson et al., 2000, 2004). The
zebrafish system offers several advantages for chemical screens, providing infor-
mation on tissue specificity and toxicity, and accounting for compound activation via
drug metabolism. Furthermore, cells are not transformed and are in their normal
physiological milieu of cell–cell and cell–extracellular matrix interactions.
Murphey et al. (2006) carried out a high-throughput chemical screen to detect small
molecules capable of perturbing the cell cycle during zebrafish development, iden-
tifying several compounds that were not previously detected in cell-based screens of
the same library. As another application of this technique, Stern et al. screened a
16,000-compound library to identify small molecules capable of suppressing the cell
proliferation defect in the crash&burn cell-cycle mutant (Stern et al., 2005). This
technology could easily be applied to other cell-cycle mutants and modified to use
cell-cycle assays other than pH3 staining. In addition, such chemical suppressor
screens could be applied to any zebrafish model of human disease (Dooley and Zon,
2000). For these reasons, we provide a detailed protocol below.
The following protocol can be repeated weekly giving a throughput of more than
1000 compounds per week for a recessive lethal mutation. In the case of homozygous
viable mutants, the throughput could be improved by using fewer embryos (3–5) per
well in 96-well plates.
Protocol:
1. For a chemical screen, large numbers of embryos at approximately the same
developmental stage need to be generated. Set up 100 heterozygote pairwise
matings with fish separated by a divider. The next morning, remove the divider,
allow the fish to mate, and collect the embryos.
2. Dilute chemicals into screening medium. The screen is conducted in 48-well
plates with a volume of 300 ml per well. Individual chemicals could be added to
each well, but to improve throughput, we devised a matrix pooling strategy: The
chemical library (courtesy of the Institute of Chemistry and Cell Biology,
Harvard Medical School) was arrayed in 384-well plates with the last four
columns empty, thus containing 320 compounds per plate. Given this plate
geometry, 8  10 matrix pools were created. A hit detected in both a horizontal
and a vertical pool identified the individual compound.
a. Transfer 80 ml of screening medium to each well of four 384-well plates
using a TECAN liquid-handling robot.
2. Analysis of Cell Proliferation, Senescence, and Cell Death in Zebrafish Embryos 33

b. Pin transfer 1 ml of each compound (arrayed at 5 mg/mL in DMSO) into each


well of screening medium by performing 10 transfers with a 100 nl 384-pin
array for each of the four 384-well plates (total of 320  4 = 1280
compounds).
c. Pooling was performed with a TECAN liquid-handling robot by pipeting the
diluted chemicals from the 384-well plates to 48-well plates. For vertical
pools, 30 mL was transferred from each of 8 wells plus an additional 60 mL
of screening medium to bring the total volume to 300 ml. For horizontal
pools, 30 mL was transferred from each of 10 wells.
3. Aliquot embryos to the 48-well plates at 50% epiboly.
a. Prior to aliquoting embryos to wells, examine them under a dissecting
microscope and discard all dead, delayed, or deformed embryos.
b. Pool embryos in a single 100 mm tissue culture dish or a 50 mL conical tube.
c. Decant the embryo medium and remove as much liquid from the embryo
suspension as possible with a transfer pipet. Pressing the transfer pipet tip to
the bottom of the tube or dish allows most liquid to be removed without
aspirating the embryos.
d. Add approximately 20 embryos to each well by scooping them with a small
chemical weighing spatula. With 20 embryos per well and a Mendelian
recessive inheritance, there is a 0.3% chance of a well having no mutants.
Since a hit requires detection in both a horizontal and a vertical pool, each
with 20 embryos, the false-positive rate for identification of complete sup-
pressors is 0.001%.
4. Place 48-well plates into an incubator at 28.5  C.
5. One to two hours later, clean out any dead embryos from each well using a long
glass Pasteur pipet bent at a 90 degree angle.
6. Incubate at 28.5  C overnight.
7. Dechorionate embryos by adding 150 mL of a 5 mg/mL pronase solution to each
well. After 10 min, gently shake plates until embryos come out of the chorions.
8. Using a transfer pipet fitted with a 10 mL tip, remove as much of the pronase/
chemical mixture as possible from each well.
9. Rinse the embryos once in fresh embryo medium and remove as in step 8.
10. Add 500 ml of PFA to each well.
11. Parafilm the edges of the plates to prevent evaporation and fix at 4  C at least
overnight but not longer than a week.
12. Using a transfer pipet, move embryos to 48-well staining grids made of acetone-
resistant plastic with a wire mesh bottom.
13. Perform pH3 staining protocol by placing staining grids into 11  8.5 cm
reservoirs containing 20–30 mL of the appropriate solution. To change solu-
tions, the grid can be lifted out of one reservoir and placed into another reservoir
with the next solution. For overnight antibody incubations, the reservoir should
be sealed with parafilm to prevent evaporation.
34 Daniel Verduzco and James F. Amatruda

14. After staining is complete, move embryos with a transfer pipet back into 48-well
plates that have been precoated with 100 mL of 1% agarose in 1 PBS. The
agarose forms a meniscus that keeps embryos in the center of the well where
they are easier to score.
15. Score for absence of mutants or for partial suppression without effect on wild
types. In addition to suppressors and enhancers, one can identify compounds
that affect both wild types and mutants, thus having a more general effect.
16. Deconvolute matrix pool to identify individual chemicals.

IV. Conclusions
Given the power of zebrafish in forward vertebrate genetics and organism-based
small molecule screens, the system will nicely complement traditional model organ-
isms for studying the cell division cycle. Many of the assays that are commonly used
to probe the cell cycle in systems such as yeast, Drosophila, and mammalian cells
can be used in the zebrafish. The protocols outlined in this chapter can be utilized to
characterize known mutants for alterations in cell proliferation or, alternatively, can
be used to screen for more cell-cycle mutants. Given that zebrafish embryos are
amenable to gene knockdown via antisense morpholino-modified oligonucleotides
and overexpression by mRNA injection, these protocols can also be used to study
cell-cycle genes in the zebrafish without generating a mutant.

V. Reagents and Supplies

Alkaline solution 0.6 g NaOH, 250 mL 200 mM EDTA pH 10, to 50 mL ddH2O


Alkaline electrophoresis
Buffer 12 g NaOH, 2 mL 500 mM EDTA pH 8, to 1 L ddH2O
Anti-BrdU Roche cat # 1170 376
Block 2% Blocking reagent (Roche cat # 1096 176), 10% fetal calf serum,
1% dimethylsulfoxide in PBST
BrdU block 0.2% Blocking reagent (Roche cat # 1096 176), 10% fetal calf serum,
1% dimethylsulfoxide in PBST. The lower concentration of
blocking reagent improves detection
DAPI 40 ,6-Diamidino-2-phenylindole
DMSO Dimethylsulfoxide
E3 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4
Mesh Small Parts, Inc. 105 mm mesh with cat # U-CMN-105D. 40 mm mesh
with cat # U-CMN-40D
PBS Phosphate-buffered saline, pH 7.5.
PBST 1 PBS with 0.1% (v/v) Tween-20
Pellet pestle & tubes Fisher, cat # K749520-0090
PFA 4% PFA buffered with 1 PBS
Propidium iodide 0.1% Sodium citrate, 0.05 mg/mL propidium iodide, 0.0002%
Triton X-100 (added fresh)
2. Analysis of Cell Proliferation, Senescence, and Cell Death in Zebrafish Embryos 35

Sigma senescence fixation Contains 20% formaldehyde, 2% glutaraldehyde, 70.4 mM


buffer 10 (cat # F1797) Na2HPO4, 14.7 mM KH2PO4, 1.37 M NaCl, and 26.8 mM KCl
Sigma senescence staining Mix the following for preparation of 10 ml of the staining mixture:
mixture (prepare just 1 mL of staining solution 10 buffer (cat # S5818)
prior to use) 125 mL of reagent B (cat # R5272)
125 mL of reagent C (cat # R5147)
0.25 mL of X-gal solution (cat # X3753)
8.50 mL of ultrapure water
Screening medium E3 supplemented with 1% DMSO, 20 mM metronidazole, 50 units/mL
penicillin, 50 mg/mL streptomycin, and 1 mM Tris pH 7.4

Acknowledgments
We thank Len Zon and members of the Zon laboratory for useful discussions. Original versions of
many of these protocols were worked out by Jennifer L. Shepard, Ryan Murphey, Howard M. Stern,
Kathryn L. Pfaff, and J.F.A. D.V. was supported by NIH Training Grant 5 T32 GM008203 and J.F.A. was
supported by grants from the Lance Armstrong Foundation, the Amon G. Carter Foundation, the Welch
Foundation, and NIH/NCI grant 1R01CA135731.

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CHAPTER 3

Analysis of Cilia Structure and


Function in Zebrafish
Jarema Malicki,* Andrei Avanesov,* Jade Li,y Shiaulou Yuany
and Zhaoxia Suny
*
Division of Craniofacial and Molecular Genetics, Tufts University, Massachusetts, USA
y
Departments of Genetics, Yale University School of Medicine, New Haven, Connecticut, USA

Abstract
I. Introduction
II. Cilia in Zebrafish Embryos and Larvae
A. Kupffer’s Vesicle
B. Pronephros
C. Sensory Organs
D. Neural Tube
III. Analytical Tools for Cilia Morphology and Motility
A. Method 1: Imaging of Kupffer’s Vesicle Cilia Following arl13b/sco-eGFP mRNA
Overexpression
B. Method 2: Modified Methods for Imaging of the Pronephric Duct and Spinal
Central Canal Using arl13b/sco-eGFP Over-Expressants
C. Alternative Method: Imaging of Cilia Using arl13b/scorpion-eGFP Transgenic Fish
D. Method 3. Detection of Protein Expression in Cilia Using Immunohistochemistry
IV. Analysis of Cilia-related Mutant Phenotypes in Zebrafish
A. Left–Right Asymmetry Defects
B. Method 4: Evaluation of Heart Positioning in Live Zebrafish Embryos
C. Alternative Method: Analysis of Left–Right Asymmetry Defects Using in situ
Hybridization
D. Kidney Cysts
E. Method 5: Detection of Kidney Cysts in Zebrafish
F. Degeneration of Sensory Neurons
G. Method 6: Evaluation of Photoreceptor Cell Layer Morphology on Plastic Sections
H. Method 7: Analysis of Photoreceptor Cells on Cryosections via
Immunohistochemistry
I. Method 8: Whole-Mount Staining and Imaging of Hair Cells in the Inner Ear
J. Method 9: Detection of Basal Bodies in Hair Cells
K. Method 10: Detection of Neuromast Hair Cells in a Living Specimen

METHODS IN CELL BIOLOGY, VOL 101 0091-679X/10 $35.00


Copyright 2011. Elsevier Inc. All rights reserved. 39 DOI 10.1016/B978-0-12-387036-0.00003-7
40 Jarema Malicki et al.

L. Method 11: Labeling of Olfactory Neurons by DiI Incorporation


V. Future Directions
Acknowledgments
References

Abstract

The cilium, a previously little studied cell surface protrusion, has emerged as an
important organelle in vertebrate cells. This tiny structure is essential for normal
embryonic development, including the formation of left-right asymmetry, limb
morphogenesis, and the differentiation of sensory cells. In the adult, cilia also
function in a variety of processes, such as the survival of photoreceptor cells, and
the homeostasis in several tissues, including the epithelia of nephric ducts. Human
ciliary malfunction is associated with situs inversus, kidney cysts, polydactyly,
blindness, mental retardation, obesity, and many other abnormalities. The genetic
accessibility and optical transparency of the zebrafish make it an excellent verte-
brate model system to study cilia biology. In this chapter, we describe the morphol-
ogy and distribution of cilia in zebrafish embryonic and larval organs. We also
provide essential protocols to analyze cilia formation and function.

I. Introduction

Cilia are thin cell surface protrusions of varying length supported by a charac-
teristic arrangement of microtubules. On cross sections, the vast majority of cilia
feature nine evenly spaced peripheral microtubule doublets. Two additional micro-
tubule singlets are frequently present in the center of the cilium. The microtuble-
based core cytoskeleton of the cilium is known as the axoneme. The arrangement of
axonemal microtubules is exceptionally well conserved in evolution, remaining
essentially unchanged in mammals, fish, and green algae (Avidor-Reiss et al.,
2004; Rosenbaum and Witman, 2002; Satir et al., 2008).
Cilia are associated with a wide spectrum of processes in embryonic development
and in adult physiology. In the embryo, cilia are necessary for the determination of
left–right (LR) asymmetry, limb morphogenesis, the formation of ventricular spaces
in the brain, the differentiation of the craniofacial skeleton, and finally the differ-
entiation of sensory neurons, such as photoreceptor cells of the retina (Basu and
Brueckner, 2008; Haycraft et al., 2005; Tissir et al., 2010; Tsujikawa and Malicki,
2004; Walczak-Sztulpa et al., 2010). In adult mammals, cilia are required for the
function of photoreceptor and olfactory sensory neurons, the removal of foreign
particles from the respiratory track, the transport of oocytes in the oviduct, the
motility of the sperm, the maintenance of kidney duct morphology, and the deposi-
tion of fat (Afzelius, 2004; Cardenas-Rodriguez and Badano, 2009).
On the cellular level, cilia appear to play two major roles. First, they function as
mechanical drivers of movement. Cilia can propagate the movement of cells or small
3. Analysis of Cilia Structure and Function in Zebrafish 41

multicellular organisms. Similarly, apical cilia drive fluid flow over the surface of
epithelial sheets. Second, cilia provide a subcellular compartment for signal trans-
duction cascades that range from phototransduction to hedgehog signaling. This
second mode of action underlies the role of cilia in signal detection and signal
transduction (reviewed in, Green and Mykytyn, 2010). Given the diversity of ciliary
functions, it is to be expected that they have received considerable attention in
organisms that range from unicellular algae to humans.
The zebrafish is a vertebrate animal model exceptionally well suited for the
studies of cilia formation and function for several reasons. First, the zebrafish
embryo develops outside the maternal organism, is transparent, and features several
organs that differentiate prominent cilia early in embryogenesis. These character-
istics make it easy to monitor the length and movement of the ciliary axoneme (Sun
et al., 2004; Tsujikawa and Malicki, 2004). Second, zebrafish cilia mutants fre-
quently feature the curved body axis, a phenotype, that is very easy to detect in
genetic screens. Consequently an impressive collection of ciliary mutants is avail-
able in zebrafish (Brand et al., 1996; Doerre and Malicki, 2002; Drummond et al.,
1998; Malicki et al., 1996a; Sun et al., 2004).
In addition to forward genetic strategies, the zebrafish embryonic development
can be analyzed using fast and inexpensive antisense approaches (Eisen and Smith,
2008; Nasevicius and Ekker, 2000). One interesting application of these antisense
methods is the use of zebrafish as a tool for testing human mutant alleles associated
with ciliary diseases (Khanna et al., 2009; Leitch et al., 2008; Zaghloul et al., 2010).
In this chapter, we briefly describe cilia in several organs of wild-type zebrafish and
point out defects that are associated with their malfunction in mutant strains. Methods
to study the ciliary structure and function in the zebrafish model are presented.

II. Cilia in Zebrafish Embryos and Larvae


Cilia are abundant and can be easily visualized in a number of organs in zebrafish
embryos (Fig. 1). For example, the length, density, and motility of cilia have been
analyzed in the Kupffer’s vesicle (KV), the pronephric duct, neurons of sensory
systems, and the neural tube (Essner et al., 2005; Kramer-Zucker et al., 2005;
Tsujikawa and Malicki, 2004). In the following we describe the distribution of cilia
and the developmental timing of their differentiation in several organs of zebrafish.
As the vast majority of zebrafish research is performed on embryos and early larvae,
the descriptions of ciliogenesis that we provide in this section focus on these stages.

A. Kupffer’s Vesicle
KV is a closed, spherical transient structure embedded on the ventral side of the
tailbud. It contains a liquid-filled lumen lined with ciliated epithelial cells that are
derived from dorsal forerunner cells (Cooper and D’Amico, 1996; D’Amico and
Cooper, 1997; Oteiza et al., 2008). Although in terms of its developmental origin
42 Jarema Malicki et al.

[(Fig._1)TD$IG]

Fig. 1 Cilia in zebrafish. (A) The zebrafish larva at approximately 120 hpf. The locations of tissues that feature prominent
cilia are indicated. (B) Confocal image of olfactory cilia stained with anti-acetylated tubulin antibody (green) at 3 dpf. F-actin is
labeled with fluorophore-conjugated phalloidin (in red) to visualize the morphology of the olfactory pit. (C) Transverse
cryosection through the retina at 3 dpf. Cilia are stained with anti-acetylated tubulin (green) and anti-Ift88 (red) antibodies,
and imaged using confocal microscopy. The photoreceptor cell layer is indicated with a bracket. IFT88 signal is enriched at the
base of cilia (red). (D) Confocal image of the anterior macula in the ear of a whole embryo at 3 dpf. Hair cells and their cilia are
3. Analysis of Cilia Structure and Function in Zebrafish 43

and structure the KV is fairly distinct from the mouse node, it plays a similar role in
the establishment of the LR asymmetry of the body plan (Bisgrove et al., 2005; Essner
et al., 2005). It is thought that the generation and sensing of directional fluid flow by
cilia in the KV and the node alike are the initial symmetry-breaking events along the
LR axis (McGrath et al., 2003; Nonaka et al., 2002, 1998). As a result, the KV
represents an excellent system to evaluate the morphology and motility of cilia
(Fig. 1J, 2B), as well as the functional consequences of ciliary defects. Cilia in the
KV are best imaged during the four to eight-somite stage (around 11–13 hour post-
fertilization (hpf) at 28.5  C), when this structure is most prominent. Both motile and
immotile cilia can be found in the KV, and most KV cells contain a single cilium
(Borovina et al., 2010; Kramer-Zucker et al., 2005; Kreiling et al., 2007; Okabe et al.,
2008). Since zebrafish development is very rapid, maternally deposited gene products
frequently persist to quite advanced developmental stages. This needs to be taken into
consideration during the analysis of zygotic mutants, since they start their life with a
robust dose of maternal gene products, which frequently mask the early developmen-
tal function of analyzed genes (Cao et al., 2010; Huang and Schier, 2009).

B. Pronephros
The functional kidney in the zebrafish embryo is the pronephros. During devel-
opment, the pronephros originates from the intermediate mesoderm. By the 20-
somite stage, a pair of pronephric ducts are already formed, one on each side of
the body axis (Drummond et al., 1998). The differentiation of the pronephric
glomerulus takes place slightly later, and glomerular filtration commences between
36–48 hpf (Drummond et al., 1998). Ciliary defects in renal epithelial cells almost
inevitably lead to the formation of kidney cysts in both zebrafish and mammals

visualized using antibodies to acetylated tubulin (green). The apical cell surface marker Crumbs is also visualized via antibody
staining (in red). (E) Confocal image of a crista in the ear of a whole embryo at 4 dpf. Hair cells are visualized as in (D).
Phalloidin is used to visualize tissue morphology (red). (F) Transverse cryosection through the trunk exposes the proneprhic duct
at 4 dpf. Its apical surface differentiates actin-rich microvilli and is easy to visualize with phalloidin (blue). A bundle of ciliary
axonemes on the apical surface of a multiciliated cell is visualized with anti-acetylated tubulin antibodies (green). The apical
surface of this cell is visualized with anti-Crumbs antibodies (red). (G) Confocal image of a lateral line neuromast in the whole
embryo. Hair cells and their cilia are visualized with anti-acetylated tubulin staining. (H) Confocal en face image of a neuromast
stained with phalloidin (green) and anti-g -tubulin antibody (red). Arrows in the inset indicate planar orientation of neuromast
hair cells. (I) Spinal canal cilia visualized via the expression of a mouse Arl13b-GFP transgene. To visualize cell membranes,
embryos were injected with mRNA encoding membrane-bound RFP. (J) Kupffer’s vesicle cilia labeled using antibodies to
acetylated alpha tubulin in a whole embryo. (K) Transverse section through the central retina at 5 dpf. An electron micrograph
showing retinal photoreceptors. Note differentiated outer segment (OS). Asterisk marks the photoreceptor cell soma. (L) An
electron micrograph showing numerous cilia and villae in the lumen of the pronephric duct. A transverse section through the
zebrafish trunk at 4 dpf. (M) A close-up view of panel (L) showing a cross-section through the ciliary shaft. The 9 + 2
arrangement of microtubules is evident. (N) A cross-section of a hair cell kinocilium in the ear of 3 dpf. An electron micrograph
showing the 9 + 2 arrangement of microtubules. (O) Section through the ear macula showing the kinocilium (arrowheads) and a
nearby bundle of sterocilia. In (C–E, and G) apical is up. Image in panel (I) courtesy of Brian Ciruna. B–D, F–H, J, and K–N are
courtesy of the present and former Malicki lab members: Motokazu Tsujikawa, Yoshihiro Omori, Chengtian Zhao, and Peter
Kovach. (E, O) reprinted with permission from (Tsujikawa and Malicki, 2004). (See Plate no. 3 in the Color Plate Section.)
44 Jarema Malicki et al.

(Fig. 3B–F) (Pazour et al., 2002; Sun et al., 2004; Yoder et al., 2002). The zebrafish
pronephric duct features two populations of ciliated cells: multiciliated cells (MCC)
and single ciliated cells (SCC), which form clustered cilia bundles and single cilia,
respectively (Liu et al., 2007; Ma and Jiang, 2007). Cilia on both cell types display
the ‘‘9 + 2’’ configuration of microtubules, i.e., they contain a central pair of micro-
tubules. While both MCCs and SCCs can be found in the anterior and middle
segment of the pronephric duct, the posterior portion of the duct consists of only
SCCs. Conveniently, cilia formation in SCCs and MCCs takes place at different time
points; although single cilia can be detected as early as 20 hpf, bundles of cilia from
MCCs are observed starting only at around 36 hpf. Due to the presence of maternal
contribution, some cilia mutants initially display normal SCC cilia on the apical
surface of pronephric duct cells at earlier time points but do not form cilia bundles
later on (Cao et al., 2010). Therefore, it is useful to investigate the pronephric cilia
both at an early stage (e.g., 20 hpf) and a later time point (e.g., 5 dpf).

C. Sensory Organs
Similar to other vertebrates, including mammals, zebrafish sensory organs feature
prominent populations of cilia. These are present on the apical surface of sensory
neurons, including photoreceptors, as well as olfactory and mechanosensory cells
(Fig. 1B–E, G, H). It is well established that in the case of photoreceptors and
olfactory cells, sensory signal transduction cascades are compartmentalized to cilia
(Kennedy and Malicki, 2009; McEwen et al., 2007; Pugh and Lamb, 2000). In the
following we briefly comment on the major features of cilia in the visual, auditory,
and olfactory systems.

1. Photoreceptors
Photoreceptor cells of the vertebrate retina acquire nearly all visual information
available to the organism. Outer segments, specialized compartments that project from
the apical surface of photoreceptor cells, mediate this function. Interestingly, the outer
segment is a highly modified cilium that features dramatically enlarged membranes
forming hundreds of parallel folds (Fig. 1K) (Rodieck, 1973; Kennedy and Malicki,
2009). Rod outer segments harbor approximately 108–109 light-sensitive opsin mole-
cules and similarly impressive quantities of other components of the visual transduc-
tion cascade (Pugh and Lamb, 2000). As found in other types of cilia, the outer
segment is supported by microtubules, which originate from the basal body located
at the apical terminus of the photoreceptor soma and span its entire length (Insinna
et al., 2008; Wen et al., 1982). The axoneme of the photoreceptor outer segment lacks
the central microtubule pair and appears to fulfill two roles: it supports the elaborate
folding of the outer segment membrane, and provides a transport route for outer
segment proteins, such as opsins. Photoreceptors of the zebrafish retina first become
postmitotic at 40–48 hpf, start to express opsins at about 50 hpf, and begin to form
outer segments by 54 hpf (Hu and Easter, 1999; Raymond et al., 1995; Schmitt and
Dowling, 1999). Based on behavioral tests, outer segments are functional by 3 dpf
3. Analysis of Cilia Structure and Function in Zebrafish 45

(Easter and Nicola, 1996) but continue to elongate for at least the next three weeks
(Branchek and Bremiller, 1984).

2. Mechanosensory Hair Cells


Similar to other vertebrates, the inner ear of zebrafish consists of interconnected
canals and chambers that in several locations feature patches of specialized cells
known as mechanosensory hair cells (Fig. 1D, E) (Bang et al., 2001; Haddon and
Lewis, 1996; Whitfield et al., 1996). Similar to photoreceptors, hair cells also
differentiate a prominent apical cilium, known as the kinocilium. The first cells that
feature hair cell morphology differentiate in the otic vesicle at two locations and
become first detectable around 19.5 hpf (Haddon and Lewis, 1996). They are posi-
tioned underneath otoliths, deposits of protein and inorganic salts, which appear as
two round structures, one anterior to the other inside the otic vesicle (Haddon and
Lewis, 1996; Malicki et al., 1996b; Riley et al., 1997). As the animal develops, new
hair cells are gradually added next to the previous ones, forming sensory patches,
known as maculae (Bang et al., 2001; Haddon and Lewis, 1996). Later in develop-
ment, around 21–25 dpf, a third macula differentiates in the third chamber of the
inner ear, the lagena (Bang et al., 2001). In addition to maculae, three other locations
known as the anterior, the lateral, and the posterior crista differentiate hair cell
populations at about 3 dpf (Haddon and Lewis, 1996). The kinocilia of each crista
project into the lumen of a different semicircular canal, and are substantially longer
than those of maculae (Haddon and Lewis, 1996). Although they seem to be immo-
tile, they feature the 9+2 configuration of microtubules. Adjacent to the kinocilium,
hair cells feature a bundle of apical protrusions, known as stereocilia. The term
‘‘stereocilia’’ is a misnomer, as these structures are supported by actin cytoskeleton
and thus are related to microvilli, not cilia.
The detection of sound waves by hair cells is thought to be mediated by a physical
displacement of stereociliary bundles (Hudspeth, 1989). The role of kinocilium in
this process is, however, unclear. In zebrafish, hair cell kinocilia are maintained
throughout the adulthood and thus could play a role in hearing (Bang et al., 2001;
Haddon and Lewis, 1996). In contrast, the hair cells in the adult mammalian auditory
organ lose their kinocilia (Kikuchi and Hilding, 1965; Kimura, 1966). In vitro studies
using explants from the adult mammalian ear show that after an induced mechanical
stress, the subsequent self-repair of the damaged tissue is accompanied by a regen-
eration of the kinociliary axoneme, which suggests a role for kinocilia in hair cell
morphogenesis (Sobkowicz et al., 1995). In line with this idea, vertebrate cilia have
been linked to cell–cell signaling pathways, one of which is the planar cell polarity
pathway (Cao et al., 2010; Huangfu et al., 2003; Jones et al., 2008).
One characteristic feature found in aquatic vertebrates, including fish, is the
presence of hair cells on the body surface, in sensory organs known as neuromasts
(Fig. 1G, H). Similar to those in the ear, hair cells of neuromasts help to detect
hydrodynamic movements (reviewed in, Montgomery et al., 2000). Neuromasts
differentiate in a stereotypical pattern on both the trunk and the head. Neuromasts
46 Jarema Malicki et al.

of the trunk and the tail belong to the posterior lateral line, while the ones on the head
form the anterior lateral line (reviewed in, Dambly-Chaudiere et al., 2003; Metcalfe,
1985; Metcalfe et al., 1985). The anterior lateral line differentiates the first neuro-
mast anterior to the ear by 30 hpf (Metcalfe, 1985). Shortly thereafter, at 36 hpf, the
posterior lateral line begins to differentiate neuromasts in the rostal–caudal
sequence: the anterior trunk neuromasts are the first to appear, whereas the tail ones
appear last. The posterior lateral line system reaches the tail by 48 hpf and includes
about six neuromasts (Metcalfe, 1985). As the larva grows, the posterior lateral line
continues to differentiate additional neuromasts (Ledent, 2002), and by 5 dpf, 10–11
neuromasts are found along the myoseptum on each side of the fish (Metcalfe et al.,
1985). As the organism matures, additional lateral line branches form and new
neuromasts are added to preexisting branches, so that eventually over a thousand
neuromasts are found in the adult zebrafish (Ledent, 2002).
Interestingly, the neuromasts of the lateral line are polarized in the plane of the
skin surface, a feature particularly evident in the localization of hair cell kinocilia
(Lopez-Schier et al., 2004; Lopez-Schier and Hudspeth, 2006). This polarity is
already prominent by 3 dpf, and is characterized by several topographical features
(Fig. 1H) (Lopez-Schier and Hudspeth, 2006). First, within each neuromast the basal
bodies of hair cell kinocilia are displaced toward the edge of the apical surface along
a single axis in either direction (Fig. 1H, arrows in the inset). Second, hair cells of
each neuromast are subdivided into two groups of approximately equal size: in one
area, their kinocilia are displaced in one direction, while in the remaining area, in the
opposite direction. Finally, the axis of polarization varies in neuromasts; while early
differentiating neuromasts polarize along the anterior–posterior axis, the ones dif-
ferentiating later display dorsoventral polarity (Lopez-Schier et al., 2004). Given the
ease of neuromast identification, their early differentiation, and accessibility, the
lateral line neuromasts provide an attractive opportunity to study the genetic bases of
planar cell polarity (Lopez-Schier and Hudspeth, 2006; Lopez-Schier et al., 2004;
McDermott et al., 2010; Raible and Kruse, 2000)

3. Olfactory Sensory Neurons


The olfactory system is also ciliated in both zebrafish and other vertebrate embryos
(Hansen and Zeiske, 1993; Kimura et al., 2009; Menco and Farbman, 1985). In
zebrafish, the openings of olfactory pits appear between 34–36 hpf, and their inside
surfaces harbor ciliated dendritic endings of olfactory sensory neurons (Hansen and
Zeiske, 1993). As olfactory pits gradually enlarge during embryogenesis, the number of
cilia increases (Fig. 1B) (Hansen and Zeiske, 1993). On the structural level, zebrafish
olfactory cilia feature both 9 + 0 and 9 + 2 microtubule configurations (Wloga et al.,
2009). Similar to photoreceptor outer segments as well as some invertebrate cilia, the
tips of zebrafish olfactory cilia contain microtubule singlets (Ward et al., 1975; Wloga
et al., 2009). Consistent with the 9 + 0 and 9 + 2 configurations, the olfactory systems
of both frog and zebrafish contain motile and nonmotile cilia (Drummond, 2009; Mair
et al., 1982). Both types are easily detectable using antibody staining of whole animals.
3. Analysis of Cilia Structure and Function in Zebrafish 47

D. Neural Tube
By about 18 hpf, the zebrafish neural keel differentiates the central canal and
begins to acquire the form of a tube (Kimmel et al., 1995). In adult vertebrates, the
inside lining of the spinal canal is provided by the ciliated ependymal epithelium
(Nakayama and Kohno, 1974; Worthington and Cathcart, 1963). Also in zebrafish
embryos and early larvae, the presence of cilia has been well documented on the
apical surfaces of cells that line the spinal canal lumen (Fig. 1I) (Borovina et al.,
2010; Kramer-Zucker et al., 2005). Despite their 9 + 0 microtubule configuration,
these cilia are motile and are required to propagate the flow of the cerebrospinal fluid
inside brain ventricles and the spinal canal (Kramer-Zucker et al., 2005;
Worthington and Cathcart, 1966). The spinal canal cilia have been proposed to play
developmental roles (Sawamoto et al., 2006). In zebrafish, the disruption of spinal
canal cilia leads to the abnormal expansion of brain ventricles that resembles
hydrocephalus (Kramer-Zucker et al., 2005). Finally, it is worth noting that basal
bodies of zebrafish neuroepithelial cells are displaced to the posterior region of the
apical surface and thus display planar polarization (Borovina et al., 2010).

III. Analytical Tools for Cilia Morphology and Motility


In this section, we describe general approaches used to detect cilia. Labeling of
the ciliary axoneme using antibodies to microtubules has been the most common
approach. There are several commercially available monoclonal markers that rec-
ognize various posttranslational modifications of tubulin (Table I). The most com-
monly used antibody recognizes acetylated a-tubulin (Fig. 1) (Duldulao et al., 2009;
Kishimoto et al., 2008; Omori and Malicki, 2006; Tsujikawa and Malicki, 2004;
Zhao and Malicki, 2007). Although this marker has been used extensively, it is not
entirely specific to cilia as acetylated a-tubulin is also present in the cytoplasm of
some cells, such as hair cells (Fig. 1D, E) (Omori and Malicki, 2006). Likewise,
axons of retinal ganglion cells also contain richly acetylated microtubules.
Antibodies that recognize other modifications of tubulin, such as glutamylation or
glycosylation, can also be used to label cilia (Table I, Wloga et al., 2009). In addition,
we have developed a rabbit polyclonal anti-Scorpion/Arl13b (anti-Sco) antibody that
is also a robust and specific marker for cilia (Duldulao et al., 2009) (Fig. 2A). Small
aliquots of anti-Sco can be requested from the authors and a detailed protocol for
immuno-staining and imaging has been described elsewhere (Drummond, 2009).
Detection of the basal body is another way to reveal the position of cilia (Fig. 1H).
A number of monoclonal antibodies to g -tubulin recognize this structure. A trans-
genic line expressing GFP-tagged centrin has been used as well (Borovina et al.,
2010; Zolessi et al., 2006). Although not useful in visualizing the axoneme, these
methods help to survey for the potential displacement of basal bodies in cilia
mutants. Antibodies and transgenic lines that have been used in the analysis of
zebrafish cilia are listed in Table I.
48 Jarema Malicki et al.

[(Fig._2)TD$IG]

Fig. 2 Localization of proteins in cilia. (A) Sco/Arl13b is highly enriched in the cilia of the pronephric
duct. A confocal image of a whole embryo stained with anti-Sco/Arl13b antibody (in green), and an anti-
acetylated tubulin antibody (a-tub, in red). (B) tg(actin::sco-eGFP) embryos show cilia-specific eGFP
signal (green). The left panel shows a fixed embryo counterstained with DAPI (in blue) to visualize nuclei.
Inset shows an enlargement. The middle and right panels show snapshots of a live embryo featuring both
motile (red arrows, note the change of position in the two pictures) and immotile (yellow arrow, note the
same position in the two images) cilia in the KV. (C-D) Seahorse localization shown by immunolabeling
with anti-Seahorse (Sea, in green) and anti-acetylated tubulin (a-tub, in red) antibodies. C shows results
from co-incubation of the two antibodies, while D shows results from sequential incubations. (See Plate
no. 4 in the Color Plate Section.)
3. Analysis of Cilia Structure and Function in Zebrafish 49

Motility is an important aspect of ciliary function. High-speed videomicroscopy


techniques allow for qualitative and quantitative analysis of cilia beat frequency,
amplitude and waveform in a variety of ciliated organs (Kramer-Zucker et al., 2005;
Okabe et al., 2008; Zhao and Malicki, 2007). These approaches utilize brightfield or
DIC microsocopy and a high-speed camera to visualize cilia movement in the KV,
the pronephric duct, the olfactory pit, and the spinal canal. It has been, however,
challenging to visualize immotile cilia via these imaging methods. A slight modi-
fication to these approaches allows for the visualization of both motile and immotile
cilia by high-speed fluorescence microscopy (Fig. 2B). An essential component of
this method is the use of embryos expressing GFP-tagged cilia markers, such as
Scorpion/Arl13b (Fig. 2B) (Duldulao et al., 2009). We outline two protocols for live
imaging of fluorescently labeled cilia that can be applied to various ciliated organs
such as the KV, the pronephric duct, and the spinal central canal.

A. Method 1: Imaging of Kupffer’s Vesicle Cilia Following arl13b/sco-eGFP mRNA


Overexpression
Microinjection of in vitro transcribed scorpion/arl13b-eGFP mRNA allows for
robust and versatile GFP-labeling of cilia as the amount and concentration of
transcript can be adjusted as desired. This method is particularly useful for imaging
cilia at early developmental stages. The following describes the method for imaging
motile and immotile cilia in KV.

1. Materials
 Standard microinjection reagents (borosilicate micropipettes, microinjection
apparatus, agar injection molds, embryo medium, phenol red, etc.) (Yuan and
Sun, 2009).
 Methods 1 and 2: scorpion/Arl13b eGFP (zebrafish scorpion/arl13b, eGFP-
tagged) RNA, in vitro transcribed from linearized DNA template as described
previously (Yuan and Sun, 2009).
 Alternative Method: Tg(bact::arl13b-GFP) transgenic fish line.
 1.5% low-melting point agarose solution (in embryo medium) in small glass petri
dish on a 50  C heat block.
 Viewing chamber made from a large microscope slide (25  60 mm) and a large
coverslip (22  40 mm) separated by two ‘‘spacer’’stacks of 3 small #1 coverslips
(22  22 mm); glued together with ‘‘Super Glue’’ (cyanoacrylate). The space
between the slide and the coverslip fits a living zebrafish embryo without crushing
it, thus enabling in vivo imaging using florescence microscopy. Prepare the cham-
bers ahead of time by gluing together two stacks of three small coverslips to a large
microscope slide; position them on the slide 3 cm apart from each other. During
the experiment, embryos will be embedded in agar directly on the surface of the
large the coverslip, which is then inverted and placed between the small coverslip
spacers and the slide (for detailed diagrams, refer to Westerfield, 2000).
50 Jarema Malicki et al.

 Fine-tipped Dumont forceps or tungsten needles (Fine Science Tools, 11252-40 or


10130-05).
 Clear nail polish.
 An inverted microscope equipped for imaging of fluorescently labeled samples
and a Coolsnap HQ2 (Photometrics) cooled-CCD camera or equivalent.
 MetaMorph (Molecular Devices) or ImageJ (NIH) software.

2. Protocol
1. Following the previously described method (Yuan and Sun, 2009), inject wild-
type zebrafish embryos at the one-cell stage with 250 pg of in vitro transcribed
scorpion-eGFP RNA. It is possible to inject embryos as late as the four-cell
stage. We found, however, that the earlier the injection the more evenly distrib-
uted the injected mRNA will be. Varying the amount of injected transcript
allows one to control expression levels.
2. At the 4–6 somite stage (11–12 hpf), dechorionate embryos manually using
fine-tipped Dumont forceps.
3. Using a glass, briefly immerse 5–10 embryos in a 1.5% low-melting point
agarose solution for 5–10 s on a 50  C heat block.
4. Using the same pipette, take up the embryos and a small amount of agarose and
place them in the middle of a large glass coverslip (22  40 mm). Quickly move
to the next step.
5. Gently adjust embryos in the agarose using a pair of fine-tipped Dumont forceps
or a tungsten needle so that the tailbud touches the coverslip. We recommend
positioning the embryo in such a way that the anterior–posterior axis of the KV
is parallel to the surface of the coverslip. This method positions the KV as
closely as possible to the surface of the coverslip, thereby minimizing the optical
depth required to image it. Work quickly as the agarose will begin to solidify
rapidly at room temperature (RT).
6. Wait 5 min for the agarose to completely solidify at RT.
7. Using forceps, pick up the coverslip. Invert it so that the side with the agar-
embedded embryos is facing down.
8. Mount the coverslip onto the viewing chamber slide, so that it spans the 3 cm
space created between two stacks of coverslips as described in materials above.
Embryos should be facing the interior of the chamber.
9. Apply a small amount of nail polish to the corners of the coverslip to prevent it
from moving. Let the nail polish dry for 5 min.
10. Use a Pipetteman to inject 100 to 200 mL of embryo medium into the interior of
the chamber.
11. Image embryos on an inverted microscope with a 25 or 40 water immersion
objective and a Coolsnap HQ2 (Photometrics) camera using GFP fluorescence.
The KV will be located at the tip of the tailbud and should appear as a spherical
structure posterior to the notochord and somites. Using this mounting method, the
KV should be easily visible as it will be located very close to the coverslip surface.
3. Analysis of Cilia Structure and Function in Zebrafish 51

12. Once you locate the KV, use the fine focus to determine an optimal Z-section.
Acquire 3 to 5 time-series of immunofluorescence images per each embryo at
different Z-planes within the KV. Collect all time series at the rate of 50 fps
(frames per second) with a 5 ms exposure time and a 320  320 pixel resolution
(fine adjustments to these settings may be required depending on your micro-
scope setup). In our experience, a length of 20 s for each time-series is optimal.
It is worth noting that this acquisition speed is insufficient for measuring beat
frequency, but will allow one to distinguish motile from immotile cilia.
13. After the acquisition of each time-series, record a short time-series in DIC or
brightfield illumination to serve as an overlay for data analysis. Be sure to mark
the antero–posterior orientation of each KV. This will aid in further analyses.
14. View each time-series at 10 fps for analysis using MetaMorph (Molecular
Devices) or ImageJ (NIH) software. Be sure to distinguish between immotile
and motile populations of cilia.
This protocol can be modified to visualize other ciliated organs such as the
pronephric duct and spinal central canal, as described below.

B. Method 2: Modified Methods for Imaging of the Pronephric Duct and Spinal Central
Canal Using arl13b/sco-eGFP Over-Expressants
1. Additional Materials
 0.003% Phenylthiourea (PTU) in embryo medium;
 3% Methycellulose: add 3 g methycellulose (Sigma, M7027) to 100 ml embryo
medium, stir overnight at 4  C, spin in a benchtop centrifuge for 10 min, store
at 4  C;
 25  MESAB solution: dissolve 0.4 g of ethyl 3-aminobenzoate methanesulfo-
nate salt (Sigma, A5040) in 100 ml of 10 mM Tris (pH 7.0), store frozen.

2. Protocol
1. Following the previously described method, inject wild-type zebrafish embryos
at the one-cell stage with 250 pg of in vitro transcribed arl13b/sco-eGFP RNA.
2. To prevent the appearance of pigmentation at later stages of development, raise
embryos in 0.003% PTU in embryo medium after 20–24 hpf.
3. At 2–5 dpf, dechorionate embryos manually by using fine-tipped Dumont
forceps.
4. Anesthetize embryos in 1 MESAB in embryo medium for 1 to 3 min.
5. Using a pipette tip, place a small drop of 3% methylcellulose in embryo medium
at the center of viewing chamber slide between the stacks of small coverslips.
6. Using a glass Pasteur pipette, remove 3 to 5 embryos from the embryo medium/
MESAB solution and place them onto the drop of methylcellulose on the
viewing chamber slide.
52 Jarema Malicki et al.

7. Gently adjust embryos/larvae in methylcellulose using a pair of fine-tipped


Dumont forceps or a tungsten needle so that the side of each embryo is parallel
to the coverslip.
8. Using forceps, mount a medium coverslip (22  40 mm) onto the viewing
chamber. Make sure that the embryos are well positioned in the interior of the
chamber.
9. If the embryo is not satisfactorily oriented after coverslip placement, the cov-
erslip can be gently pushed to improve the orientation. This can be done without
damage to the embryo due to the viscosity of the methylcellulose solution.
10. Apply a small amount of nail polish to the corners of the coverslip to prevent it
from moving. Let the nail polish dry for 5 min.
11. Image embryos on an inverted fluorescence microscope equipped with a 25 or
40 water immersion objective and a Coolsnap HQ2 (Photometrics) camera
using GFP fluorescence. We recommend locating the pronephric duct by look-
ing directly posterior to the otic vesicle. It will appear as a tubule lined with
motile cilia. The pronephric duct continues posteriorly along the body axis
following closely the gut to the cloaca.
The spinal cord can easily be located by searching through the dorsal portion of
the embryo. It will appear as a tube lined with cells featuring single motile cilium.
The spinal cord extends from the head of the embryo down to the tail, following the
notochord closely. This mounting method enables the visualization of the proneph-
ric duct and the spinal cord, since they will be located very close to the coverslip.
12. Follow Steps 12–14 of the previous protocol for further imaging and analysis.

C. Alternative Method: Imaging of Cilia Using arl13b/scorpion-eGFP Transgenic Fish


An alternative method is to utilize a transgenic line that constitutively expresses
Arl13b/Scorpion-eGFP. Since injected RNA transcripts gradually degrade as the
embryo develops, the level of expression may fade by 3 dpf depending on the
stability of that particular transcript and the translated product. To circumvent this
difficulty and enable analysis at later stages in embryonic development, we gener-
ated a transgenic line. A similar line using mouse Arl13b was also generated by the
Ciruna group (Borovina et al., 2010).
Similar to microinjection of arl13b/sco-eGFP RNA, arl13b/scorpion-eGFP
transgenic embryos can be used to image a variety of ciliated organs including the
KV (Fig. 2B), the pronephric duct and spinal central canal. The two protocols
described above can be adapted to the use of the scorpion/arl13b-GFP transgenic
line by simply omitting the microinjection step.

D. Method 3. Detection of Protein Expression in Cilia Using Immunohistochemistry


An increasing number of proteins have been found in the cilium. To examine
whether a protein of interest localizes to the cilium, co-immunostaining with
anti-acetylated tubulin (clone 6-11B-1, Sigma) and an antibody against the protein
3. Analysis of Cilia Structure and Function in Zebrafish 53

of interest, followed by confocal imaging, is often the method of choice. We found,


however, that the extreme high affinity of the anti-acetylated tubulin antibody for
cilia may lead to artifacts. An example is shown in Figure 2C. When zebrafish
embryos fixed with methanol were probed with combined primary antibodies (affin-
ity purified anti-Seahorse, 1:100; anti-acetylated tubulin, 1:20,000), Seahorse
appears to be enriched on pronephric ductcilia. However, when antibody incubations
are carried out sequentially, the ciliary localization of Seahorse disappears (Fig. 2C).
Further control experiments using anti-Seahorse antibody in the absence of anti-
acetylated tubulin antibody detected no enrichment of this protein on the cilium (not
shown), suggesting that the ciliary enrichment of Seahorse shown in Figure 2C is a
false positive result. A similar false positive signal was detected for a number of
other antibodies when used in combination with an anti-acetylated tubulin antibody
(Sun lab, unpublished). Co-immunolabeling for acetylated tubulin thus needs to be
performed keeping the above considerations in mind. Appropriate dilution of anti-
bodies, sequential staining, and careful controls are essential to circumvent this
problem. Below, we describe a protocol for sequential labeling.

1. Materials
 Anti-acetylated tubulin, clone 6-11B-1, Sigma T6793 (Table I).
 Dent’s fixative: one part of DMSO mixed with four parts of methanol.
 30% hydrogen peroxide.
 PBT: PBS with 0.5% Tween-20.
 Blocking solution: PBT + 10% serum. Use serum that matches the species in
which secondary antibodies were generated.
 Secondary antibodies: Jackson ImmunoResearch (West Grove, PA).
 Mounting medium: we use Vectorshield Hardset Mounting Medium (Vector
Laboratories, H-1400).

2. Protocol
1. Fix embryos with Dent’s fixative overnight. For embryos younger than 30 hpf,
use precooled Dent’s fixative and incubate at –20  C. For older embryos, use
Dent’s fixative at RT and incubate at RT.
2. Bleach pigment by incubating embryos in 1:2 hydrogen peroxide/methanol
solution for 2 hours to overnight at RT.
3. Wash with methanol three times, 5 min each.
4. Rehydrate embryos by incubating in 50:50 PBT/methanol and PBT sequentially
for 5 min each.
5. Block embryos in the blocking solution for 30 min.
6. Incubate embryos in the blocking solution and a 1:20,000 dilution of anti-
acetylated tubulin antibody at RT for 2 h or at 4  C overnight.
7. Wash embryos with PBT five times, 20 min each.
8. Incubate embryos with a fluorophore-conjugated anti-mouse IgG at the con-
centration recommended by the manufacturer for 2 h at RT.
54 Jarema Malicki et al.

9. Wash embryos with PBT five times, 20 min each.


10. Incubate embryos with an antibody developed in a non-mouse host (because the
cilia marker 6-11-B anti-acetylated tubulin antibody is a mouse monoclonal
antibody) against the protein of interest in blocking solution at RT for 2 h or at
4  C overnight.
11. Wash embryos with PBT five times, 20 min each.
12. Incubate embryos with an appropriate secondary antibody.
13. Wash embryos with PBT five times, 20 min each.
14. Place stained embryos in mounting medium. Using Dumont forceps, carefully
remove as much yolk and muscle as possible. For pronephric duct staining, add
coverslip and press the coverslip firmly against the slide. The embryos will be
crushed, but usually the pronephric duct will stay intact. You may need to
perform tests to determine the proper way to dissect the tissue of your choice.
Allow the mounting medium to harden by leaving slides at RT for 30 min.
The specificity of an antibody against the protein of interest needs to be tested
rigorously. One of the most convincing controls is the absence or decrease of signal in
mutants or morphants. The optimal dilution of the antibody should also be tested
empirically, since too high a concentration will lead to high background whereas too
low a concentration will not provide sufficient signal intensity. IgG purification or
affinity purification frequently improves the signal to noise ratio. Many vendors for
custom antibody production provide purification services. Alternatively, one can purify
antibodies in house following well-established protocols (Harlow and Lane, 1988).

IV. Analysis of Cilia-related Mutant Phenotypes in Zebrafish


A. Left–Right Asymmetry Defects
The transparency and external development of the zebrafish embryo allow for
direct inspection of morphological phenotypes in live embryos. In addition, tools
such as in situ hybridization, immunohistochemistry, and histology can also be
utilized to analyze mutant phenotypes in depth. One common phenotype associated
with cilia defects is abnormal left-right asymmetry. The breaking of bilateral sym-
metry is believed to be triggered by cilia-driven fluid flow in the KV, similar to that
described in the mouse node (Essner et al., 2002). Defects in nodal or KV cilia
disrupt the establishment of the LR asymmetry of the body plan, leading to mis-
placement of multiple internal organs, such the heart (Nonaka et al., 1998).

B. Method 4: Evaluation of Heart Positioning in Live Zebrafish Embryos


1. Materials
 Zebrafish embryos at 30 hpf.
 Dissecting microscope.
 Dumont forceps.
3. Analysis of Cilia Structure and Function in Zebrafish 55
[(Fig._3)TD$IG]

Fig. 3 Phenotypes of zebrafish cilia mutants. (A) Side view of an arl13b/scohi459 mutant embryo (sco) and its wild-type
sibling (wt) at 36 hpf. (B) A scohi459 mutant at 3 dpf. Arrow points to a kidney cyst. (C–F) Plastic transverse sections through the
zebrafish trunk at 50 hpf. Tissue architecture is visualized via hematoxylin/eosin staining (Method 5). (C, D) Sections through
the region of the glomerulus (arrowhead) and the pronephric tubule (arrow) in the wild-type (C) and scohi459 mutant (D) animals.
(E, F) Sections through the duct region (arrows) in the wild-type (E) and scohi459 mutants (F). (G–I) Ventral views of embryos at
30 hpf that display heart position (red circles) at the left side (G, L), middle (H, M), and the right side (I, R) of the body axis.
Arrowheads indicate the midline. (J, K) Confocal images showing cilia (stained with anti-acetylated tubulin antibody, in red) in
the pronephric duct (stained with anti-Cdh17 antibody in green) in an arl13b/scohi459 mutant embryo (K, sco), compared to a
wide-type sibling (J, wt) at 33 hpf. Scale bar: 10 mm. Arrow in (J) points to cilia bundle from MCCs while the arrowhead points to
cilia from SCCs. (L-N) Plastic (JB4) sections of wild-type and IFT mutant retinae stained using Methylene Blue/Azure II
(Method 6). (L) The wild-type retina features elongated photoreceptor cell nuclei (arrow). The black tissue next to photoreceptor
cells is the retinal pigmented epithelium. Lightly stained features (arrowhead) are outer segments. (M, N) The photoreceptor cell
layer of animals mutant for IFT-related genes, flr and ovl, display defective photoreceptor morphology and the lack of outer
segments. Cell death in the photoreceptor cell layer results in the appearance of empty spaces (arrows in M, N). The asterisk
shows optic nerve. (O, P) Confocal images of transverse cryosections though the retina. Double cones are visualized with the
Zpr-1 antibody in a wild-type (O) and elitp49 mutant (P) retinae (green signal). Mueller glia are identified using anti-carbonic
anhydrase antibody (red). (P) Mutations in the gene encoding an IFT particle component, elipsa (eli), lead to a degeneration of
photoreceptor cells. Older photoreceptors in the central retina are lost first. (Q, R) Transverse plastic sections through the ear of
the wild type (Q) and its oval mutant sibling (R) at 5 dpf. Larvae were embedded in JB4, sectioned, and stained with Methylene
Blue/Azure II. Arrow points to hair cell nuclei, which are absent in ovl mutant individuals. (S, T) DiI labeling (Method 11, red
signal) of whole embryos stains olfactory pits of a wild-type (S) but not ovl mutant (T) animals. A–F, J, and K are reprinted with
permission from Duldulao et al., 2009; G-I from Cao et al., 2009; L-P from Doerre and Malicki, 2002; and Q-T from Tsujikawa
and Malicki, 2004. (See Plate no. 5 in the Color Plate Section.)
56 Jarema Malicki et al.

2. Protocol
Owing to the transparency of the zebrafish embryo at 30 hpf, the heart is detect-
able using a dissecting microscope (Fig. 3G-I). Use a pair of tweezers to turn the
embryo to a ventral side up position, which allows for easier visualization of the
beating heart. The heart of most wild-type embryos at this stage is located on the left
side of the embryo, whereas cilia mutants often feature hearts that are either posi-
tioned at the center or on the right side (Fig. 3G–I). If mutants develop other obvious
morphological phenotypes, such as body curvature, separate mutant and wild-type
individuals based on these phenotypes first, then count and record the heart position
of every embryo. Perform statistical analysis using results from multiple crosses to
determine whether abnormal heart position is observed in significantly higher
percentages in mutant embryos, compared to their wild-type siblings. It is worth
noting that some zebrafish strains have a high incidence of spontaneous LR defects.
Therefore, it is critical to inspect background LR frequency in a particular strain so
that experiments are properly interpreted.
In zebrafish, the products of many genes associated with cilia formation and/or
function are maternally deposited, which can mask the early functions of these genes
(Cao et al.). As a result, some cilia mutants will not show a LR asymmetry defect. It
is possible to use a morpholino oligo targeting the translation initiation site to block
expression of maternally deposited mRNA. In such an experiment proper controls
need to be included, such as a rescue of the morphant phenotype, a second nonover-
lapping morpholino, a control morpholino with mismatched bases to rule out tox-
icity or off-target effects. Alternatively, a maternal-zygotic mutant can be generated
to study a true null mutant by genetically removing both the maternal and the zygotic
contribution of the analyzed gene (Ciruna et al., 2002).

C. Alternative Method: Analysis of Left–Right Asymmetry Defects Using in situ


Hybridization
In addition to observing the position of internal organs in zebrafish embryos, one
can also analyze the distribution of molecular markers important in the establish-
ment of LR asymmetry using in situ hybridization. Southpaw (spaw) is one of the
earliest markers of LR asymmetry in zebrafish embryos (Long et al., 2003). In wild-
type embryos, spaw is expressed in the left lateral plate mesoderm, while embryos
with LR asymmetry defects show left-sided, right-sided, bilateral, or absent spaw
expression (Long et al., 2003). Note that the expression of spaw is dynamic and its
asymmetric expression is most obvious between the 18–22-somite stages. Other
useful markers for LR asymmetry include pitx2, lefty1, and lefty2 (Bisgrove et al.,
1999; Campione et al., 1999; Zhao and Malicki, 2007). A detailed protocol for in situ
hybridization in zebrafish embryos can be found at: http://zfin.org/ZFIN/Methods/
ThisseProtocol.html (see also Westerfield, 2000).
3. Analysis of Cilia Structure and Function in Zebrafish 57

D. Kidney Cysts
Cilia mutants almost inevitably develop kidney cysts and ventral body curvature
(Kramer-Zucker et al., 2005; Sun et al., 2004) (Fig. 3A-F, J, K). Here we describe a
method to analyze kidney cysts formation in zebrafish embryos.

E. Method 5: Detection of Kidney Cysts in Zebrafish


1. Materials
 Dissecting microscope.
 Day 2–5 zebrafish embryos.
 Embryo medium.
 3% methycellulose (as in Method 2).
 25 MESAB solution (as in Method 2).
 Bouin’s fixative: Polysciences, 16045-1.
 JB-4 plus embedding kit: Polysciences, 18570.
 JB-4 infiltration and embedding solutions: prepare following manufacturer’s
instructions.
 Harris’ hematoxylin: Sigma, HHS16.
 ammonia water: mix 500 ml water with 1.5 ml ammonium hydroxide (VWR,
VW3571-1), make fresh before use.
 stock solution of Eosin Y: dissolve 1 g Eosin Y (Mallinckrodt, 15086-94-9) in
100 ml deionized H2O.
 stock solution of Phloxine-B: dissolve 1 g Phloxine-B (Sigma, 18472-87-2) in
100 ml deionized H2O.
 Eosin-Phloxine solution: add 100 ml stock Eosin and 10 ml stock Phloxine to
780 ml deionized H2O, add 4 ml glacial acetic acid, bring volume to 1000 ml with
deionized H2O.

2. Protocol
Kidney cysts are frequently located in the glomerular–tubular region. Given the
transparency of both the embryo and the pronephros, the latter is usually invisible in
the wild type when using a dissecting scope. The cyst, on the other hand, has a bubble-
like appearance, slightly anterior and medial to the pectoral fin (Fig. 3B, arrow). The
cysts are often first visible by 2 dpf, and they tend to enlarge as the embryo develops.
The kidney duct is also often dilated. The presence of kidney cysts and dilated ducts
can be verified via histological sections as detailed below (Fig. 3C-F).

Fixing, embedding and sectioning


1. Anaesthetize embryos in embryo medium with 1 MESAB.
2. Fix embryos in Bouin’s fixative at RT overnight.
3. Wash embryos with PBT five times, 5 min each.
4. Incubate embryos in JB-4 infiltration solution at RT for 1 h.
58 Jarema Malicki et al.

5. Replace JB-4 infiltration solution with a fresh aliquot; incubate at RT for 1 h to


overnight.
6. Replace with fresh embedding solution.
7. Transfer embryos into a mold. Do not use a silicone-coated mold, as it might
inhibit the polymerization of JB4 resin. Fill the mold with embedding solution.
Orient embryos with a pipette tip or a needle.
8. To facilitate the polymerization of the resin, cover the mold with parafilm.
9. The embedding solution will polymerize in approximately 40 min at RT.
10. Section embryos at 4 mm with a microtome. Typically, 8–10 sections can be
collected on each slide.

Hematoxylin/Eosin staining
1. Stain slides in Harris’ hematoxylin for 10 min. For even staining, quickly dip
slides in the solution several times and then let them sit in hematoxylin for
10 min.
2. Dip slides in deionized H2O briefly.
3. Dip slides in ammonia water 2–3 times. Sections should turn bright blue.
4. Dip slides in deionized H2O.
5. Stain with Eosin-Phloxine for 5 min at RT.
6. Dip in deionized H2O.
7. Check with a dissecting scope to make sure that staining is sufficiently intense.
Repeat the staining procedure if necessary.
8. Air dry at RT or dry in an incubator at 65  C.
9. Add mounting medium (Permount, Fisher, SP15-100) and coverslip.
10. Observe sections using a microscope; nuclei should appear blue while cyto-
plasm should have a pink appearance.

F. Degeneration of Sensory Neurons


Mutations of cilia-related genes, such as those encoding IFT proteins, lead to a
degeneration of neurons in the visual, auditory, and olfactory systems (Doerre and
Malicki, 2002; Gross et al., 2005; Krock and Perkins, 2008; Omori et al., 2008;
Tsujikawa and Malicki, 2004). Below we describe several methods used to monitor
ciliated cells in sense organs.

Visual System
Cilia-related defects in photoreceptor cells can be monitored using several
approaches. The simplest one is to evaluate the gross appearance of mutant retina
by analyzing plastic histological sections (Fig. 3L-N). Using a microscope equipped
with Nomarski optics to view sections stained to highlight cell nuclei, one can
distinguish photoreceptor outer segments (arrowhead in Fig. 3L), a feature
3. Analysis of Cilia Structure and Function in Zebrafish 59

frequently abnormal in cilia mutants (Doerre and Malicki, 2001). In severely


affected mutant retinae, the loss of photoreceptors results in the appearance of
obvious holes in the outer retina (arrows in Fig. 3M–N).
Immunohistochemical analysis of protein expression is an informative way to
follow histological studies. The Zpr-1 antibody stains the entire cell body of red–
green double cone photoreceptor cells. This antibody can be used both on whole
embryos and on frozen sections (Larison and Bremiller, 1990). Zpr-1-positive cells
are found throughout the entire photoreceptor cell layer by 3 dpf (Fig. 3O) (Larison
and Bremiller, 1990). In addition, staining of retinae with anti-opsin antibodies is
very informative, as the transport of opsin is almost invariably affected in cilia
mutants. Defects in IFT-dependent processes, for example, lead to opsin accumula-
tion in the cell body (Doerre and Malicki, 2002; Tsujikawa and Malicki, 2004).
Several anti-opsin antibodies are available for zebrafish (Table I, see also Avanesov
and Malicki, 2010).
Lastly, another useful approach to the analysis of ciliary (i.e., outer segment)
defects in zebrafish photoreceptors is electron microscopy. Outer segments differ-
entiate a very distinctive array of parallel membrane folds (Fig. 1K), which can be
visualized on electron micrographs in exquisite detail. This membrane architecture
is often disrupted or absent in cilia mutants (Doerre and Malicki, 2002; Krock and
Perkins, 2008). Electron microscopy of photoreceptors is performed using standard
approaches (Doerre and Malicki, 2001; Schmitt and Dowling, 1999).
In rare but interesting cases, cilia-related mutations may affect a subset of photo-
receptors (Zhao and Malicki, unpublished). For example, rods may degenerate but
not cones, or vice versa. To distinguish subtypes of photoreceptors, one can use the
opsin antibodies mentioned above. In situ hybridization with antisense opsin RNA
antiopsin probes can also be used for the same purpose. Alternatively, transgenes that
express GFP in a subset of photoreceptors are available (see, for example, Fadool,
2003). More information on photoreceptor cell markers, including transgenes, has
been provided elsewhere (Avanesov and Malicki, 2010).

G. Method 6: Evaluation of Photoreceptor Cell Layer Morphology on Plastic Sections


1. Materials
 PBST: PBS + 0.1% Tween-20.
 Fixative: 4% PFA (Sigma, P6148) in PBST.
 Ethanol: Sigma, 279741-1L.
 JB4 embedding kit (as in Method 5).
 Methylene Blue/Azure II staining solution: Methylene blue (Sigma, MB-1),
0.13%; Azure II (Sigma, A2507), 0.02%; Glycerol, 10%; Methanol, 10%;
45 mM phosphate buffer, pH 6.9).
 Deionized H2O.
 Frosted microscope glass slides: Fisher, 12-544-3.
 Permount: Fisher, SP15-500.
60 Jarema Malicki et al.

Table I
Markers of cilia and ciliated cells in zebrafish

Name/specificity Marker type/ Localization References/Sources


concentration

CILIARY AXONEME
Acetylated tubulin Ab, mono Ubiquitous cilia marker (Duldulao et al., 2009; Tsujikawa and
1:1000–1:20,000 Malicki, 2004)
Sigma, clone 6-11B-1
AXO49 Ab, mono Cilia in the nasal epithelium, (Callen et al., 1994; Wloga et al., 2009)
(polyglycylated tubulin) 1:2000 neuromast hair cells, medial
section of pronephros
GT335 Ab, mono Cilia in olfactory placode, PND, (Pathak et al., 2007; Zhao and Malicki,
(glutamylated tubulin) 1:400 hair cells, cilia in other unpublished, Wloga et al., 2009;
organs Wolff et al., 1992)
TAP952 Ab, mono Cilia in olfactory placode, otic (Callen et al., 1994; Wloga et al., 2009)
(monoglycylated tubulin) 1:2000 vesicle, PND, spinal cord,
hypochord, neuromast hair
cells
Scorpion (sco)/Arl13b Ab, poly Cilia in olfactory placode, CNS, (Duldulao et al., 2009)
1:200–1:2000 PND, KV
arl13b/sco-eGFP mRNA Cilia in PND, and other organs (Duldulao et al., 2009)
bact::arl13b(hennin)-GFP Transgene Cilia in PND, CNS, KV (Borovina et al., 2010)

BASAL BODIES
Gamma tubulin Ab, mono Ubiquitous basal body marker (Duldulao et al., 2009; Tsujikawa and
1:200 Malicki, 2004)
Sigma, clone GTU88
centrina-GFP mRNA CNS, possibly other organs (Borovina et al., 2010)
GFP-centrinb mRNA Retinal neuroepithelium, (Zolessi et al., 2006)
possibly in other organs

PHOTORECEPTORS
opn1sw1::GFP Transgene UV cones (Takechi et al., 2003)
UV opsin Ab, poly UV cone outer segments (Doerre and Malicki, 2001; Vihtelic
1:1000 et al., 1999)
UV opsin RNA probe UV cones (Hisatomi et al., 1996; Takechi et al.,
2003)
Zpr1 Ab, mono Red–green double cones (Larison and Bremiller, 1990)
(FRet 43) 1:250 ZIRC
Red opsin Ab, poly Red cone outer segments (Doerre and Malicki, 2001; Vihtelic
1:200 et al., 1999)
Red opsin RNA probe Red cones (Chinen et al., 2003; Raymond
et al., 1995; Takechi and Kawamura,
2005)
Blue opsin Ab, poly Blue cone outer segments (Doerre and Malicki, 2001; Vihtelic
1:200 et al., 1999)
Blue opsin RNA probe Blue cones (Chinen et al., 2003; Raymond et al.,
1995; Vihtelic et al., 1999)
Green opsin Ab, poly Green cone outer segments (Doerre and Malicki, 2001; Vihtelic
1:500 et al., 1999)

(Continued)
3. Analysis of Cilia Structure and Function in Zebrafish 61

Table I (Continued)

Name/specificity Marker type/ Localization References/Sources


concentration

Green opsin RNA probe Green cones (Chinen et al., 2003; Raymond et al.,
1995; Vihtelic et al., 1999)
Zpr3 (FRet11) Ab, mono Rods (Schmitt and Dowling, 1996)
1:300 ZIRC
Rod opsin Ab, poly Rod outer segments (Doerre and Malicki, 2001; Vihtelic
1:1000 et al., 1999)
Rod opsin RNA probe Rods (Chinen et al., 2003; Raymond et al.,
1995)
xops1.3::opsin-GFP-CT44 Transgene Rod outer segments (Perkins et al., 2002)
xops::EGFPa Transgene Rods (Fadool, 2003)
MECHANOSENSORY HAIR CELLS
Acetylated tubulin Ab, mono Cilia and somata of hair cells (Jing and Malicki, 2009; Tsujikawa and
1:1000 - 1:20 000 Malicki, 2004; Zhao and Malicki,
2007) Sigma, clone 6-11B-1
HCS-1 Ab, mono Somata of hair cells (Gale et al., 2000; Lopez-Schier and
1:20 - 1:250 Hudspeth, 2006; Schibler and
Malicki, 2007)
brn3c::GFP Transgene Somata of hair cells (Xiao et al., 2005)
OLFACTORY SENSORY NEURONS
Olfactory marker protein RNA probe Olfactory sensory neurons (Tsujikawa and Malicki, 2004; Yoshida
et al., 2002)
DiI Lipophilic Tracer Olfactory sensory neurons (Dynes and Ngai, 1998; Tsujikawa and
Malicki, 2004), Invitrogen N22880

KV, Kupffer’s vesicle; PND, pronephric duct.


a
derived from xenopus.
b
derived from zebrafish.

2. Protocol

Fixing, embedding and sectioning


Use JB-4 embedding and sectioning protocol as described in Method 5. We prefer
transverse sections. Ideally, they should contain the optic nerve, which can serve as a
point of reference to facilitate comparisons among samples (asterisk in Fig. 3N).

Methylene blue-Azure II staining


1. Dip slides with sections into staining solution for 10 s at RT.
2. Remove and rinse immediately with a generous amount of tap water. If staining is
on the faint side, then restain as above. It is suggested to test the timing of this
staining technique on dispensable sections first.
3. Allow slides to dry completely. Apply 20–40 mL of Permount and add a coverslip.
4. Slides may be stored at RT indefinitely.
62 Jarema Malicki et al.

H. Method 7: Analysis of Photoreceptor Cells on Cryosections via Immunohistochemistry


1. Materials
 Anti-opsin antibodies, Zpr-1, see also Table I.
 PTU solution (as in Method 2).
 PBST: PBS + 0.1% Tween-20.
 Fixative: 4% PFA (Sigma, P6148) in PBST.
 25  MESAB (as in Method 2).
 30% Sucrose/PBST solution. Add 0.05% (w/v) of Sodium Azide and store at 4  C.
 Frozen Section Medium NEG50: Richard-Allan Scientific, 6502.
 3 mL dispensable transfer polyethylene pipettes: e.g., BD Falcon, 357524
 Dissecting needle.
 Superfrost plus microscope slides precleaned: Fisher, 12-550-15. The choice of
slides is essential to avoid losing sections during washes.
 Grease pen: Electron Microscopy Sciences, 71319.
 Empty tip Boxes with the perforated holding surface: e.g., USA Scientific, 1111-
1800.
 Blocking solution: PBS + 0.5% Triton X-100 + 10% serum. Use serum that
matches the species in which secondary antibodies were generated.
 VectaShield Mounting Media: Vector Labs, H-1000.
 Optional: antigen retrieval solution: 100 mM Sodium Citrate in deionized H2O.
 Optional: Alexa Fluor 546 Phalloidin: Invitrogen, A22283. Prepare stock solution
in methanol according to manufacturer’s instructions.

Fixing, embedding and sectioning


1. To visualize outer segments, pigmentation needs to be eliminated. To block
pigment formation, raise embryos in the presence of PTU starting at 24 hpf or
earlier (Method 2, Step 2)
2. When embryos/larvae reach the desired stage, sacrifice them by over anesthe-
tization in MESAB, and immediately transfer to fixative.
3. Fix embryos for 2 h at RT or overnight at 4  C. Use an orbital shaker. Subsequent
steps are at RT unless stated otherwise.
4. Remove fix and wash three times in PBST, 5 min each.
5. Cryoprotect in sucrose solution overnight at 4  C, or at RT until specimens sink.
6. Replace sucrose with Frozen Section Medium. This is a very viscous substance.
Use Pasteur pipette.
7. Cut polyethylene pipette perpendicular to its long axis into 6–10 mm wide rings.
Place the rings on a glass slide and fill them halfway with Frozen Section
Medium.
8. Using Pasteur pipette transfer specimen into the ring ‘‘mold’’ described in the
previous step. Some Frozen Section Medium will transfer too, which will fill up
the mold completely.
3. Analysis of Cilia Structure and Function in Zebrafish 63

9. Use dissecting needle to orient specimen. To obtain transverse sections, heads of


specimen must point straight down. Cool slides with specimen molds to -20  C
in the cryostat chamber. Medium will solidify and turn white in about 5–10 min.
10. Prior to sectioning, remove polyethylene molds by cutting through their walls
with a razorblade.
11. Mount frozen blocks on a cryostat specimen holder using additional Frozen
Section Medium and collect 12–30 mm thick sections on a prechilled slide
inside the cryostat chamber set to 20  C. When brought to RT, sections will
thaw and adhere to the slide.
12. Allow slides to dry at RT for 4 h. Then proceed to the next step.

Immunohistochemistry
1. Optional Step: Detection of some antigens may require special treatment such as
placing slides into boiling-hot 10% sodium citrate solution for 10 min (this
procedure is known as antigen retrieval).
2. Using a grease pen draw a hydrophobic border around at the edge of the slide.
Allow the grease to dry for 2–3 min.
3. Briefly rehydrate slides in PBST. This will also dissolve the Frozen Section
Medium. Make sure to be gentle, as it is easy to lose sections during this step.
4. In the meantime prepare humidified chamber. For this purpose, we use a tip rack
with a cover (see Materials section above for product information). Fill the
bottom compartment of the tip rack with tap water. This will serve as a home-
made humidifying chamber for antibody incubations.
5. Open the tip rack cover. Place slides on the top of the perforated, tip holding
surface.
6. Overlay the slide with 100–200 mL of the blocking solution. The grease border
will keep the blocking solution on the slide. Close the rack cover. Incubate the
slide with the blocking solution for 30–60 min at RT.
7. Dilute primary antibody(ies) to appropriate concentration(s) in blocking
solution.
8. Remove the blocking solution by positioning slides vertically and collecting
excess solution with a Kimwipe. Apply the primary antibody solution (as in
Step 6). Incubate overnight at 4  C.
9. Next day wash slides three times in PBST, 5 min each.
10. Incubate with secondary antibody(ies) at RT for 2 h. Optional: Add phalloidin
to counterstain F-actin (dilute stock solution 1:40).
11. Wash slides several times in PBST.
12. Use Kimwipe to remove excess PBST and apply mounting medium. Place a
coverslip and seal with nail polish.
13. Image using confocal or conventional fluorescence microsocopy.
14. The preparations may be stored at 4  C for at least a week, although the quality
of signal deteriorates over time.
64 Jarema Malicki et al.

Auditory System
Mutations of zebrafish IFT genes, and presumably also other cilia-related loci,
lead to the loss of kinocilia and subsequently the degeneration of mechanosensory
hair cells (Tsujikawa and Malicki, 2004). This can be monitored using similar
approaches to those described for photoreceptor cells. The overall health of hair
cells is easy to evaluate on transverse sections of maculae. On plastic sections,
maculae appear as thickenings of the epithelium that lines the otic vesicle
(Fig. 3Q). The maculae contain two major cell types: the supporting cells and hair
cells. The latter are oval in shape and positioned next to the apical surface (Bang
et al., 2001; Schibler and Malicki, 2007). To evaluate the ear by simple histology on
plastic sections, we use the protocol described in Method 6. Prior to embedding,
otoliths have to be removed as described in Method 8 below.
Anti-acetylated tubulin antibodies stain hair cell bodies in addition to their kino-
cilia (Fig. 1 D, E, G). The signal is particularly prominent at the apical surface of the
cell. Therefore, these antibodies can also be used to evaluate hair cell morphology. In
addition, the HCS-1 antibody, which has recently been shown to recognize otoferlin,
is a very good marker of hair cell somata in zebrafish (Gale et al., 2002; Goodyear
et al., 2010; Schibler and Malicki, 2007).
Immunohistochemistry can be conveniently performed on whole animals through
at least 5 dpf either alone or in combination with phalloidin staining. Phalloidin is
commonly used to stain filamentous actin-rich structures. As hair cell sterocilia are
supported by dense actin bundles, phalloidin is very useful to visualize the morphol-
ogy of stereociliary bundles. Double staining with phalloidin and anti-tubulin anti-
bodies makes it possible to evaluate the planar polarity of hair cells. This is done by
comparing the relative positions of the kinocilium and the stereociliary bundle. The
directionality of each hair cell can be visualized on en face images of maculae by
drawing a line connecting the kinocilium and the center of the stereociliary bundle.
In a polarized epithelium, all lines will have the same direction. As in the case of
plastic sections, additional steps must be taken to remove otoliths in order to properly
visualize hair cells (see Method 8 below).
In addition to whole-mount staining, auditory cells and their features can be
analyzed on frozen sections. The use of frozen sections is a necessity for reagents
that produce high background in whole-mount applications, such as anti-g tubulin
antibodies. Hair cells can also be visualized with a brn3c::GFP reporter transgene
(Xiao et al., 2005). This transgenic line produces a robust signal in whole animals,
which obviates the need to the section. Below we describe a method for staining of
hair cells in whole embryos. To analyze the staining of hair cells on frozen sections,
follow the protocol described for photoreceptors (Method 7).

I. Method 8: Whole-Mount Staining and Imaging of Hair Cells in the Inner Ear
1. Materials
 Mouse anti-acetylated tubulin or mouse anti-HCS-1 (Table I).
 PBST (PBS + 0.1% Tween-20).
3. Analysis of Cilia Structure and Function in Zebrafish 65

 25  MESAB (as in Method 2).


 Fixative: 4% PFA (Sigma, P6148) in PBST.
 PBST + 1% Triton X-100.
 Blocking solution: PBS + 0.5% Triton X-100 + 10% serum. Use serum that
matches the species in which secondary antibodies were generated.
 Water dipping objectives: e.g., Leica 40 dipping lens, working dist. 3.3 mm, NA
0.8; or Leica 63 dipping lens, working dist. 2.2 mm, NA 0.9.
 Optional: Alexa Fluor 546 Phalloidin: Invitrogen, A22283.

2. Additional Materials

Agarose embedding supplies


 Embryo medium (as in Method 5).
 Low melting point-agarose (Sigma, A9414-10G), 1% in embryo medium (keep
liquid on 50  C heat block).
 Regular agarose (Invitrogen, 16500–500), 1% in embryo medium.
 Custom made molds to imprint the surface of agarose. Blastomere transplantation
molds can be used here. They produce wells with the square, 1  1 mm opening,
and 30 degree tilted bottom. Molds can be made by local machine shops or
purchased from Adaptive Science Tools.
 Large (90 mm) and small (60 mm) disposable petri dishes.
 Dissecting needle.
 Razor blade.

3. Protocol

Fixing and staining


1. Sacrifice embryos at desired stage by over anesthetization in MESAB, and
immediately transfer to fixative.
2. Fix embryos or larvae for 4 h at RT or overnight at 4  C in freshly prepared
fixative on an orbital shaker. Subsequent steps are at RT on the orbital shaker
unless stated otherwise.
3. Wash once in PBST for 5 min.
4. Incubate in 1% Triton X-100 in PBST for 8 h or longer. This treatment dissolves
otoliths. Alternatively, otoliths can be dissolved by incubating larvae in 120 mM
EDTA in 0.1 M phosphate buffer (up to 3 d for dissected adult ears).
5. Wash once in PBST for 5 min.
6. Incubate in the blocking solution for 30 min.
7. Incubate specimen overnight at 4  C in the blocking solution containing appro-
priate concentration of the primary antibody (Table I).
8. Wash four times in PBST, 30 min each.
66 Jarema Malicki et al.

9. Incubate specimen overnight at 4  C in the blocking solution containing appro-


priate concentration of the secondary antibody. Optional: Add phalloidin to
counterstain F-actin (dilute stock solution 1:40).
10. Wash three times in PBST, 30 min each. Wash once in PBS.

Embedding in agarose for imaging


1. Pour 1% regular agarose into a large petri dish and place a mold on a top of it. The
mold will float on agarose surface. Let the agarose harden completely.
2. Removing the mold from the agarose will leave imprinted wells on the agarose.
Using a razor blade, cut and gently retrieve the agarose block that contains the
wells. Place it into a 60 mm petri dish in the orientation that exposes well openings.
3. To properly orient the embryo, place it on its lateral side over the well opening so
that the yolk mass fits inside the well. This will position the embryo flat on the
agarose surface.
4. Once embryos are positioned properly, immobilize them using 1% low melting
point agarose. If necessary, adjust embryo orientation before the agarose hardens.
5. Flood embryos in agarose with egg water and view using a water-dipping objec-
tive. Embryos can be stored for several days prior to imaging, although the quality
of signal will gradually deteriorate.
6. This procedure can be also used in the analysis of living specimen. For this
application, lower heat block temperature to 40  C or less.

J. Method 9: Detection of Basal Bodies in Hair Cells


1. Materials
 Mouse anti-g tubulin: Sigma, GTU88, T6557
 PBST: PBS + 0.1% Tween-20.
 Fixative: 4% PFA (Sigma, P6148) in PBST.
 25  MESAB (as in Method 2)
 PBD: PBST + 1% v/v DMSO + 1% w/v BSA.
 Serum that matches the species in which secondary antibodies were generated.
 0.25% Trypsin (Fisher, T-360) in PBS. Make fresh each time.
 Agarose embedding supplies (as in Method 8).
 Optional – Alexa Fluor 546 Phalloidin: Invitrogen, A22283. Prepare stock solu-
tion in methanol according to manufacturer’s instructions.

2. Protocol
1. Sacrifice embryos at desired stage by anesthetization in MESAB, and imme-
diately transfer to fixative.
2. Fix embryos in fresh fixative for 2 h at RT or overnight at 4  C. Use the orbital
shaker. Subsequent steps at RT unless stated otherwise.
3. Wash twice in PBST, 5 min each.
3. Analysis of Cilia Structure and Function in Zebrafish 67

4. Permeabilize using 0.25% Trypsin in PBS (15 min for embryos at 30 hpf,
30 min at 4 dpf).
5. Wash twice in PBST, 5 min each.
6. Incubate in PBST containing 0.2% Triton X-100 for 30 min.
7. Wash twice in PBST, 5 min each.
8. Block for 30 min by incubating in PBD containing 10% serum.
9. Incubate in the primary antibody diluted in PBD (1:300) overnight at 4  C.
10. Wash four times in PBD, 30 min each.
11. Incubate in the secondary antibody (diluted 1:300 in PBD) overnight at 4  C.
Optional: Add phalloidin to counterstain F-actin (dilute stock solution 1:40).
12. Wash three times in PBD, 30 min each.
13. Wash once in PBS to remove detergent.
14. Proceed to agarose mounting (see Method 8)

Lateral Line
Neuromast hair cells can be labeled with the same reagents as used to stain cilia in
the ear. Since neuromasts are protruding from the surface of the skin, sectioning of
the tissue may not be necessary. Some protocols, such as anti-g -tubulin staining
described above, work very well for the lateral line. Neuromasts are easily identified
using the vital dye DASPEI, which stains hair cells. We describe a DASPEI staining
method below.

K. Method 10: Detection of Neuromast Hair Cells in a Living Specimen


1. Materials
 DASPEI: Invitrogen, D426.
 E3 medium: 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4.
 MESAB (as in Method 2).
 Methylcellulose (as in Method 2).
 Agarose mounting implements (as in Method 8).

2. Protocol
1. Transfer larvae to 1 mM DASPEI solution in E3 medium.
2. incubate for 20 min.
3. rinse thoroughly in E3.
4. anaesthetize in MESAB, and mount in methylcellulose or agarose.
5. view using fluorescence microscopy.

Olfactory System
As olfactory cilia are located on the surface of the animal, they are easily acces-
sible to staining reagents. The length of cilia can be evaluated by antibody staining
68 Jarema Malicki et al.

and confocal microscopy in whole animals (Omori et al., 2008). Phalloidin is useful
as a counterstain in such experiments to highlight the appearance of the surrounding
tissues. Similar to photoreceptors and mechanosensory hair cells, olfactory sensory
neurons degenerate following the loss of cilia in IFT mutants (Tsujikawa and
Malicki, 2004). The survival of these neurons can be monitored using in situ
hybridization with probes to the olfactory marker protein gene or via DiI labeling
(Dynes and Ngai, 1998; Hansen and Zeiske, 1993; Tsujikawa and Malicki, 2004).
Below, we describe DiI labeling.

L. Method 11: Labeling of Olfactory Neurons by DiI Incorporation


1. Materials
 Neuro Trace DiI Tissue Labeling Paste: Invitrogen, N22880.
 DMSO: Sigma, D5879.
 Ethanol: Sigma, 279741-1L.
 Embryo medium (recipe as in Method 5).
 Incubator set at 30  C.
 Regular agarose, 1% (as in Method 8).

2. Protocol
1. Prepare DiI/DMSO stock by mixing ca. 60 mg of DiI paste (one scoop with a
200 mL tip) in 500–1000 mL of DMSO.
2. Prepare embryo medium containing 7% DiI/DMSO stock and 1% ethanol. Warm
up to 30  C.
3. Incubate live embryos in the solution from Step 2 at 30  C for 10 min.
4. Wash embryos in embryo medium at 28  C. Embryos will not look healthy but
will remain alive.
5. To observe olfactory pits, larvae can be positioned vertically, heads up, in agarose
wells. In this case, wells can be made by punching holes in an agarose bed with a
Pasteur pipette. Mounted embryos can be imaged by confocal or conventional
fluorescence microscopy.

V. Future Directions
The importance of cilia function in the KV, the kidney duct, and sensory organs is
now well appreciated. However, cilia not restricted to these organs and many basic
questions remain unanswered. For example, when and where do cilia first start to
form in the context of organogenesis? What are the distribution patterns of motile
and immotile cilia in different organs during development? What is the normal
frequency and waveform of cilia movement in different tissues? Are the morphology,
length, and the motility of cilia regulated by physiological parameters? Furthermore,
3. Analysis of Cilia Structure and Function in Zebrafish 69

although IFT trafficking and calcium influx are intimately associated with cilia
formation and function, they are still challenging to image in live embryos. This
is an important technical issue that merits further attention. With the development of
more advanced imaging tools, we will gain a deeper and more comprehensive
understanding of the function of this miniature cell surface organelle in development
and adult physiology.

Acknowledgments
We thank Dr. Chengtian Zhao, and Peter Kovach of the Malicki lab, as well as members of Yale Center
for PKD research for helpful discussions, Nicole Semanchik for superb technical assistance and SueAnn
Mentone for assistance on histology. This work was supported by NIDDK (RO1 DK069528 and P50
DK057328 (project #3) awards (to ZS), and NEI research grant RO1 EY018176 (to JM).

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CHAPTER 4

Cellular Dissection of Zebrafish


Hematopoiesis
David L. Stachura*,y and David Traver*,y
*
Division of Biological Sciences, Section of Cell and Developmental Biology, University of California San
Diego, La Jolla, California, USA
y
Department of Cellular and Molecular Medicine, University of California San Diego School of Medicine,
La Jolla, California, USA

Abstract
I. Introduction
II. Zebrafish Hematopoiesis
A. Primitive Hematopoiesis
B. Definitive Hematopoiesis
C. Adult Hematopoiesis
III. Hematopoietic Cell Transplantation
A. Embryonic Donor Cells
B. Adult Donor Cells
IV. Enrichment of Hematopoietic Stem Cells
V. In vitro Culture and Differentiation of Hematopoietic Progenitors
A. Stromal Cell Culture Assays
B. Clonal Methylcellulose-based Assays
VI. Conclusions
References

Abstract

The zebrafish is an excellent model system to study vertebrate blood cell devel-
opment due to a highly conserved hematopoietic system, optical transparency, and
amenability to both forward and reverse genetic approaches. The development of
functional assays to analyze the biology of hematopoietic mutants and diseased
animals remains a work in progress. Here we discuss recent advances in zebrafish
hematology, prospective isolation techniques, cellular transplantation, and culture-
based assays that now provide more rigorous tests of hematopoietic stem and

METHODS IN CELL BIOLOGY, VOL 101 0091-679X/10 $35.00


Copyright 2011. Elsevier Inc. All rights reserved. 75 DOI 10.1016/B978-0-12-387036-0.00004-9
76 David L. Stachura and David Traver

progenitor cell function. Together with the proven strengths of the zebrafish, the
development and refinement of these assays further enable efforts to better under-
stand the development and evolution of the vertebrate hematopoietic system.

I. Introduction

Over the past two decades, the development of forward genetic approaches in the
zebrafish has provided unprecedented power in understanding the molecular basis of
vertebrate blood development. Establishment of cellular and hematological
approaches to better understand the biology of resulting blood mutants, however,
has lagged behind these efforts. In this chapter, recent advances in zebrafish hema-
tology will be reviewed, with an emphasis on prospective strategies for isolation of
both embryonic and adult hematopoietic stem cells (HSCs) and the development of
assays to rigorously test their function.

II. Zebrafish Hematopoiesis


Developmental hematopoiesis, in both mammals and teleosts, occurs in four
sequential waves (Fig. 1). The first two waves are termed ‘‘primitive,’’ and each
generates transient precursors that respectively give rise to embryonic macrophages
and erythrocytes (Keller et al., 1999; Palis et al., 1999). The next two waves consist
of definitive hematopoietic precursors, defined as multipotent progenitors of adult
cell types. The first to arise are erythromyeloid progenitors (EMPs), which give rise
to erythroid and myeloid lineages (Bertrand et al., 2005b, 2007; Palis et al., 1999,
2001), followed by multipotent HSCs, which are endowed with the potential to both
self-renew and generate all adult hematopoietic cell types [reviewed in Cumano and
Godin (2007)].

A. Primitive Hematopoiesis
Primitive hematopoiesis has been extensively studied in the mouse, where prim-
itive macrophages and erythroid cells are generated in the extraembryonic yolk sac
(YS) (Bertrand et al., 2005b; Palis et al., 1999). In the zebrafish, primitive macro-
phages develop in an anatomically distinct area known as the rostral blood island
(RBI) (Fig. 1A). Transcripts for tal1 (also known as scl), lmo2, gata2a, and fli1a are
found in the RBI between the three- and five-somite stages (ss) (Brown et al., 2000;
Liao et al., 1998; Thompson et al., 1998). This is quickly followed by expression of
spi1 (also known as pu.1) in a subset of these precursors (Bennett et al., 2001;
Lieschke et al., 2002). Between 11 and 15 somites, spi1+ macrophages are detect-
able, and they migrate toward the head midline (Herbomel et al., 1999; Lieschke
et al., 2002; Ward et al., 2003) and across the yolk ball (Fig. 1C). Some of these
precursors enter circulation, while others migrate into the head (Herbomel et al.,
4. Cellular Dissection of Zebrafish Hematopoiesis 77
[(Fig._1)TD$IG]

Fig. 1 Model of hematopoietic ontogeny in the developing zebrafish embryo. (A) Different regions of lateral plate mesoderm
give rise to anatomically distinct regions of blood cell precursors. Anatomical regions of embryo responsible for generation of
hematopoietic precursors (red), vasculature (blue), and pre-HSCs (green) are highlighted. Cartoon is a five-somite stage embryo,
dorsal view. (B) Timing of mouse and zebrafish hematopoietic development. In mouse (left), primitive hematopoiesis initiates in
the yolk sac (YS; yellow), producing primitive erythroid cells and macrophages. Later, definitive EMPs emerge in the YS. HSCs
are specified in the aorta, gonad, and mesonephros (AGM, teal) region. These HSCs eventually seed the fetal liver (orange), the
main site of embryonic hematopoiesis. Adult hematopoiesis occurs in the thymus (blue), spleen (green), and bone marrow (red).
Zebrafish hematopoiesis is similar: temporal analogy to mouse hematopoiesis shown in (B, right), spatial locations shown in (C).
Numbers in (C) correspond to timing of distinct precursor waves. (C) Embryonic hematopoiesis occurs through four independent
waves of precursor production. First, primitive macrophages arise in cephalic mesoderm, migrate onto the yolk ball, and spread
throughout the embryo (purple, 1). Then, primitive erythrocytes develop in the intermediate cell mass (ICM; yellow, 2). The first
definitive progenitors are EMPs, which develop in the posterior blood island (PBI; orange, 3). Later, HSCs arise in the AGM
region (teal, 4), migrate to the CHT (later name for the PBI, orange), and eventually seed the thymus and kidney (blue; red).
Similar hematopoietic events in mouse and fish are color-matched between right and left panels of (B). Hematopoietic sites (B)
and locations (C) are also color matched. (hpf: hours postfertilization, dpf: days postfertilization, wpf: weeks postfertilization, E:
embryonic day, RBI: rostral blood island.) (See Plate no. 6 in the Color Plate Section.)
78 David L. Stachura and David Traver

1999). By 28–32 h postfertilization (hpf), macrophages are found in circulation


and dispersed throughout the embryo.
Primitive erythroid cell generation begins in the murine YS blood islands at day
7.5 postcoitum (E7.5) (Fig. 1B) [reviewed in Palis et al. (2010)]. These blood islands
consist of nucleated erythroid cells that express embryonic globin genes surrounded
by endothelial cells. Although it was previously believed that mammalian primitive
erythroid cells uniquely remained nucleated (similar to the nucleated erythrocytes of
birds, fish, and amphibians), it is now accepted that mammalian primitive red blood
cells do, in fact, enucleate into reticulocytes and prenocytes (Fraser et al., 2007;
Kingsley et al., 2004; McGrath et al., 2008). The zebrafish has an equivalent
anatomical site to mammalian blood islands, known as the intermediate cell mass
(ICM), where two stripes of mesodermal cells expressing tal1, lmo2, and gata1a
converge to the midline of the zebrafish embryo and are surrounded by endothelial
cells that become the cardinal vein. (Al-Adhami and Kunz, 1977; Detrich et al.,
1995) (Fig. 1A and B). Although the ICM is intraembryonic in zebrafish, it has a
cellular architecture similar to the mammalian YS blood islands (Al-Adhami and
Kunz, 1977; Willett et al., 1999).
The development of transgenic zebrafish expressing fluorescent markers under
the control of early mesodermal, prehematopoietic promoters (see Table I) now
allow testing of primitive fate potentials by prospective isolation strategies and the
functional assays outlined in this chapter.

B. Definitive Hematopoiesis
Similar to primitive hematopoiesis, definitive hematopoiesis initiates through two
distinct precursor subsets. In the mouse, multilineage hematopoiesis is first evident
in the YS (Bertrand et al., 2005b; Palis et al., 1999, 2001; Yoder et al., 1997a, 1997b)
and placenta (Gekas et al., 2005; Ottersbach and Dzierzak, 2005) by E9.5.
Multilineage precursors in both tissues can be isolated and distinguished by the
expression of CD41, an integrin molecule that labels early hematopoietic progeni-
tors. CD41+ cells differentiate into both myeloid and erythroid lineages, but con-
spicuously lack lymphoid potential (Bertrand et al., 2005a; Yokota et al., 2006).
These studies suggest that the definitive hematopoietic program in the developing
mouse begins with committed EMPs. Recent studies in the zebrafish have demon-
strated evolutionary conservation of EMPs as the first definitive precursor formed,
and expanded upon findings in the mouse YS. EMPs can be isolated from the
zebrafish posterior blood island (PBI) between 26 and 36 hpf (Fig. 1C) by their
co-expression of fluorescent transgenes driven by the lmo2 and gata1a promoters
(Fig. 2A) (Bertrand et al., 2007). In vitro differentiation experiments (Fig. 2B and C)
and in vivo transplantation assays have shown these cells capable of only erythroid
and myeloid differentiation (Bertrand et al., 2007). Studies performed in mindbomb
mutant zebrafish lacking Notch signaling showed that EMP specification and dif-
ferentiation are not affected by loss of the Notch pathway (Bertrand et al., 2010b),
4. Cellular Dissection of Zebrafish Hematopoiesis 79

whereas HSCs are absent (Burns et al., 2005), further distinguishing these two
definitive progenitors.
The zebrafish allows the discrimination of EMPs from (HSCs, both of which have
similar cell-surface markers and differ only in their differentiation and self-renewal
potentials (Bertrand et al., 2005a,; 2005b). Unlike the murine system, these two
progenitors arise in separate anatomical locations (Bertrand et al., 2007), and are
therefore easily distinguishable. Importantly, fate-mapping studies in the zebrafish

Table I
List of relevant transgenic zebrafish lines currently available for hematopoietic studies, indicating the promoter:gene
expressed and the cell population(s) identified

Transgene Tissue Transgene Tissue

lmo2:GFP (Zhu et al., Prehematopoietic, vasculature kdrl:EGFP (Cross et al., 2003) Pre-hematopoietic,
2005) vasculature
lmo2:mCherry* Prehematopoietic, vasculature kdrl:DsRed (Jin et al., 2007b) Pre-hematopoietic,
vasculature
lmo2:DsRed (Lin et al., Prehematopoietic, vasculature fli1a:EGFP (Lawson and Pre-hematopoietic,
2005) Weinstein, 2002) vasculature
itga2b:GFP (Lin et al., EMPs, HSCs, thromobocytes fli1a:DsRed (Jin et al., 2007b) Pre-hematopoietic,
2005) vasculature
itga2b:mCherry* EMPs, HSCs, thromobocytes gata3:AmCyan (Bertrand et al., Kidney
2008)
itga2b:CFP* EMPs, HSCs, thromobocytes rag2:EGFP (Langenau et al., Immature B and T cells
2003)
ptprc:DsRed Pan-leukocyte lck:EGFP (Langenau et al., Mature T cells
(Bertrand et al., 2004)
2008)
ptprc:CFP* Pan-leukocyte il7r:mCherry* Lymphoid precursors, T cells
ptprc:AmCyan* Pan-leukocyte mhcII:GFP* (Wittamer et al., B cells, macrophages,
2011) dendritic cells
gata1a:GFP (Long et al., Red blood cells mhcII:AmCyan* B cells, macrophages,
1997) dendritic cells
gata1a:DsRed Red blood cells lyz:EGFP (Hall et al., 2007) Neutrophils
(Traver et al., 2003b)
cmyb:GFP HSCs, neural lyz:DsRed (Hall et al., 2007) Neutrophils
(Bertrand et al.,
2008)
mpx:EGFP Neutrophils runx1P1:GFP (Lam et al., 2008) EMPs
(Renshaw et al.,
2006)
gata2a:EGFP Eosinophils runx1P2:GFP (Lam et al., 2008) HSCs
(Traver et al., 2003b)
Ighm:EGFP* B cells ccr9a:cfp* Lymphoid precursors
Ighz:EGFP* B cells mpeg1:GAL4 (Ellett et al., 2010) Embryonic Macrophages

This list is not comprehensive; other transgenic animals are being constantly generated, but these are a few of the essential tools currently being
used in zebrafish hematopoiesis laboratories.
*
Unpublished transgenic animals generated in the Traver laboratory.
80 David L. Stachura and David Traver

[(Fig._2)TD$IG]

Fig. 2 Functional in vitro differentiation studies demonstrate that gata1+lmo2+ cells are committed erythromyeloid progeni-
tors (EMP). (A) Purified EMP at 30 hpf (lmo2+gata1a+, black gate) have the immature morphology of early hematopoietic
progenitors. As a comparison, purified primitive erythroblasts are shown (lmo2lowgata1a+, red gate). Magnification,
1000. (B) Short-term in vitro culture of lmo2+gata1a+ cells atop ZKS cells demonstrate erythroid (E), granulocytic
(G), and monocytic/macrophage (M) differentiation potentials. Cultured cells were stained with May-Gr€ unwald/Giemsa and
for myeloperoxidase (MPX) activity. lmo2lowgata1a+ cells only differentiated into erythroid cells (not shown).
4. Cellular Dissection of Zebrafish Hematopoiesis 81

have demonstrated EMPs to arise from posterior mesodermal derivatives that


express the lmo2 gene. This finding, in combination with lineage tracing studies
demonstrating EMPs to completely lack T lymphoid potential, indicate that
EMPs and HSCs are unique populations and independently derived during
development.
The final wave of hematopoiesis culminates with the formation of HSCs, which
self-renew and give rise to all definitive blood cell lineages, including lymphocytes.
It has been demonstrated that HSCs arise in an area of the mid-gestation mouse
bounded by the aorta, gonads, and mesonephros (AGM) at E10–10.5 (Fig. 1B)
(Cumano and Godin, 2007; Dzierzak, 2005). Many studies have also suggested that
transplantable HSCs are present in the YS on E9 (Lux et al., 2008; Weissman et al.,
1978; Yoder et al., 1997a, 1997b) and later in the placenta by E11 (Gekas et al., 2005;
Ottersbach and Dzierzak, 2005). While these results suggest that HSCs may arise in
distinctly different locations in the developing mouse embryo, it is now clear that
HSCs originate from arterial endothelium. Recent studies in the E10.5 mouse
(Boisset et al., 2010) and 36–52 hpf zebrafish embryo (Bertrand et al., 2010a;
Kissa and Herbomel, 2010) demonstrated directly the birth of HSCs from aortic
endothelium via confocal imaging. A commonality among all vertebrate embryos
thus seems to be the generation of HSCs from hemogenic endothelium lining the
aortic floor (Ciau-Uitz et al., 2000; de Bruijn et al., 2002; Jaffredo et al., 1998; North
et al., 2002; Oberlin et al., 2002). Similar studies will need to be performed to
determine whether or not additional embryonic sites can likewise generate HSCs
autonomously, including the YS and placenta. In all locations, however, it is clear
that HSCs are present only transiently; by E11, the fetal liver (FL) is populated by
circulating HSCs (Houssaint, 1981; Johnson and Moore, 1975) and becomes the
predominant site of blood production during midgestation, producing the first full
complement of definitive, adult-type effector cells. Shortly afterward, hematopoie-
sis is evident in the fetal spleen, and occurs in bone marrow throughout adulthood
(Keller et al., 1999).
The zebrafish possesses an anatomical site that closely resembles the mammalian
AGM (Fig. 1B and C). Between the dorsal aorta and cardinal vein between 28 and
48 hpf, cmyb+ and runx1+ blood cells appear in intimate contact with the dorsal aorta
(Burns et al., 2002; Kalev-Zylinska et al., 2002; Thompson et al., 1998). Lineage
tracing of CD41+ HSCs derived from this ventral aortic region show their ability to
colonize the thymus (Bertrand et al., 2007; Kissa et al., 2008) and pronephros
(Bertrand et al., 2008; Murayama et al., 2006), which are the sites of adult hema-
topoiesis (Jin et al., 2007a; Murayama et al., 2006). After 48 hpf, blood production
appears to shift to the caudal hematopoietic tissue (CHT) (Fig. 1C), and later to the
pronephros, which serves as the definitive hematopoietic organ for the remainder of
life.
The development of transgenic zebrafish expressing fluorescent markers under
the control of definitive hematopoietic promoters such as itga2b (also known as
cd41), cmyb, and runx1 (see Table I) now allows testing of fate potentials by
prospective isolation strategies and functional assays outlined later in this chapter.
82 David L. Stachura and David Traver

C. Adult Hematopoiesis
Previous genetic screens in zebrafish were successful in identifying mutants that
affected primitive erythropoiesis. The screening criteria used in these screens scored
visual defects in circulating blood cells during early embryogenesis; mutants defec-
tive in definitive hematopoiesis but displaying normal primitive blood cell devel-
opment were therefore likely missed. Current screens aimed at identifying mutants
with defects in the generation of definitive HSCs in the AGM should reveal new
genetic pathways required for multilineage hematopoiesis. Recent studies in zebra-
fish show that nearly all adult hematopoietic cells derive from HSCs born from aortic
endothelium (Bertrand et al., 2010a), consistent with findings in the murine system
(Chen et al., 2009; Zovein et al., 2008). Therefore, mutational screens designed to
identify defects in hemogenic endothelium may yield information about the full
repertoire of hematopoietic regulation, specification, maintenance, and differentia-
tion over the organism’s lifespan. Understanding the biology of mutants isolated
using these approaches, however, first requires the characterization of normal,
definitive hematopoiesis and the development of assays to study the biology of
zebrafish blood cells more precisely. To this end, we have established several tools
to characterize the definitive blood-forming system of adult zebrafish.
Blood production in adult zebrafish, like other teleosts, occurs in the kidney,
which supports both renal functions and multilineage hematopoiesis (Zapata,
1979). Similar to mammals, T lymphocytes develop in the thymus (Trede and
Zon, 1998; Willett et al., 1999) (Fig. 3A), which exists in two bilateral sites in
zebrafish (Hansen and Zapata, 1998; Willett et al., 1997). The teleostean kidney
is a sheath of tissue that runs along the spine (Fig. 3B, E); the anterior portion, or
head kidney, shows a higher ratio of blood cells to renal tubules than does the
posterior portion (Zapata, 1979), termed the trunk kidney (Fig. 3B, C). All mature
blood cell types are found in the kidney and morphologically resemble their mam-
malian counterparts (Fig. 3G, Fig. 4), with the exceptions that erythrocytes remain
nucleated and thrombocytes perform the clotting functions of platelets
(Jagadeeswaran et al., 1999). Histologically, the zebrafish spleen (Fig. 3D) has a
simpler structure than its mammalian counterpart in that germinal centers have not
been observed (Zapata and Amemiya, 2000). The absence of immature precursors in
the spleen, or any other adult tissue, suggests that the kidney is the predominant
hematopoietic site in adult zebrafish. The cellular compositions of whole kidney
marrow (WKM), spleen, and blood are shown in Fig. 3F-H. Morphological examples
of all kidney cell types are presented in Fig. 4.
Analysis of WKM by fluorescence activated cell sorting (FACS) showed that
several distinct populations could be resolved by light scatter characteristics
(Fig. 5A). Forward scatter (FSC) is directly proportional to cell size, and side
scatter (SSC) proportional to cellular granularity (Shapiro, 2002). Using com-
bined scatter profiles, the major blood lineages can be isolated to purity from
WKM following two rounds of cell sorting (Traver et al., 2003b). Mature erythroid
cells were found exclusively within two FSClow fractions (Populations R1 and R2,
4. Cellular Dissection of Zebrafish Hematopoiesis 83
[(Fig._3)TD$IG]

Fig. 3 Histological analyses of adult hematopoietic sites. (A) Sagittal section showing location of the thymus (T), which is
dorsal to the gills (G). (B) Midline sagittal section showing location of the kidney, which is divided into the head kidney (HK),
and trunk kidney (TK), and spleen (S). The head kidney shows a higher ratio of blood cells to renal tubules (black arrows), as
shown in a close up view of the HK in (C). (D) Close up view of the spleen, which is positioned between the liver (L) and the
intestine (I). (E) Light microscopic view of the kidney (K), over which passes the dorsal aorta (DA, white arrow). (F) Cytospin
preparation of splenic cells, showing erythrocytes (E), lymphocytes (L), and an eosinophil (Eo). (G) Cytospin preparation of
kidney cells showing cell types as noted above plus neutrophils (N) and erythroid precursors (O, orthochromic erythroblast). (H)
Peripheral blood smear showing occasional lymphocytes and thrombocytes (T) clusters amongst mature erythrocytes. (A–D)
Hematoxylin and eosin stains, (F–H) May-Gr€ unwald/Giemsa stains.
84 David L. Stachura and David Traver

[(Fig._4)TD$IG]

Fig. 4 Proposed model of zebrafish definitive hematopoietic differentiation. Isolated, cytospun, and stained blood cells from
the zebrafish kidney, thymus, and peripheral tissues and their proposed upstream progenitors. Proposed lineage relationships are
based on those demonstrated in clonogenic murine studies. Multipotent and lineage restricted progenitors likely reside in the
kidney marrow, but their existence has never been experimentally proven due to a paucity of in vitro assays.

Fig. 5A, D), lymphoid cells within a FSCint SSClow subset (Population R3, Fig. 5A,
E), immature precursors within a FSChigh SSCint subset (Population R4, Fig. 5A, F),
and myelomonocytic cells within only a FSChigh SSChigh population (Population R5,
Fig. 5A, G). Interestingly, two distinct populations of mature erythroid cells exist
(Fig. 5A, R1, R2 gates). Attempts at sorting either of these subsets reproducibly
resulted in approximately equal recovery of both (Fig. 5D). This likely resulted due
to the elliptical nature of zebrafish red blood cells, because sorting of all other
populations yielded cells that fell within the original sorting gates upon re-analysis.
Examination of splenic (Fig. 5B) and peripheral blood (Fig. 5C) suspensions showed
each to have distinct profiles from WKM, each being predominantly erythroid. It
should be noted that, due to differences in the fluidics and beam size, erythroid cells
are not discretely detectable on BD FACScan, FACS Caliber, or FACS Aria I and II
flow cytometers. However, FACS Vantage and LSR-II flow cytometers have a
different fluidics system and are well suited to these analyses. Sorting of each scatter
population from spleen and blood showed each to contain only erythrocytes, lym-
phocytes, or myelomonocytes in a manner identical to those in the kidney. Immature
precursors were not observed in either tissue. Percentages of cells within each scatter
population closely matched those obtained by morphological cell counts,
4. Cellular Dissection of Zebrafish Hematopoiesis 85
[(Fig._5)TD$IG]

Fig. 5 Each major blood lineage can be isolated by size and granularity using FACS. (A) Scatter profile for WKM. Mature
erythrocytes are found within R1 and R2 gates, lymphocytes within the R3 gate, immature precursors within the R4 gate, and
myeloid cells within the R5 gate. Mean percentages of each population within WKM are shown. Scatter profiling can also be
utilized for analyzing spleen (B) and peripheral blood (C). Purification of each WKM fraction by FACS (D–G). (D) Sorting of
populations R1 or R2 yields both upon re-analysis. This appears to be due to the elliptical shape of erythrocytes (right panel). (E)
Isolation of lymphoid cells. (F) Isolation of precursor fraction. (G) Isolation of myeloid cells. FACS profiles following one round
of sorting are shown in left panels, after two rounds in middle panels, and morphology of double-sorted cells shown in right
panels (E–G).

demonstrating that this flow cytometric assay is accurate in measuring the relative
percentages of each of the major blood lineages.
Many transgenic zebrafish lines have been created using proximal promoter
elements from genes that demonstrate lineage-affiliated expression patterns in the
mouse. These include gata1a:GFP (Long et al., 1997), gata2a:EGFP (Jessen et al.,
1998; Traver et al., 2003b), rag2:EGFP (Langenau et al., 2003), lck:EGFP
(Langenau et al., 2004), spi1:EGFP (Hsu et al., 2004; Ward et al., 2003), and
itga2b:EGFP (Lin et al., 2005; Traver et al., 2003b) stable transgenic lines. In the
adult kidney, we have demonstrated that each of these animals expresses green
86 David L. Stachura and David Traver

fluorescent protein (GFP) in the expected kidney scatter fractions (Traver et al.,
2003b). For example, all mature erythrocytes express GFP in gata1a:GFP trans-
genic animals, as do erythroid progenitors within the precursor population. High
expression levels of Gata2 are seen only within eosinophils, Rag2 and Lck only
within cells in the lymphoid fraction, and Spi1 in both myeloid cells and rare
lymphoid cells. The development of itga2b:EGFP transgenic animals has demon-
strated that rare thrombocytic cells are found within the kidney, with thrombocyte
precursors appearing in the precursor scatter fraction and mature thrombocytes in the
lymphoid fraction. Without fluorescent reporter genes, rare populations such as
thrombocytes cannot be resolved by light scatter characteristics alone. By combining
the simple technique of scatter separation with fluorescent transgenesis, specific
hematopoietic cell subpopulations can now be isolated to a relatively high degree of
purity for further analyses.
FACS profiling can also serve as a diagnostic tool in the examination of zebrafish
blood mutants. The majority of blood mutants identified to date are those displaying
defects in embryonic erythrocyte production (Traver et al., 2003a). Most of these
mutants are recessive and many are embryonic lethal when homozygous. Most have
not been examined for subtle defects as heterozygotes. Several heterozygous
mutants such as retsina, riesling, and merlot showed haploinsufficiency as evi-
denced by aberrant kidney erythropoiesis (Traver et al., 2003b). All mutants dis-
played anemia with concomitant increases in erythroid precursors. These findings
suggest that many of the gene functions required to make embryonic erythrocytes are
similarly required in their adult counterparts at full gene dosage for normal function.

III. Hematopoietic Cell Transplantation

In mammals, cellular transplantation has been used extensively to functionally


test putative hematopoietic stem and progenitor cell populations, precursor/progeny
relationships, and cell autonomy of mutant gene function. To address similar issues
in zebrafish, several different varieties of hematopoietic cell transplantation (HCT)
have been developed (Fig. 6).

A. Embryonic Donor Cells


Although scatter profiling has proven very useful in analyzing and isolating
specific blood lineages from the adult kidney, it cannot be used to enrich blood cells
from the developing embryo. To study the biology of the earliest blood-forming cells
in the embryo, we have made use of transgenic zebrafish expressing fluorescent
proteins. As discussed above, hematopoietic precursors appear to be specified from
mesodermal derivatives that express lmo2, kdrl (also known as flk1), and gata2a. The
proximal promoter elements from each of these genes have been shown to be
sufficient to recapitulate their endogenous expression patterns. Using germline
transgenic animals expressing GFP under the control of each of these promoters,
4. Cellular Dissection of Zebrafish Hematopoiesis 87
[(Fig._6)TD$IG]

Fig. 6 Methods of hematopoietic cell transplantation in the zebrafish. See the text for experimental
details.

blood cell precursors can be isolated by flow cytometry from embryonic and larval
animals for transplantation into wild-type recipients. For example, GFP+ cells in
lmo2:EGFP embryos can be visualized by FACS by 8–10 somites (Traver, 2004).
These cells can be sorted to purity and tested for functional potential in a variety of
transplantation (Fig. 6) or in vitro culture assays (see Fig. 2).
We have used two types of heterochronic transplantation strategies to address two
fundamental questions in developmental hematopoiesis. The first is whether cells
that express Lmo2 at 8–12 somites have hemangioblastic potential, i.e., can generate
both blood and vascular cells. We reasoned that purified cells should be placed into a
relatively naive environment to provide the most permissive conditions to assess
their full fate potentials. Therefore, we attempted transplantation into 1000 cell stage
blastulae recipients. Transplanted cells appear to survive this procedure well and
GFP+ cells could be found over several days later in developing embryos and larvae.
By isolating GFP+ cells from lmo2:EGFP animals also carrying a gata1a:DsRed
transgene, both donor-derived endothelial and erythroid cells can be independently
visualized in green and red, respectively. Using this approach we have shown that
Lmo2+ cells from 8 to 12 ss embryos can generate robust regions of donor
88 David L. Stachura and David Traver

endothelium and intermediate levels of circulating erythrocytes (D. Traver, C. E.


Burns, H. Zhu, and L. I. Zon, unpublished results). We are currently generating
additional transgenic lines that express DsRed or mCherry under ubiquitous pro-
moters to test the full fate potentials of Lmo2+ cells upon transplantation.
Additionally, although these studies demonstrate that Lmo2+ cells can generate at
least blood and endothelial cells at the population level, single-cell fate-mapping
studies need to be performed to assess whether clonogenic hemangioblasts can be
identified in vivo.
The second question addressed through transplantation is whether the earliest
identifiable primitive blood precursors can generate the definitive hematopoietic cells
that arise later in embryogenesis. It has been previously reported that the embryonic
lethal vlad tepes mutant dies from erythropoietic failure due to a defect in the gata1a
gene (Lyons et al., 2002). This lethality can be rescued by transplantation of WKM
from wild-type adults into mutant recipients at 48 hpf (Traver et al., 2003b). We
therefore tested whether cells isolated from 8 to 12ss lmo2:EGFP embryos could give
rise to definitive cell types and rescue embryonic lethality in vlad tepes recipients.
Following transplantation of GFP+ cells at 48 hpf, approximately half of the cells in
circulation were GFP+ and the other half were DsRed+ 1 day posttransplantation.
Three days later, analyses of the same animals showed that the vast majority of cells
in circulation were DsRed+, apparently due to the differentiation of Lmo2+ precursors
to the erythroid fates. Compared to untransplanted control animals which all died by
12 days postfertilization (dpf), some mutant recipients survived for 1–2 months
following transplantation. We observed no proliferation of donor cells at any time
point following transplantation, however, and survivors analyzed over 1 month post-
transplantation showed no remaining cells in circulation (D. Traver, C. E. Burns, H.
Zhu, and L. I. Zon, unpublished results). Therefore, these data indicate that mutant
survivors were only transiently rescued by short-lived, donor-derived erythrocytes.
Thus, within the context of this transplantation setting, it does not appear that Lmo2
+ hematopoietic precursors can seed definitive hematopoietic organs to give rise to
enduring repopulation of the host blood forming system.

1. Protocol for Isolating Hematopoietic Cells from Embryos


This simple physical dissociation procedure is effective in producing single cell
suspensions from early embryos (8–12 ss) as well as from embryos as late as 48 hpf.
1. Stage and collect embryos. We estimate that approximately 200 cells can be
isolated per 10–12 ss lmo2:EGFP embryo. It is recommended that as many
embryos are collected as possible since subsequent transplantation efficiency
depends largely upon cell concentration. At least 500–1000 embryos are
recommended.
2. Transfer embryos into 1.5 ml eppendorf centrifuge tubes. Add embryos until
they sediment to the 0.5 ml mark. Remove embryo medium since it is not
optimal for cellular viability.
4. Cellular Dissection of Zebrafish Hematopoiesis 89

3. Wash 2X with 0.9X Dulbecco’s PBS (Gibco; 500 ml 1X Dulbecco’s PBS +


55 ml ddH2O).
4. Remove 0.9X PBS and add 750 ml ice-cold staining medium (SM; 0.9X
Dulbecco’s PBS + 5% FCS). Keep cells on ice from this point onward.
5. Homogenize with blue plastic pestle and pipette a few times with a p1000 tip.
6. Strain resulting cellular slurry through a 40 mm nylon cell strainer (Falcon
2340) atop a 50 ml conical tube. Rinse with additional SM to flush cells through
the filter.
7. Gently mash remaining debris atop strainer with a plunger removed from a 28-
gauge syringe.
8. Rinse with more SM until the conical tube is filled to the 25 ml mark (this helps
to remove the yolk).
9. Centrifuge for 5 min @ 200g at 4  C. Remove supernatant until 1–2 ml remain.
10. Add 2–3 ml SM; resuspend by pipetting.
11. Strain again through 40 mm nylon mesh into a 5 ml Falcon 2054 tube. It is
important to filter the cell suspension at least twice before running the sample
by FACS. Embryonic cells are sticky and will clog the nozzle if clumps are not
properly removed beforehand.
12. Centrifuge again for 5 min @ 200g at 4  C. Repeat steps 10–12 if necessary.
13. Remove supernatant and resuspend with 1–2 ml SM depending upon number of
embryos used.
14. Propidium iodide (PI) may be added at this point to 1 mg/ml to exclude dead
cells and debris on the flow cytometer. When using, however, bring samples
having PI only and GFP only to set compensations properly. Otherwise, the
signal from PI may bleed into the GFP channel resulting in false positives.
Alternatively, add 1:1000 Sytox Red (excited by 633 nm laser), or Sytox Blue
(excited by the 405 nm laser) for dead cell discrimination, as they have no
spectral overlap with GFP.
Embryonic cells are now ready for analysis or sorting by flow cytometry. It is often
difficult to visualize GFP+ cells when the expression is low or the target population is
rare, so one should always prepare age-matched GFP-negative embryos in parallel
with transgenic embryos. It is then apparent where the sorting gates should be drawn
to sort bona fide GFP+ cells. If highly purified cells are desired, one must perform
two successive rounds of sorting. In general, sorting GFP+ cells once yields popula-
tions of approximately 50–70% purity. Two rounds of cell sorting generally yields
>90% purity as observed with 10ss lmo2:EGFP cells (Traver, 2004). Cells should be
kept ice-cold during the sorting procedure.

2. Transplanting Purified Cells into Embryonic Recipients


After sorting, centrifuge cells for 5 min at 200g and 4  C. Carefully remove all
supernatant. Resuspend cell pellet in 5–10 ml of ice-cold SM containing 3U heparin
and 1U DnaseI to prevent coagulation and lessen aggregation. Preventing the cells
90 David L. Stachura and David Traver

from aggregating or adhering to the glass capillary needle used for transplantation is
critical. Mix the cells by gently pipetting with a 10 ml pipette tip. Keep on ice. For
transplantation, we use the same needle-pulling parameters used to make needles for
nucleic acid injections, the only difference being the use of filament-free capillaries
to maintain cell viability. We also use the standard air-powered injection stations
used for nucleic acid injections.

3. Transplanting Cells into Blastula Recipients


1. Stage embryonic recipients to reach the 500–1000 cell stage at the time of
transplantation.
2. Prepare plates for transplantation by pouring a thin layer of 2% agarose made in
E3 embryo medium into a 6 cm Petri dish. Drop transplantation mold [similar to
the embryo injection mold described in Chapter 5 of The Zebrafish Book
(Westerfield, 2000) but having individual depressions rather than troughs] atop
molten agarose and let solidify.
3. Dechorionate blastulae in 1–2% agarose-coated Petri dishes by light pronase
treatment or manually with watchmaker’s forceps.
4. Transfer individual blastulae into individual wells of transplantation plate that has
been immersed in 1X HBSS (Gibco). Position the animal pole upward.
5. Using glass, filament-free, fine-pulled capillary needles (1.0 mm OD) backload
3–6 ml of cell suspension after breaking needle on a bevel to an opening of
20 mm. Load into needle holder and force cells to injection end by positive
pressure using a pressurized air injection station.
6. Gently insert needle into the center of the embryo and expel cells using either
very gentle pressure bursts or slight positive pressure. Transplanting cells near
the marginal zone of the blastula leads to higher blood cell yields since embryonic
fate maps show blood cells to derive from this region in later gastrula stage
embryos.
7. Carefully transfer embryos to agarose-coated Petri dishes using glass transfer
pipettes.
8. Place into E3 embryo medium and incubate at 28.5  C. Many embryos will not
survive the transplantation procedure, so clean periodically to prevent microbial
outgrowth.
9. Monitor by fluorescence microscopy for donor cell types.

4. Transplanting Cells into 48 hpf Embryos


1. All procedures are performed as above except that dechorionated 48 hpf embryos
are staged and used as transplant recipients.
2. Fill transplantation plate with 1X HBSS containing 1X penicillin/streptomycin
and 1X buffered tricaine, pH 7.0. Do not use E3, as it is suboptimal for cellular
viability. Anesthetize recipients in tricaine and then array individual embryos into
4. Cellular Dissection of Zebrafish Hematopoiesis 91

individual wells of transplantation plate. Position head at bottom of well, yolk


side up.
3. Load cells as above. Insert injection needle into the sinus venosus/duct of Cuvier
and gently expel cells by positive pressure or gentle pressure bursts. Take care not
to rupture the YS membrane. A very limited volume can be injected into each
recipient. It is thus important to use very concentrated cell suspensions in order to
reconstitute the host blood-forming system. If using WKM as donor cells, con-
centrations of 5  105 cells/ml can be achieved if care is taken to filter and
anticoagulate the sample.
4. Allow animals to recover at 28.5  C in E3. Keep clean and visualize daily by
microscopy for the presence of donor-derived cells.

B. Adult Donor Cells


Whereas the first HSCs transdifferentiate from embryonic aortic endothelium,
multilineage hematopoiesis is not fully apparent until the kidney becomes the site of
blood cell production. The kidney appears to be the only site of adult hematopoiesis,
and we have previously demonstrated that it contains HSCs capable of the long-term
repopulation of embryonic (Traver et al., 2003b) and adult (Langenau et al., 2004)
recipients. For HSC-enrichment strategies, both high-dose transplants and limiting
dilution assays are required to gauge the purity of input cell populations. In embry-
onic recipients, we estimate that the maximum number of cells that can be trans-
planted is approximately 5  103, and the precise quantitation of transplanted cell
numbers is difficult. To circumvent both issues, we have developed HCT into adult
recipients.
For transplantation into adult recipients, myeloablation is necessary for successful
engraftment of donor cells. We have found g-irradiation to be the most consistent
way to deplete zebrafish hematopoietic cells. The minimum lethal dose (MLD) of
40 Gy specifically ablates cells of the blood-forming system and can be rescued by
transplantation of one kidney equivalent (106 WKM cells). Thirty-day survival of
transplanted recipients is approximately 75% (Traver et al., 2004). An irradiation
dose of 20–25 Gy is sublethal, and the vast majority of animals survive this treatment
despite having nearly total depletion of all leukocyte subsets 1 week following
irradiation (Traver et al., 2004). We have shown that this dose is necessary and
sufficient for transfer of a lethal T cell leukemia (Traver et al., 2004), and for long-
term (>6 month) engraftment of thymus repopulating cells (Langenau et al., 2004).
We do not yet know the average relative chimerism of donor to host cells when
transplantation is performed following 20–25 Gy. That this dose is sufficient for
robust engraftment, for long-term repopulation, and yields extremely high survival
suggests that 20–25 Gy may be the optimal dose for myeloablative conditioning
prior to transplantation. Improvement in short-term engraftment and long-term
survival of transplant recipients will also likely require matching of MHC loci
between donor and host genotypes.
92 David L. Stachura and David Traver

1. Protocols for Isolating Hematopoietic Cells from Adult Zebrafish


Anesthetize adult animals in 0.02% tricaine in fish water.
For blood collection, dry animal briefly on tissue then place on a flat surface with
head to the left, dorsal side up. Coat a 10 ml pipette tip with heparin (3 U/ml) then
insert tip just behind the pectoral fin and puncture the skin. Direct the tip into the
heart cavity, puncture the heart, and aspirate up to 10 ml blood by gentle suction.
Immediately perform blood smears or place into 0.9X PBS containing 5% FCS and
1 U/ml heparin. Mix immediately to prevent clotting. Blood from several animals
may be pooled in this manner for later use by flow cytometry. Red cells may be
removed using a red blood cell hypotonic lysis solution (Sigma; 8.3 g/l ammonium
chloride in 0.01 M Tris–HCl, pH 7.5) on ice for 5 min. Add 10 volumes of ice-cold
SM then centrifuge at 200g for 5 min at 4  C. Resuspended blood leukocytes can
then be analyzed by flow cytometry or cytocentrifuge preparations.
For collection of other hematopoietic tissues, place fish on ice for several minutes
following tricaine. Make a ventral, midline incision using fine scissors under a
dissection microscope.
For spleen collection, locate spleen just dorsal to the major intestinal loops and
tease out with watchmaker’s forceps. Place into ice cold SM. Dissect any nonsplenic
tissue away and place on a 40 mm nylon cell strainer (Falcon 2340) atop a 50 ml
conical tube. Gently mash the spleen using a plunger removed from a 28-gauge
insulin syringe and rinse with SM to flush cells through the filter. Up to 10 spleens
can be processed through each filter. Centrifuge at 200g for 5 min at 4  C. Filter
again through 40 mm nylon mesh if using for FACS.
For kidney collection, remove all internal organs using forceps and a dissection
microscope. Take care during dissection because ruptured intestines or gonads will
contaminate the kidney preparation. Using watchmaker’s forceps, tease the entire
kidney away from the body wall starting at the head kidney and working toward the
rear. Place into ice-cold SM. Aspirate vigorously with a 1 ml pipetteman to separate
hematopoietic cells (WKM) from renal cells. Filter through 40 mm nylon mesh, wash,
centrifuge, and repeat. Perform last filtration step into a Falcon 2054 tube if using for
FACS. It is important to filter the WKM cell suspension at least twice before running
the sample. PI may be added at this point to 1 mg/ml to exclude dead cells and debris
on the flow cytometer. When using, however, compare to samples having PI only and
GFP only (if using) to set compensations properly. Otherwise, the signal from PI may
bleed into the GFP channel resulting in false positives. Alternatively, add 1:1000 Sytox
Red (excited by the 633 nm laser) or Sytox Blue (excited by the 405 nm laser) for dead
cell discrimination, as they have no spectral overlap with GFP.

2. Transplanting Whole Kidney Marrow


After filtering and washing the WKM suspension three times, centrifuge cells for
5 min at 200g and 4  C. Carefully remove all supernatant. Resuspend cell pellet in 5–
10 ml of ice-cold SM containing 3U heparin and 1U DnaseI to prevent coagulation
4. Cellular Dissection of Zebrafish Hematopoiesis 93

and lessen aggregation. Preventing the cells from aggregating or adhering to the
glass capillary needle used for transplantation is critical. Mix the cells by gently
pipetting with a 10 ml pipette tip. Keep on ice. For blastulae and embryo transplan-
tation, perform following previous protocols. Between 5  102 and 5  103 cells can
be injected into each 48 hpf embryo if the final cell concentration is approximately
5  105 cells/ml.

3. Transplanting Cells into Irradiated Adult Recipients


For irradiation of adult zebrafish, we have used a 137Cesium source irradiator
typically used for the irradiation of cultured cells (Gammacell 1000). We lightly
anaesthetize five animals at a time and then irradiate in sealed Petri dishes filled with
fish water (without tricaine). We performed careful calibration of the irradiator using
calibration microchips to obtain the dose rate at the height within the irradiation
chamber nearest to the 137Cesium point source. We found the dose rate to be uniform
among calibration chips placed within euthanized animals in Petri dishes under
water, under water alone, or in air alone, verifying that the tissue dosage via total
body irradiation (TBI) was accurate.
Transplantation into circulation is most efficiently performed by injecting cells
directly into the heart. We perform intracardiac transplantation using pulled fila-
ment-free capillary needles as above, but we break the needles at a larger bore size of
approximately 50 mm. The needle assembly can be handheld and used with a
standard gas-powered microinjection station. We have also had limited success
transplanting cells intraperitoneally using a 10 ml Hamilton syringe. Engraftment
efficiency for WKM is only marginal using this method, but transplantation of T cell
leukemia or solid tumor suspensions is highly efficient following irradiation at
20 Gy (Traver et al., 2004).

4. Irradiation
1. Briefly anaesthetize adult zebrafish in 0.02% tricaine in fish water.
2. Place five fish at a time into 60 mm  15 mm Petri dishes (Falcon) containing
fish water. Wrap dish with Parafilm and irradiate for length of time necessary to
achieve desired dose.
3. Return irradiated animals to clean tanks containing fish water. We have success-
fully transplanted irradiated animals from 12 to 72 h following irradiation. Using
a 20 Gy dose, the nadir of host hematopoietic cell numbers occurs at approxi-
mately 72 h postirradiation.

5. Transplantation
1. Prepare cells to be transplanted as above, taking care to remove particulates/
contaminants by multiple filtration and washes. When using WKM as donor cells,
we typically make final cell suspensions at 2  105 cells/ml. Keep cells on ice.
94 David L. Stachura and David Traver

2. Anaesthetize an irradiated animal in 0.02% tricaine in fish water.


3. Transfer ventral side up into a well cut into a sponge wetted with fish water. Under
a dissection microscope, remove scales covering the pericardial region with fine
forceps.
4. Fill injection needle with 20 ml of cell suspension. Force cells to end of needle
with positive pressure and adjust pressure balance to be neutral. Hold needle
assembly in one hand while placing gentle pressure on the abdomen of the
recipient with the index finger of the other hand. This will position the heart
adjacent to the skin and allow visualization of the heartbeat. Insert needle through
the skin and into the heart. If the needle is positioned within the heart, and the
pressure balance is neutral, blood from the heart will enter the needle and the
meniscus will rise and fall with the heartbeat. Inject approximately 5–10 ml by
gentle pressure bursts.
5. Return recipient to fresh fish water. Repeat for each additional recipient. Do not
feed until the next day to lessen chance of infection.

IV. Enrichment of Hematopoietic Stem Cells


The development of many different transplantation techniques now permits the
testing of cell autonomy of mutant gene function, oncogenic transformation, and
stem cell enrichment strategies in the zebrafish. For HSC enrichment strategies,
fractionation techniques can be used to divide WKM into distinct subsets for func-
tional testing via transplantation. The most successful means of HSC enrichment in
the mouse has resulted from the subfractionation of whole bone marrow cells with
monoclonal antibodies (mAbs) and flow cytometry (Spangrude et al., 1988). We
have attempted to generate mAbs against zebrafish leukocytes by repeated mouse
immunizations using both live WKM and purified membrane fractions followed by
standard fusion techniques. Many resulting hybridoma supernatants showed affinity
to zebrafish WKM cells in FACS analyses (Fig. 7). All antibodies showed one of two
patterns, however. The first showed binding to all WKM cells at similar levels. The
second showed binding to all kidney leukocyte subsets but not to kidney erythro-
cytes, similar to the pattern shown in the left panel of Fig. 7A. We found no mAbs
that specifically bound only to myeloid cells, lymphoid cells, etc when analyzing
positive cells by their scatter profiles. We reasoned that these nonspecific binding
affinities might be due to different oligosaccharide groups present on zebrafish
blood cells. If the glycosylation of zebrafish membrane proteins were different from
the mouse, then the murine immune system would likely mount an immune response
against these epitopes. To test this hypothesis, we removed both O-linked and N-
linked sugars from WKM using a deglycosylation kit (Prozyme), and then incubated
these cells with previously positive mAbs. All mAbs tested in this way showed a
time-dependent decrease in binding, with nearly all binding disappearing following
2 h of deglycosylation (Fig. 7A). It thus appears that standard immunization
approaches using zebrafish WKM cells elicit a strong immune response against
4. Cellular Dissection of Zebrafish Hematopoiesis 95
[(Fig._7)TD$IG]

Fig. 7 Potential methods of stem cell enrichment. (A) Mouse monoclonal antibodies generated against zebrafish WKM cells
react against oligosaccharide epitopes. De-glycosylation enzymes result in time-dependent loss of antibody binding (bold
histograms) compared to no enzyme control (left panel and grey histograms). (B) Differential binding of lectins to WKM scatter
fractions. Peanut agglutinin splits both the lymphoid and precursor fraction into positive and negative populations (left panels).
Potato lectin shows a minor positive fraction only within the lymphoid fraction (right panels). (C) Hoechst 33342 dye reveals a
side population (SP) within WKM. 0.4% of WKM cells appear within the verapamil-sensitive SP gate (left panel). Only the
lymphoid fraction, where kidney HSCs reside, contains appreciable numbers of SP cells (right panels).
96 David L. Stachura and David Traver

oligosaccharide epitopes. This response is likely to be extremely robust, because we


did not recover any mAbs that reacted with specific blood cell lineages. Similar
approaches by other investigators using blood cells from frogs or other teleost
species have yielded similar results (L. du Pasquier, M. Flajnik, personal commu-
nications). In an attempt to circumvent the glycoprotein issue, new series of immu-
nizations using deglycosylated kidney cell membrane preparations may be effective.
Previous studies have shown that specific lectins can be used to enrich hemato-
poietic stem and progenitor cell subsets in the mouse (Huang and Auerbach, 1993;
Lu et al., 1996; Visser et al., 1984). In preliminary studies, we have shown that FITC-
labeled lectins such as peanut agglutinin (PNA) and potato lectin (PTL) differen-
tially bind to zebrafish kidney subsets. As shown in Fig. 7B, PNA binds to a subset of
cells both within the lymphoid and precursor kidney scatter fractions. Staining with
PTL also shows that a minor fraction of lymphoid cells binds PTL, whereas the
precursor (and other) scatter fractions are largely negative (Fig. 7B). We are cur-
rently testing both positive and negative fractions in transplantation assays to deter-
mine whether these different binding affinities can be used to enrich HSCs.
We have previously demonstrated that long-term HSCs reside in the adult kidney
(Traver et al., 2003b). We therefore isolated each of the kidney scatter fractions from
gata1a:EGFP transgenic animals and transplanted cells from each into 48 hpf
recipients to determine which subset contains HSC activity. The only population
that could generate GFP+ cells for over 3 weeks in wild type recipients was the
lymphoid fraction. This finding is in accord with mouse and human studies that have
shown purified HSCs to have the size and morphological characteristics of inactive
lymphocytes (Morrison et al., 1995).
Another method that has been extremely useful in isolating stem cells from whole
bone marrow is differential dye efflux. Dyes such as rhodamine 123 (Mulder and
Visser, 1987; Visser and de Vries, 1988) or Hoechst 33342 (Goodell et al., 1996)
allow the visualization and purification of a ‘‘side population’’ (SP) that is highly
enriched for HSCs. This technique appears to take advantage of the relatively high
activity of multidrug resistance transporter proteins in HSCs that actively pump each
dye out of the cell in a verapamil-sensitive manner (Goodell et al., 1996). Other cell
types lack this activity and become positively stained, allowing isolation of the
negative SP fraction by FACS. Our preliminary studies of SP cells in the zebrafish
kidney demonstrated a typical SP profile when stained with 2.5 mg/ml of Hoechst
33342 for 2 h at 28  C (Fig. 7C). This population disappears when verapamil is
added to the incubation. Interestingly, the vast majority of SP cells appear within the
lymphoid scatter fraction (Fig. 7C). Further examination of whether this population
is enriched for HSC activity in transplantation assays is warranted.
Finally, there are many other methods to enrich hematopoietic stem and progenitor
cells from WKM including sublethal irradiation, cytoreductive drug treatment, and
use of transgenic lines expressing fluorescent reporter genes (see Table I). We have
shown following 20 Gy doses of g-irradiation that nearly all hematopoietic lineages
are depleted within 1 week (Traver et al., 2004). Examination of kidney cytocen-
trifuge preparations at this time shows that the vast majority of cells are immature
4. Cellular Dissection of Zebrafish Hematopoiesis 97

precursors. That this dose does not lead to death of the animals demonstrates that
HSCs are spared and are likely highly enriched 5–8 days following exposure. We
have also shown that cytoreductive drugs such as cytoxan and 5-fluorouracil have
similar effects on kidney cell depletion, although the effects were more variable than
those achieved with sublethal irradiation (A. Winzeler, D. Traver, and L. I. Zon
unpublished). Because HSCs are contained within the kidney lymphoid fraction,
they can be further enriched by HSC-specific or lymphocyte-specific transgenic
markers. Possible examples of transgenic promoters are lmo2, gata2a, or cmyb to
mark HSCs and ccr9a, il7r, rag2, lck, or B-cell receptor genes to exclude lympho-
cytes from this subset (see Table I).

V. In vitro Culture and Differentiation of Hematopoietic


Progenitors

Hematopoiesis is one of the best-studied models of developmental differentiation


because of the multitude of experimental methods developed over the past 60 years to
assess the proliferation, differentiation, and maintenance of its cellular constituents.
Stem and progenitor cell transplantation into lethally irradiated animal recipients (Ford
et al., 1956; McCulloch and Till, 1960) were the first in vivo assays to be developed,
followed shortly thereafter by the clonal growth of bone marrow progenitors in vitro
(Bradley and Metcalf, 1966). Although these techniques have been substantially
refined over the past decades, they still remain the foundation for analyzing the
hierarchical organization of vertebrate hematopoietic stem and progenitor cells.
In vitro cultures to assess hematopoietic stem and progenitor cell biology gener-
ally fall into two categories: growth of progenitor cells on a supportive stromal cell
layer, and clonal growth of cells in a semisolid medium with the addition of sup-
plemental cytokines or growth factors. Most stromal culture assays largely derive
from the modification and refinement of Dexter cultures (Dexter et al., 1977a,
1977b), whereby stromal cells from hematopoietic organs support the differentiation
of HSCs and their downstream progenitors. These early studies were instrumental for
the development of cobblestone-area-forming-cell (CAFC) (Ploemacher et al.,
1991) and long-term culture initiating cell (LTC-IC) assays, which have been uti-
lized to examine murine (Lemieux et al., 1995) and human (Sutherland et al., 1991)
multilineage hematopoietic differentiation. The development of stromal cells from
the calvaria of macrophage colony stimulating factor (M-CSF)-deficient mice
(Nakano et al., 1994, 1996) were instrumental for examining hematopoietic differ-
entiation of embryonic stem (ES) cells down the hematopoietic pathway, and for
differentiation of hematopoietic precursors into multiple mature blood cell types.
With refinement, these OP9 cells have proven to be an efficient tool to study T-cell
lineage commitment and development (Schmitt et al., 2004; Schmitt and Zuniga-
Pflucker, 2002), once an extremely difficult process to study.
To assess the progenitor capacity of normal and mutant zebrafish hematopoietic
cells functionally, we created primary zebrafish kidney stromal (ZKS) cells derived
98 David L. Stachura and David Traver

from the main site of hematopoiesis in the adult fish. Culture of hematopoietic
progenitor cells on these stromal cells resulted in their continued maintenance
(Stachura et al., 2009) and differentiation (Bertrand et al., 2007, 2010b; Stachura
et al., 2009). It also allowed investigation and rescue of a genetic block in erythroid
maturation, confirming the utility of these assays (Stachura et al., 2009). Finally, the
ZKS culture system has been utilized to investigate the molecular events underlying
the progression of T-lymphoblastic lymphoma (T-LBL) to acute T-lymphoblastic
leukemia (T-ALL) (Feng et al., 2010).

A. Stromal Cell Culture AssaysFS


To create a suitable in vitro environment for the culture of zebrafish hematopoietic
cells, we isolated the stromal fraction of the zebrafish kidney, the main site of
hematopoiesis in the adult fish (Zapata, 1979). The benefit of utilizing hematopoi-
etic stromal layers is two-fold. First, performing culture assays in the zebrafish has
been hampered by a paucity of defined and purified hematopoietic cytokines. Most
zebrafish cytokines have poor sequence homology to their mammalian counterparts,
and as a consequence, have not been well described, characterized, or rigorously
tested. Secondly, some hematopoietic cell types, especially T cells, require physical
cell-cell interaction for their differentiation.

1. Generation of ZKS Cells


To create ZKS cells, kidney was isolated from AB* wild-type fish as described
above (also see Stachura et al., 2009). The kidney tissue was sterilized by washing
for 5 min in 0.000525% sodium hypochlorite (Fisher Scientific), then rinsed in
sterile 0.9X Dulbecco’s PBS. Tissue was then mechanically dissociated by trituration
and filtered through a 40 mm filter (BD Biosciences). Flow-through cells (WKM)
were discarded, and the remaining kidney tissue was cultured in vacuum plasma-
treated vented flasks (Corning Incorporated Life Sciences) at 32  C and 5% CO2.

2. Maintenance and Culture of ZKS Cells


ZKS cells are maintained in the following tissue culture medium:

500 ml L-15
350 ml DMEM (high glucose)
150 ml Ham’s F-12
150 mg Sodium bicarbonate
15 ml HEPES (1 M stock)
20 ml Penicillin/streptomycin (5000 U/ml penicillin, 5000 mg/ml streptomycin stock)
10 ml L-glutamine (200 mM stock)
100 ml Fetal bovine serum (FBS)
2 ml Gentamicin sulfate (50 mg/ml stock)
4. Cellular Dissection of Zebrafish Hematopoiesis 99

Medium is made by first adding sodium bicarbonate to the mixture of L-15,


DMEM, and Ham’s F-12. Warm the medium to 37  C, and allow the sodium
bicarbonate to dissolve. Then, add other medium components and filter-sterilize
with 0.22 mm vacuum apparatus.
All medium components are available from Mediatech. We utilize FBS from the
American Type Culture Collection, but one can use FBS from other sources. It is
important to note, however, that different manufacturing lots of FBS investigated in
the laboratory differ wildly in their support of hematopoietic progenitor differenti-
ation and proliferation. Once a manufacturing lot is tested and shown to be support-
ive, we recommend buying a large quantity to minimize experimental variation.
ZKS cells are maintained at 32  C and 5% CO2 in a humidified incubator. Cells
are grown in vacuum plasma treated 75 cm2 vented flasks (T-75; Corning
Incorporated Life Sciences) in 10 ml of medium until 60–80% confluent before
passaging. Medium is then removed, and 2 ml trypsin–EDTA (0.25%; Invitrogen) is
added to cover the stromal cells. Allow cells to incubate for 5 min at 32  C. Add 8 ml
medium to cells to stop trypsinization, pipetting up and down to achieve a single cell
suspension. Spin cells for 5 min at 300g, aspirate supernatant and resuspend pellet
gently in 10 ml of medium. Take 1 ml of the cell solution, add 9 ml of medium, and
move to a new flask. Cells should not be split more than 1:10 to passage, as they are
somewhat density-dependent.
ZKS cells may be frozen and thawed at a later time. Even though we have never
experienced senescence or a decrease in hematopoietic differentiation capacity of
ZKS cells in culture, it is useful to perform critical experiments with similar passages
of cells to avoid experimental variation. To freeze ZKS cells, first trypsinize a T-75
flask. Spin cells for 5 min at 300g, aspirate supernatant, and resuspend pellet gently
in 2 ml of medium. Prepare freezing medium (500 ml of tissue culture-certified
DMSO and 1.5 ml of FBS) and aliquot 500 ml into four cryopreservation tubes
keeping everything on ice. Add 500 ml of cells to each tube, invert to gently mix,
and place tubes into isopropanol-jacketed freezing chamber. Place freezing chamber
at –80  C for 24 h. Remove tubes from freezing chamber and place into liquid
nitrogen for long-term storage.
To thaw ZKS cells at a later date, remove tube from nitrogen, and quickly warm in
37  C water bath. Wear eye protection; if nitrogen seeped into the freezing tubes, the
rapid warming may cause the tube to violently rupture. Remove liquid from tube, add
slowly to 10 ml of medium in a 15 ml conical tube, and spin at 300g for 5 min.
Carefully aspirate all of the medium to remove all traces of DMSO. Resuspend cells
in 10 ml of fresh medium and place into T-75 flask. Early the next morning change
the medium and determine whether the cells are ready to be passaged or require
another day to recover from thawing.
As with all tissue culture, strict attention to sterility and cleanliness should be
adhered to at all times. All procedures should be performed in a tissue culture
laminar flow hood, and it is recommended that vented, filtered flasks be utilized
to prevent airborne contamination during culture in incubators.
100 David L. Stachura and David Traver

3. Protocols for in vitro Proliferation and Differentiation Assays


Purify prospective progenitors by FACS as described above. Plate cells on con-
fluent ZKS at a density of 1  104 cells/well in a 12-well tissue culture plate, using
2 ml complete medium per well. Lower density of progenitors is not recommended;
if using fewer cells, reduce the size of the tissue culture well and volume of medium.
If testing or investigating the effects of growth factors or small molecules, add them
to the medium, being sure to have a vehicle only control as well as different con-
centrations of your experimental factor. 24-well tissue culture plates are extremely
useful in this regard, as one can easily plate out a multitude of experimental condi-
tions on one plate.
A. Morphological assessment of hematopoietic cells after in vitro culture
a. Gently aspirate hematopoietic cells from the ZKS cultures, taking care not to
disturb the stromal underlayer.
b. Cytocentrifuge up to 200 ml of the hematopoietic cells at 250g for 5 min onto
glass slides using a Shandon Cytospin 4 (Thermo Fischer Scientific). It is
possible to concentrate the cells before cytocentrifugation at 300g for 10 min.
Cytocentrifugation of over 200 ml of cell suspensions is not recommended.
c. Perform May-Gr€ unwald/Giemsa staining by allowing slides to air-dry briefly.
Then, submerge slide in May-Gr€ unwald staining solution (Sigma Aldrich) for
10 min. Transfer slide to 1:5 dilution of Giemsa stain (Sigma Aldrich) in
dH2O for an additional 20 min. Rinse slide in dH2O, and allow to air dry.
Coverslip slide with cytoseal XYL mounting medium (Richard-Allan
Scientific) and Corning no.1 18 mm square cover glass (Corning). Allow
slides to completely dry before visualization on upright microscope, espe-
cially if using an oil-immersion lens.
B. Proliferation assessment of hematopoietic cells after in vitro culture
a. Gently aspirate hematopoietic cells as above.
b. Count cells with use of a bright line hemacytometer (Hausser Scientific)
using trypan blue dye (Invitrogen) exclusion to assess viability.
C. RT-PCR analysis of hematopoietic cells after in vitro culture
a. Gently aspirate hematopoietic cells as above.
b. Isolate RNA from hematopoietic cells using either Trizol (Invitrogen) or
RNAeasy kit (Qiagen).
c. Generate cDNA with oligo dT primers and superscript RT-PCR kit
(Invitrogen).
d. Perform PCR with the desired zebrafish DNA primers.
D. Cell labeling and cell division determination of hematopoietic cells after in vitro
culture
a. Prior to plating cells on ZKS monolayer, wash cells twice with 0.1% bovine
serum albumin (BSA) to remove FBS from the medium.
4. Cellular Dissection of Zebrafish Hematopoiesis 101

b. Resuspend cells in 0.1% BSA and 2 ml/ml of 5 mM carboxyfluorescein succi-


nimidyl ester (CFSE; Invitrogen) at room temperature for 10 min, in the dark.
c. Wash cells with complete medium supplanted with an additional 10% FBS
twice.
d. Save 1/10 of the culture and perform FACS (Day 0 time point). Culture
remaining cells in complete medium as described above.
e. For analysis, remove hematopoietic cells from culture at desired time points as
described above and FACS. CFSE is read in the FL-1 channel (on FACS caliber)
or with most GFP filters (FACS Aria I and II, LSR-II), and will decrease in
fluorescence intensity as cells divide. Compare divisions to Day 0 time point
with FloJo software (TreeStar, Ashland, OR, USA). We recommend using the
BD LSR-II flow cytometer, as the different scatter profile of mature cells is
easily distinguished and directly comparable to profiles shown in Fig. 5.

B. Clonal Methylcellulose-based Assays


Although stromal in vitro culture methods have been instrumental for the inves-
tigation of hematopoiesis, culturing bulk populations of progenitor cells on stroma
cannot distinguish between homogeneous multipotent progenitor populations or
heterogeneous lineage-restricted populations without performing limiting dilution
assays. The development of clonal in vitro cultures by Metcalf and colleagues
allowed not only the growth of murine bone marrow progenitors (Bradley and
Metcalf, 1966), but also the study and quantitation of progenitor numbers during
hematological disease (Bradley et al., 1967) and exposure to irradiation
(Robinson et al., 1967). These assays were utilized to investigate the ontogeny of
the developing murine hematopoietic system (Moore and Metcalf, 1970), and
refined to study human hematopoietic progenitors dysregulated during leukemo-
genesis (Moore et al., 1973a, 1973b). Importantly, the utilization of clonal assays
was instrumental for the identification and validation of CSFs, secreted proteins that
stimulate the differentiation of specific hematopoietic lineages. The ability to iso-
late, recombinantly produce, and test these factors was a huge advance in hemato-
logical research, allowing the sensitive analysis of progenitor differentiation, pro-
liferation, and restriction in the murine and human blood system.
This capability to grow progenitors in vitro to test their differentiation capacity in
an unbiased manner has greatly advanced the current understanding of hematopoi-
etic lineage restriction. The isolation of putative lineage-restricted daughter cells by
FACS coupled with in vitro clonal analysis was pivotal in identifying multipotent
(Akashi et al., 2000; Kondo et al., 1997), oligopotent (Akashi et al., 2000), and
monopotent progenitor (Mori et al., 2008; Nakorn et al., 2003) intermediates down-
stream of HSCs in the murine system.
While zebrafish likely possess multipotent, oligopotent, and monopotent progen-
itor cells, their existence has never been proven and remains speculative (see Fig. 4).
To investigate whether these cells exist in zebrafish, we developed assays to
102 David L. Stachura and David Traver

[(Fig._8)TD$IG]

Fig. 8 Recombinantly generated and purified Gcsf and Epo encourage myeloid and erythroid differentiation, respectively,
from zebrafish hematopoietic progenitors in clonal methylcellulose assays. (A) Experimental schematic for isolation and culture
of mpx:GFP, gata1a:DsRed cells from the precursor (blue) fraction of adult WKM. (B) Brightfield images (top row), mpx:
GFP fluorescence (second row), gata1a:DsRed fluorescence (third row), and merged images (bottom row) of colonies grown in
various growth factor conditions from the precursor fraction of WKM (conditions listed along top row of images). All images in
B taken at 100. Scale bars in top left panels are 50 mm. Arrowheads in top right panels denote mixed colonies. (D) Colonies
isolated from precursor fraction methylcellulose cultures cytospun and stained with May Gr€unwald/Giemsa. Tight colonies were
isolated from cultures with only carp serum and Epo (left column), while ruffled and spread colonies were isolated from cultures
containing carp serum and Gcsf (right column). All images were taken at 1000, and scale bar in bottom right is 20 mm. (E) RT-
PCR analysis of colonies isolated from precursor fraction of methylcellulose cultures. Colony morphology is listed on left, and
genes assayed are listed along top of gel images. (See Plate no. 8 in the Color Plate Section.)
4. Cellular Dissection of Zebrafish Hematopoiesis 103

investigate progenitors in the zebrafish in a clonal manner by modifying existing


methylcellulose culture techniques, exogenously adding the recently identified zeb-
rafish recombinant cytokines erythropoietin (Epo) (Paffett-Lugassy et al., 2007) and
granulocyte Gcsf (Liongue et al., 2009) to quantitate the number of myeloid and
erythroid progenitors in adult kidney marrow scatter fractions (Fig. 8)
(Stachura et al., 2011). This level of precision allows the further testing of prospec-
tive hematopoietic progenitors in normal and mutant zebrafish, allowing more
careful investigation of lineage determination and its conservation among vertebrate
animals. In addition, it allows comparison of hematopoietic progenitor cells and
their response to cytokines, furthering our understanding of cytokine signaling.
Furthermore, the ability to examine blocks in hematopoietic differentiation, aberrant
gene expression, and proliferative regulation is now possible in mutant fish already
(and currently being) generated. Finally, it also allows the rapid screening of small
molecules, blocking antibodies, and other drug compounds that may affect lineage
differentiation, maturation, and proliferation.

1. Methylcellulose
To develop a clonal assay to further enumerate and characterize progenitor cells in
the zebrafish, we utilized methylcellulose; a semi-solid, viscous cell culture medium
used in murine and human progenitor studies. The nature of methylcellulose culture
allows individual progenitor cells to develop isolated colonies within the medium,
where they can be enumerated after several days in culture. In addition, the use of
methylcellulose allows examination of colony morphology and subsequent isolation
for further characterization by morphological examination and gene expression.

2. Methylcellulose Stock Preparation


Prepare 2.0% methylcellulose by adding 20 g of methylcellulose powder (Sigma
Aldrich) to 450 ml of autoclaved H2O and boiling for 3 min. Allow mixture to cool to
room temperature before adding 2 L-15 medium powder (Mediatech). Then,
adjust the weight of the methylcellulose mixture to 1000 g with sterile water.
Methylcellulose should be allowed to thicken at 4  C overnight before being ali-
quoted and stored at –20  C.

3. Methylcellulose Clonal Assays


Complete methylcellulose medium:

10 ml 2.0% methylcellulose stock


4.9 ml DMEM (high glucose)
2.1 ml Ham’s F-12
2 ml FBS
300 ml HEPES (1 M stock)
200 ml Penicillin/streptomycin (5000 U/ml and 5000 mg/ml stock, respectively)
200 ml L-glutamine (200 mM stock)
40 ml Gentamicin sulfate (50 mg/ml)
104 David L. Stachura and David Traver

To perform experiments in triplicate, add 3.5 ml of complete methylcellulose to


sterile round bottom 14 ml tubes (Becton Dickinson) with 5 ml syringes and 16
gauge needles for each condition. Cells of interest (prospective progenitors) should
be isolated, counted, and resuspended in 100 ml of ZKS medium and added to
complete methylcellulose, along with cytokines, small molecules, or other agents
to be investigated.
For myeloid differentiation, add 1% carp serum and 0.3 mg/ml recombinant
zebrafish Gcsf to methylcellulose medium. For erythroid differentiation, add 1%
carp serum and 0.1 mg/ml recombinant zebrafish Epo to methylcellulose medium.
Cytokines and additives should not total more than 10% of the total volume, as the
medium will not be viscous enough to discern individual colonies.
To observe separable, individual colonies, cells should be resuspended at
1  104–5  104 cells/ml. Tightly cap tubes, and gently vortex solution to mix. In
triplicate, aliquot 1 ml of solution into 35 mm Petri dishes (Becton Dickinson). Swirl
Petri dishes to distribute the methylcellulose culture evenly, and place plates in a
humidified 15 cm dish (made by placing a plate of sterile dH2O inside the 15 cm
dish) at 32  C and 5% CO2. Plates should be removed 7 days after plating for
microscopic examination, colony isolation, and gene expression analyses.
As with all tissue culture, strict attention to sterility and cleanliness should be
adhered to at all times. All procedures should be performed in a tissue culture
laminar flow hood.

4. Enumeration of Colony Forming Units (CFUs)


CFUs are a measurement of how many progenitors are present in a given popu-
lation of cells; if an individual cell has the capability to proliferate and divide into
mature blood cells under certain growth conditions, it will make an individual
colony. For example, if 100 putative myeloid progenitor cells are plated under
conditions suitable for myeloid differentiation and one myeloid colony arises,
1:100 of the cells plated was a myeloid CFU.
To perform enumeration of CFUs, observe and count colonies on an inverted
microscope after 7 days in culture. Counting with a 5 or 10 objective (50–100
magnification) is recommended, with the aperture closed down slightly to grant high
contrast, which aids in the visualization of colonies. Be careful not to disturb the
dish; even though methylcellulose is viscous, excessive movement of the plates will
cause colonies to move, complicating further analysis. Depending on the cytokines,
growth factors, and other culture additives, colony shape, size, and color will be
different; be sure to note and record all of this information. Inclusion of a transgenic
marker will aid in identification of colony type and morphology as shown in Figure 8
B and C, whereby erythroid colonies express DsRed driven by the erythroid-specific
gata1a promoter, and myeloid colonies express GFP driven by the myeloid-specific
mpx promoter.
If cultures are to be returned to incubator do not remove Petri plate lids, and take
care to wipe down surfaces with 70% ethanol before starting experiment.
4. Cellular Dissection of Zebrafish Hematopoiesis 105

5. Picking and Analyzing Colonies from Methylcellulose


Hematopoietic colonies can be carefully plucked from methylcellulose cultures
with a pipetteman, preferably a p20 with a fine tip. Pay attention to pick only
individual colonies, placing them into 1.5 ml eppendorf tubes with 200 ml of PBS.
Pipette up and down gently in the PBS to remove traces of methylcellulose from your
tip, and to break up the colony. It is possible to pool colonies of similar morphology
for analyses, especially when large cell numbers are required. Colonies may be
cytospun and stained with May-Gr€ unwald/Giemsa (Sigma Aldrich) as described
above. In addition, colonies may be subjected to RT-PCR analysis for mature lineage
gene transcripts as described above.

VI. Conclusions
Over the past decade, the zebrafish has rapidly become a powerful model system
to elucidate the molecular mechanisms of vertebrate blood development through
forward genetic screens. In this chapter, we have described the cellular characteri-
zation of the zebrafish blood forming system and provided detailed protocols for the
isolation, transplantation, and culture of hematopoietic cells. Through the develop-
ment of lineal subfractionation techniques, transplantation technology, and in vitro
hematopoietic assays, a hematological framework now exists for the continued study
of the genetics of hematopoiesis. By adapting these experimental approaches that
have proven to be powerful in the mouse, the zebrafish is uniquely positioned to
address fundamental questions regarding the biology of hematopoietic stem and
progenitor cells.

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CHAPTER 5

Zebrafish Lipid Metabolism: From


Mediating Early Patterning to the
Metabolism of Dietary Fat and Cholesterol
Jennifer L. Anderson,* Juliana D. Carten* and Steven A. Farber
Carnegie Institution for Science, Department of Embryology, Baltimore, Maryland, USA

Abstract
Abbreviations
I. Introduction
A. The Need for Whole Animal Studies of Lipid Metabolism
B. Larval Zebrafish as a Model of Vertebrate Lipid Metabolism
II. Lipid Metabolism in Developing Zebrafish
III. Yolk Metabolism During Early Vertebrate Development
IV. Lipid Signaling During Early Zebrafish Development
A. Lipid Modifications Influence Primordial Germ Cell Migration
V. Visualizing Lipid Metabolism in Larval and Adult Zebrafish
A. Lipophilic Dyes
B. BODIPY Fatty Acid Analogs
C. BODIPY Cholesterol
D. Fluorescent Reporters of Lipid Metabolism
VI. Triple Screen: Phospholipase, Protease and Swallowing Function Assays
VII. Zebrafish Models of Human Dyslipidemias
VIII. Summary
Acknowledgments
References

Abstract
Lipids serve essential functions in cells as signaling molecules, membrane compo-
nents, and sources of energy. Defects in lipid metabolism are implicated in a number of
pandemic human diseases, including diabetes, obesity, and hypercholesterolemia.

*
These two authors contributed equally to the work.

METHODS IN CELL BIOLOGY, VOL 101 0091-679X/10 $35.00


Copyright 2011. Elsevier Inc. All rights reserved. 111 DOI 10.1016/B978-0-12-387036-0.00005-0
112 Jennifer L. Anderson et al.

approaches for disease prevention and treatment. Numerous studies have shown that
the zebrafish is an excellent model for vertebrate lipid metabolism. In this chapter,
we review studies that employ zebrafish to better understand lipid signaling and
metabolism.

ABBREVIATIONS
LCFA, long chain fatty acid; LD, lipid drop; MCFA, medium chain fatty acid; MTP,
microsomal triglyceride transfer protein; SCFA, short chain fatty acid, TAG,
triacylglycerol

I. Introduction
Lipids play essential roles in cells as signaling molecules, membrane compo-
nents, and sources of fuel. Given their necessity for proper cellular function, it is
not surprising that defects in lipid metabolism underlie a number of human
diseases, including obesity, diabetes, and atherosclerosis (Joffe et al., 2001;
McNeely et al., 2001; Watanabe et al., 2008). In 2007–08, one-third of US adults
and 18% of children were classified as obese (Flegal et al., 2010; Ogden et al.,
2010), with obesity and type 2 diabetes on the rise worldwide (Misra and Khurana,
2008). The globalization of the high-fat western diet and the concurrent rise in the
incidence of lipid disorders has provided an impetus to better understand lipid
metabolism in the context of metabolic dysfunction. This need to investigate the
role of lipids in metabolic disease has brought into focus unanswered questions in
the field. For instance, although the genes involved in cholesterol and fatty acid
(FA) uptake in intestinal cells have been identified, their exact mechanisms of
action are highly debated or largely unknown (Klett and Patel, 2004; Nassir et al.,
2007; Nickerson et al., 2009; Shim et al., 2009; Stahl et al., 1999). Such gaps in
our understanding of these genes and how they function hinder the development of
effective therapeutics for lipid disorders and reveal a need to create better
approaches to address them.
In this chapter, we will review novel approaches undertaken to study lipid signal-
ing and metabolism using the zebrafish model organism, with an emphasis on
presenting a diverse array of techniques employed to visualize lipid metabolism
during various stages of zebrafish development.

A. The Need for Whole Animal Studies of Lipid Metabolism


In vitro studies have laid much of the groundwork for our biochemical under-
standing of vertebrate lipid metabolism and continue to comprise many of the studies
done in the field today; however, a number of caveats arise when attempting to study
lipid metabolism in vitro. Such studies are often performed in transformed cultured
cells, such as liver HepG2, intestinal Caco2, and adipocyte 3LT3 cells. These cell
5. Zebrafish Lipid Metabolism 113

lines, comprised of a single cell type, cannot duplicate the cellular heterogeneity of
an entire organ, such as the intestine, which is composed of stem, enteroendocrine,
immune, and goblet cells. These multiple cell types are known to influence each
other through paracrine signaling that can have global effects on lipid uptake and
processing. Furthermore, bile, intestinal mucus, and the gut microbiota are all known
to greatly influence dietary lipid processing and absorption in the intestine (Backhed
et al., 2004; Field et al., 2003; Kruit et al., 2006; Martin et al., 2008; Moschetta et al.,
2005; Pack et al., 1996; Titus and Ahearn, 1992; Turnbaugh et al., 2008), and are
absent in cultured cell models. For these reasons, employing whole animal in vivo
strategies, in addition to cultured cell work, is vital to better understand how met-
abolic dysfunction arises and manifests itself in an organism.
The importance of utilizing whole animal models to identify drugs to ameliorate
metabolic dysfunction is exemplified by how the cholesterol absorption inhibitor
ezetimibe (Zetia, Vitorin; Merck-Schering Plough) was developed (Van Heek et al.,
1997), and how its mechanism of action was defined (Van Heek et al., 2000). Studies
of bile duct-cannulated rats treated with ezetimibe revealed that the bioactive com-
pound responsible for the diminished effect on cholesterol absorption was a glucur-
onidated form of ezetimibe. Since in vitro studies cannot recreate the complex
interplay of neural, chemical, and hormonal cues known to regulate metabolic
processes in vivo, there is a need for whole animal approaches to study lipid
metabolism as it plays out in a multicellular context.

B. Larval Zebrafish as a Model of Vertebrate Lipid Metabolism


The larval zebrafish is well suited for whole animal studies of lipid metabolism.
Larval zebrafish possess many of the same gastrointestinal organs present in humans
(e.g., the liver, intestine, exocrine and endocrine pancreas, and gallbladder)
(Lieschke and Currie, 2007; Pack et al., 1996; Schlegel and Stainier, 2006;
Wallace and Pack, 2003; Wallace et al., 2005) as well as the specialized cell types
involved in lipid absorption and processing (e.g., intestinal enterocytes, fat-storing
adipocytes, hepatocytes in the liver, and acinar cells of the pancreas) (Wallace et al.,
2005). These digestive organs and the cell types present in them are formed using
similar developmental programs as in mammals with hhex (Wallace et al., 2001),
pdx1 (Yee et al., 2005), and shha (Wallace and Pack, 2003) genes playing critical
roles in liver and pancreas organogenesis in zebrafish. Notch and its ligands, Delta
and Jagged, play a role in the developing pancreas, both by maintaining undifferen-
tiated precursors and regulating acinar, exocrine, and endocrine differentiation
(Apelqvist et al., 1999; Esni et al., 2004; Hald et al., 2003; Jensen et al., 2000;
Murtaugh et al., 2003; Zecchin et al., 2004). Due to the optical transparency of
larvae, these organs and their multiple cell types can all be directly observed through
the body wall without the need for invasive surgical manipulations.
Work from our lab and others has shown that zebrafish express many of the
genes needed to transport and metabolize lipids, such as the lipoprotein gene
114 Jennifer L. Anderson et al.

microsomal triglyceride transfer protein (MTP) (Marza et al., 2005) and the FA
transport protein (slc27a) and acyl-CoA synthetase (acsl) gene families (Thisse
et al., 2005; Thisse et al., 2004; Miyares, unpublished). Zebrafish express the
putative cholesterol transporter Niemann-Pick C1-Like 1 (npc1l1) (Farber, unpub-
lished) and treatment of larvae with the human drug ezetimibe, which works
through an NPC1L1-dependent pathway and is used to treat hypercholesterol-
emia, blocks intestinal cholesterol absorption (Clifton et al., 2010).
Apolipoprotein C2, a gene needed for lipoprotein assembly in humans, is
expressed and required during zebrafish larval development; larvae injected with
an apoc2 morpholino exhibit an unabsorbed yolk phenotype (Pickart et al., 2006).
Additionally, the enzymes needed to synthesize lipid-signaling molecules, such as
prostaglandins and thromboxanes, are highly conserved in zebrafish and these
enzymes can be inhibited by commonly used nonsteroidal anti-inflammatory
drugs (Grosser et al., 2002). We have presented here only a small sampling of
the numerous studies that document the homologies between human and zebrafish
lipid metabolism and validate the zebrafish as a model for investigating vertebrate
lipid metabolism.

II. Lipid Metabolism in Developing Zebrafish


During the first four days of development, a zebrafish embryo relies entirely on its
yolk sac for the nutrients needed to sustain its growth and survival. Yolk lipids are the
source of essential fat-soluble vitamins and triacylglycerol (TAG), as well as cho-
lesterol, a required component of cell membranes and a precursor for bile acids
(Babin et al., 1997; Bownes, 1992; Munoz et al., 1990). Lipids enter the developing
embryo at the yolk and embryo interface, an area termed the yolk syncytial layer
(YSL). In the YSL, lipoproteins (e.g., apoE, apoA1, apocII, and vitellogenin) and a
host of lipid-modifying enzymes (e.g., Mtp) transport lipids from the yolk ball to the
embryo (Babin et al., 1997; Marza et al., 2005). Once the circulatory system forms,
yolk, hepatic, and intestinal lipids are transported by lipoproteins to specific target
tissues throughout the organism via the bloodstream.
By 5–6 days postfertilization (dpf), the yolk is depleted and larvae must eat to
acquire lipids. Both in the wild and the laboratory, zebrafish consume a lipid-rich
diet (10% by weight) high in TAG, phospholipids, and sterols (Enzler et al., 1974;
Spence et al., 2008). Prior to absorption by the intestine, these yolk lipids must be
processed and solubilized by the digestive enzymes and bile that make up the
intraluminal intestinal milieu. As in humans, zebrafish bile is produced by hepato-
cytes and secreted into an extensive network of intrahepatic ducts, which drains into
the gall bladder. In response to hormonal stimulation triggered by food consumption,
bile is released into the intestinal lumen to emulsify dietary fat and facilitate its
absorption by intestinal enterocytes. The main components of bile are bile acids,
phospholipids, and salts, which together promote the formation of micelles by
5. Zebrafish Lipid Metabolism 115

inserting themselves between lipid bilayers and reducing the surface tension to allow
for membrane curvature (Tso and Fujimoto, 1991; Verkade and Tso, 2001). After
dietary fat is emulsified, TAG and phospholipids must be broken down by luminal
lipases to release free FA or mono- and di-acylglycerols, which can then enter the
specialized absorptive cells (enterocytes) that line the gut (Thomson et al., 1993).
The exocrine pancreas is the main source of these fat-splitting enzymes in larval
zebrafish (Hama et al., 2009) and known in mammals to secrete lipase- and protease-
rich pancreatic juice into the gall bladder (Layer and Keller, 1999). After being
emulsified and broken down, TAG can be absorbed by enterocytes, the main absorp-
tive cell type of the zebrafish intestine. These cells are highly reminiscent of
mammalian enterocytes (Buhman et al., 2002), with the characteristic microvilli
and basal nuclei (Fig. 1).
Following food consumption, zebrafish accumulate cytoplasmic lipid drops (LD)
in their enterocytes (Walters, unpublished). From there, fats are likely burned via
oxidative pathways in the mitochondria or peroxisomes or packaged into chylomi-
crons, which are secreted from the basolateral surface of enterocytes into lymphatic
or blood vessels (Field, 2001; Levy et al., 2007). In chickens, chylomicron produc-
tion and secretion is highly conserved, with the exception that lipoproteins are
secreted from the intestine directly into the portal vein and thus are termed porto-
microns (Bensadoun and Rothfeld, 1972; Griffin et al., 1982). This difference has
led some to propose that a similar portomicron process occurs in fish (Robinson and
Mead, 1973); however, careful ultrastructural studies and isotopic lipid labeling
experiments suggest that fish and mammals produce chylomicrons containing
largely similar lipoproteins (Sire et al., 1981; Skinner and Rogie, 1978). While it
remains to be seen how closely the zebrafish system will model human intestinal
lipoprotein metabolism, it is likely that many of the mechanisms of lipoprotein
production are conserved.

III. Yolk Metabolism During Early Vertebrate Development

To mobilize lipids stockpiled in the yolk sac, rodents express the same genes
required for lipoprotein production as those expressed in fully differentiated intes-
tinal enterocytes and liver hepatocytes (e.g., MTP and the apolipoproteins apoE,
apoB, and apo-A-IV and apo-A-I (Elshourbagy et al., 1985; Farese et al., 1996;
Plonne et al., 1996)). Without these genes, the rapid transport of essential nutrients
from the yolk to developing embryonic tissues is impaired and development cannot
proceed normally. Not surprisingly, mice null for MTP (Raabe et al., 1998) and apoB
(Farese et al., 1996) are embryonic lethal, and DGAT-2 null mice, which lack the
enzyme needed to synthesize diacylglycerol, exhibit stunted embryonic growth and
die perinatally (Stone et al., 2004).
The zebrafish YSL expresses a number of lipoprotein-encoding mRNAs that are
also expressed later in the larval intestine and liver, including MTP (Marza et al.,
2005), apoC2 (Farber Lab, unpublished), intestinal FA binding protein
116 Jennifer L. Anderson et al.

[(Fig._1)TD$IG]

Fig. 1 The intestinal enterocytes of zebrafish and mammals exhibit a high degree of morphological
similarity. Electron micrograph of enterocytes from a larval zebrafish (6 dpf). Zebrafish enterocytes
exhibit the typical characteristics of mammalian polarized intestinal cells including apical microvilli,
which extend into the intestinal lumen (L) and form the brush border, as well as basal nuclei (N).
Organelles and subcellular details including mitochondria (M), Golgi bodies (G), and endoplasmic
reticulum (ER) are apparent throughout the enterocytes. For comparison, see mouse EMs in
Buhman et al. (2002).

(Sharma et al., 2004), and apoE and apoA-I (Babin et al., 1997). Due to the parallel
gene expression patterns observed between the embryonic YSL and larval digestive
organs, we hypothesize that yolk utilization during early zebrafish embryogenesis is
a regulated process highly analogous to intestinal and hepatic lipoprotein-mediated
lipid transport.
5. Zebrafish Lipid Metabolism 117

To study the role of lipid metabolism genes in the YSL during early zebrafish
development, we utilize synthetic antisense morpholinos (MO) to attenuate gene
expression and assay subsequent yolk metabolism. MOs are widely used in the
zebrafish community to knock down mRNA levels (Heasman, 2002; Nasevicius
and Ekker, 2000), and numerous studies have phenocopied mutants by targeting
particular mRNA transcripts using this method (Dutton et al., 2001; Karlen and
Rebagliati, 2001; Urtishak et al., 2003). By injecting MOs targeting lipid-specific
genes into the yolk of 1–4 cell stage embryos, followed by yolk injection of fluo-
rescent lipid analogs, such as BODIPY-labeled FA (Fig. 2A), we can directly assess
the necessity of a given gene for yolk metabolism during early larval development.

[(Fig._2)TD$IG]

Fig. 2 BODIPY lipid analogs enable studies of yolk metabolism during early zebrafish development.
To assay the function of apoc2 during zebrafish development, embryos were injected with an apoc2
morpholino at the 1–4 cell stage followed by injection of a fluorescent fatty acid (BODIPY-C12) at 24 h
postfertilization (hpf). (A) At 48 hpf, embryos injected with BODIPY-C12 retain the fluorescent analog
primarily in their yolk. (B, C) apoc2 morphants exhibit an enlarged yolk phenotype (arrowhead) at 48 and
72 hpf, indicating that apoc2 is necessary for yolk utilization during larval development. (D, E) To
determine the metabolic consequences of apoc2 deficiency, 1 dpf wild-type and apoc2 morphant larvae
were injected with BODIPY-C12 and assayed using fluorescent thin layer chromatography (TLC) 2 days
later. (D) TLC analysis shows that BODIPY-C12 is incorporated primarily into triacylglycerol (TAG),
diacylglycerol (DG), and phosphatidylcholine (PC) in both wild-type and apoc2 morphant larvae. (E)
apoc2 morphants exhibit defects in PC and lysophosphatidylcholine (LPC) metabolism. Total lipid
fluorescence was quantified from TLC plates run with total lipids extracted from wild-type and apoc2
morphants. Triacylglycerol (TAG), diacylglycerol (DG), BODIPY C12:0 (C12), phospholipids (PL), PC,
and LPC.* p < 0.05.
118 Jennifer L. Anderson et al.

Following sequential injections of the morpholino and BODIPY-labeled FA,


embryos are allowed to develop and total larval lipids are extracted at 2–3 dpf. We
assay the incorporation of the fluorescent FA into metabolites using thin layer
chromatography (TLC). This approach allows one to identify metabolic abnormal-
ities in morphants that may not exhibit obvious morphological phenotypes.
Furthermore, we can assay the effects of essential genes at earlier stages of larval
development prior to lethality caused by insufficient transcript amounts.
We are currently using this technique to better understand how the apolipoprotein
apoc2 functions in yolk utilization during early zebrafish development. This gene
was first identified in a MO screen targeting secreted proteins of the unknown
function. In a screen of approximately 100 MOs, we identified one with an unab-
sorbed yolk phenotype (Fig. 2B and 2C). TLC analysis revealed that these larvae
exhibit metabolic defects in phospholipid production, as morphants incorporated
less BODIPY-C12 into phosphatidylcholine (PC) and lysophosphatidylcholine
(LPC) (Fig. 2D and 2E). Unabsorbed yolk and phospholipid metabolic deficiency
in apoc2 morphants suggest that this gene has a function in the yolk or YSL during
zebrafish development that is unrelated to its known role in the activation of lipo-
protein lipase in peripheral tissues (Jong et al., 1999).

IV. Lipid Signaling During Early Zebrafish Development

It is not surprising that many lipid-signaling molecules are derived from mem-
brane phospholipids. Such a system allows for rapid transmission of extracellular
signals via membrane, and constituents to the intracellular environment to activate
appropriate signaling cascades and cellular responses. The synthesis of membrane-
derived signaling lipids is often dependent on phospholipases, enzymes that cleave
phospholipids in response to specific cellular signals. Phospholipase A2 (PLA2) is
one such enzyme that catalyzes the hydrolysis of the second fatty acyl bond of
glycerophospholipids to liberate lysophospholipid and free FA, both lipid-signaling
precursors. In the last decade, PLA2 activity and its products have been implicated in
a wide range of cellular phenomena including inflammation, membrane remodeling,
and cancer.
Lipid signaling events during early development are not well elucidated and have
only been recently explored in zebrafish. Studies examining PLA2 enzymatic activ-
ity throughout larval development using whole embryo lysates revealed varying
activity levels during different stages of development, with larvae exhibiting a
significant PLA2 activity during somitogenesis (Fig. 3A). Pharmacological inhibi-
tion of PLA2 activity causes developmental arrest at epiboly (Farber et al., 1999).
Further experiments identified the primary source of phospholipase activity to be the
Ca2+-dependent cytosolic PLA2 (cPLA2) type, a crucial mediator of stimulus-
induced eicosanoid release. cPLA2 activity releases the polyunsaturated FA arachi-
donic acid from membranes, allowing it to participate in the synthesis of eicosa-
noids, a potent class of lipid-signaling molecules that exhibit both paracrine and
5. Zebrafish Lipid Metabolism 119
[(Fig._3)TD$IG]

Fig. 3 Phospholipase activity is required for proper zebrafish development. (A) Cytosolic phospho-
lipase A2 (cPLA2) mRNA levels and PLA2 enzymatic activity were quantified from whole embryo
lysates during early stages of zebrafish development. cPLA2 expression peaks during somitogenesis
(10 h) while enzymatic activity steadily increases as development proceeds. PLA2 activity is required to
generate prostaglandins. (B) Blocking prostaglandin production during early zebrafish development
through inhibition of prostaglandin–endoperoxide synthase (Ptgs1) via morpholino knockdown results
in developmental arrest at epiboly. Developmental arrest can be rescued by adding back the exogenous
enzyme product (PGE2). Reproduced with permission by Development (Speirs et al., 2010). (C) Embryos
exposed to the PGE2 analog (16,16-dimethyl-PGE2; dmPGE2) exhibit increased expression of runx11
and cmyb1 as evidenced by in situ hybridization. These genes are expressed in the ventral wall of the
dorsal aorta in a region analogous to the mammalian aorta–gonad–mesonephros and are required for
mammalian hematopoietic stem cell development. Reprinted by permission from Macmillan Publishers
Ltd.: Nature (North et al., 2007). Prostaglandin E2 regulates vertebrate haematopoietic stem cell homeo-
stasis. Nature 447, 1007–11, copyright 2007.
120 Jennifer L. Anderson et al.

autocrine influences on cells and tissues (Burke and Dennis, 2009; Clark et al., 1991)
and are commonly associated with provoking inflammatory and immune responses.
The developmental arrest observed in cPLA2 morphants suggests that cPLA2 activ-
ity and eicosanoid signaling are essential for early embryonic patterning, pointing to
a novel role for lipid signaling during embryogenesis.
Arachidonic acid can act as a substrate for a number of other lipid modifying
enzymes (e.g., cyclooxygenase) that are critical for cell movements required to
pattern the early zebrafish embryo. Cha et al. (2006) found that PGE2, an eicosanoid
downstream of cyclooxygenase, is essential for the morphogenic movements of
convergence and extension (Cha et al., 2006; Cha et al., 2005). Moreover, the arrest
of epiboly that results from the inhibition of cyclooxygenase 1 (also known as
Prostaglandin–endoperoxide synthases, Ptgs1) using an antisense morpholino oli-
gonucleotide can be completely rescued by the addition of PGE2 (Speirs et al., 2010)
(Fig. 3B).
The importance of PGE2 during zebrafish development was further demonstrated
in a small molecule screen performed in zebrafish larvae (36 hpf) by North et al.
(2007). Using this approach PGE2 was found to impact hematopoietic stem cell
proliferation as evidenced by a dramatic increase in the expression of hematopoietic
markers in larvae treated with a PGE2 analog (North et al., 2007) (Fig. 3C). Taken
together, these data provide evidence for the importance of PLA2-derived lipid
mediators during zebrafish development.

A. Lipid Modifications Influence Primordial Germ Cell Migration


In addition to uncovering a role for lipid signaling during the early movements of
gastrulation, work on the zebrafish has shown that lipid modifications influence
another critical cellular movement during early embryonic development: primordial
germ cell (PGC) migration. PGC migration in zebrafish embryos can be visualized
in embryos as early as 80% epiboly by injecting in vitro transcribed, capped GFP-
nanos mRNA (which consists of the coding sequence of GFP fused to the 3’UTR of
the nanos gene) into zebrafish embryos at the one-cell stage. GFP-nanos message
and protein are stabilized preferentially in PGCs such that they maintain their
fluorescence throughout early development, facilitating the detailed study of PGC
migratory behavior (Doitsidou et al., 2002).
Because lipid metabolism is highly conserved across vertebrates, human drugs
can be used to block particular steps in metabolic pathways to determine what
pathway or metabolites are necessary for a developmental process, such as PGC
migration. In Drosophila, loss of 3-hydroxyl-3-methylglutaryl-CoA reductase
(HMGCoAR) results in PGC migration defects (Van Doren et al., 1998).
Similarly, studies in zebrafish have shown that pharmacologic inhibition of
HMGCoAR by atorvastatin (Lipitor) results in abnormal development and PGC
migration (Thorpe et al., 2004). HMGCoAR is a rate-limiting step in the synthesis
of cholesterol and is the target of statins (Fig. 4). Zebrafish embryos soaked in
5. Zebrafish Lipid Metabolism 121
[(Fig._4)TD$IG]

Fig. 4 Cholesterol biosynthesis and protein lipidation are highly conserved in vertebrates. Due to the
high genetic conservation of these pathways, human drugs can be used to block specific biochemical steps
to determine the roles that cholesterol and protein lipidation play in zebrafish development. Reprinted
from Development Cell with permission from Elsevier (Thorpe et al., 2004).

statins, either mevinolin (Lovastatin) or simvastatin (Zocor), exhibit developmental


arrest, blunted axis elongation, misshapen somites, and head and axial necrosis (Fig. 5).
Additionally, PGC migration is profoundly perturbed. Embryos treated with a more
hydrophobic statin (Lipitor) exhibit PGC migration defects and only mild morphologic
abnormalities.
To determine which downstream products of HMGCoAR mediate these develop-
mental defects, a ‘‘block and rescue’’ approach was taken through injection of
putative biochemical pathway intermediates downstream of HMGCoAR following
statin-mediated inhibition. Injection of mevalonate, the product of HMGCoAR’s
reduction of ß-hydroxy-ß-methylglutaryl-CoA, completely rescued all the pheno-
types associated with the statin treatment (Fig. 6). Similar experiments using inter-
mediates downstream of mevalonate (Fig. 7) indicated that the prenylation pathway,
responsible for adding polyunsaturated lipids to proteins, was likely mediating the
effect of statins on PGC migration. High doses of the selective farnesyl transferase
inhibitors L-744 or FTI-2153 (Crespo et al., 2001; Sun et al., 1999) had no effect on
PGC migration. However, embryos treated with geranylgeranyl transferase I (GGTI)
inhibitor (GGTI-2166) exhibited a strong PGC migration phenotype with only a
122 Jennifer L. Anderson et al.

[(Fig._5)TD$IG]

Fig. 5 Embryos treated with statins exhibit developmental defects. In comparison to untreated
embryos (A), embryos soaked in a low dose of mevinolin (0.06 mM). (B) exhibit mild developmental
defects, as evidenced by tail kinks. (C) Exposure to higher doses of mevinolin (1.2 mM) results in blunted
axis elongation, necrosis and developmental arrest. (D) Simvastatin treated embryos (2.0 mM) exhibit
similar developmental defects. (E) Dose response of mevinolin on developmental arrest (mean + SEM,
n = 3). (F) Dose response of simvastatin on developmental arrest (mean + SEM, n = 3). Reprinted from
Development Cell with permission from Elsevier (Thorpe et al., 2004).

slight disruption of the notochord. These data suggest that protein prenylation,
specifically by GGTI, is required for correct PGC migration (Thorpe et al., 2004).

V. Visualizing Lipid Metabolism in Larval and Adult Zebrafish

A. Lipophilic Dyes
Lipophilic dyes, known as lysochromes, are one of the first tools used to visualize
lipids in cells. These dyes are capable of labeling a variety of lipids and lipid-
containing structures including TAG, FA, and lipoproteins. Dyes, such as oil red
O (ORO), sudan black B, and nile red, were initially used to label LDs in tissue
sections and cultured cells and continue to be used today. Marza and colleagues
(Marza et al., 2005) utilized sudan black B to identify LDs in histological sections of
fed adult zebrafish. The authors found that feeding a high-fat meal increased the
expression of MTP in intestinal epithelial cells. This protein is required for proper
5. Zebrafish Lipid Metabolism 123
[(Fig._6)TD$IG]

Fig. 6 Isoprenoid intermediates rescue the developmental defects caused by statin treatment.
Embryos at early cell stages were injected with mevalonate and then soaked overnight in mevinolin,
simvastatin, or atorvastatin. (A) Embryos treated with mevinolin show severe developmental defects
at 24 hpf. (B) Embryos injected with the isoprenoid intermediate mevalonate (1–16 cell stages) and
then treated with mevinolin exhibit normal morphology. (C) Mevalonate injection rescues the somatic
defects observed in embryos treated with different statins. Embryo morphology was scored at 24 hpf.
Data represent the MEAN  SEM from 3–4 experiments. Reprinted from Development Cell with
permission from Elsevier (Thorpe et al., 2004).

assembly and secretion of hepatic and intestinal ApoB-containing lipoproteins,


chylomicrons, and very low-density lipoproteins (VLDL) (Gordon et al., 1995).
Their observation that LDs are coincident with an upregulation of MTP expression
is consistent with MTP’s known function in humans (Marza et al., 2005).
Lysochromes can also be used to visualize endogenous lipid stores in whole fixed
zebrafish to generate an overall picture of neutral lipid localization during develop-
ment. Schlegel and Stainier used ORO to assess the consequence of MTP knock-
down (via MO) on lipid absorption in whole zebrafish larvae (Schlegel and Stainier,
2006). MTP morphants exhibited decreased yolk consumption and an inability to
absorb dietary neutral lipids, resulting in death by 6 dpf. Although lysochromes such
as sudan black B and ORO consistently label neutral lipids in tissue sections and
fixed larvae, fixation techniques are laborious and staining procedures have been
shown to cause artificial fusion of adjacent LDs and mislocalization of the LD
marker, adipose differentiation-related protein (Adrp/Perilipin2) (Fukumoto and
Fujimoto, 2002). More recent techniques to visualize LDs have focused on staining
these drops in vivo.
124 Jennifer L. Anderson et al.

[(Fig._7)TD$IG]

Fig. 7 Statin treatment causes abnormal primordial germ cell (PGC) migration in zebrafish embryos.
Compared to wild-type (A), embryos treated with statins (B) display ectopic PGCs. The arrows indicate
ectopic PGCs that have failed to migrate to the developing gonad. (C) The PGC migratory defect observed
following statin treatment is prevented by injections of isoprenoid intermediates. Embryos at early stages
(1–16 cell) were injected with gfp-nos mRNA and mevalonate and then soaked overnight in mevinolin,
simvastatin or atorvastatin. At 24 hpf, embryos were scored for ectopic PGCs, with a score of 1 indicating
a wild-type single gonadal cluster and score of 4 indicating no discernable PGC cluster. Data represent the
mean  SEM from 3–4 experiments. (D) The PGC migratory defect observed following statin treatment
is prevented by increasing the levels of isoprenoid synthesis intermediates. Embryos injected at the 1–16
cell stage with farnesol, geranylgeraniol or mevalonate and then soaked overnight with atorvastatin
(10 mM) show normal PGC migration. Data represents MEAN  SEM, *p < 0.01 difference from
atorvastatin alone. Reprinted from Development Cell with permission from Elsevier (Thorpe et al., 2004).
5. Zebrafish Lipid Metabolism 125

Greenspan et al. (1985) first showed the utility of nile red (9-diethylamino-5H-
benzo[a]phenoxazine-5-one) to label intracellular LD in live cultured peritoneal
macrophages and smooth muscle cells. Nile red is an uncharged heterocyclic mol-
ecule that only fluoresces in a hydrophobic environment. Labeled neutral lipids
fluoresce a yellow-gold to red color, depending on their relative hydrophobicity,
with no detectable damage or deformation of dye-infused tissues (Fowler and
Greenspan, 1985). More recently, Jones et al. (2008) used nile red to visualize
neutral lipid deposits in live zebrafish larvae. They initially demonstrated that daily
exposure of larvae to nile red-containing embryo media for 4 days (from 3 to 7 dpf)
consistently labeled lipid-rich tissues. The authors then sought to test the effects of
known pharmacological inhibitors of triglyceride metabolism on total larval lipid
content. Treatment with nicotinic acid, a potent pharmacological inhibitor of adi-
pocyte lipolysis (Carlson, 1963), resulted in an increase in total triglyceride content
and decreased cholesterol levels. Treatment with resveratrol, a compound known to
inhibit FA synthase (Tian, 2006), resulted in a decrease in total triglyceride content
as detected by nile red staining. Total triglyceride content was further decreased
when resveratrol was supplemented with norepinephrine (Jones et al., 2008).
While fluorescent dyes and stains are useful for identifying lipid deposits in cells
and tissues, issues arise regarding the distribution and affinity properties of these
compounds. Nonspecific labeling of tissues devoid of lipid deposits may be
observed and staining and washing procedures must then be carefully optimized
to minimize this effect. Additionally, nile red staining does not distinguish between
FA and cholesterol in vivo, although some discrimination based on staining intensity
of tissue sections is possible (Fowler and Greenspan, 1985).

B. BODIPY Fatty Acid Analogs


The wide variety of fluorescent lipid analogs commercially available allows one to
fully exploit the optical clarity of zebrafish larvae to study lipid metabolism. One
type of analog widely used in cultured cells to visualize lipid dynamics is BODIPY-
labeled FA. These analogs consist of an acyl chain of variable length attached to the
BODIPY (4,4-difluoro-4-bora-3a, 4a-diaza-S-indacene) fluorescent moiety. First
synthesized by Treibs and Kreuzer in 1968 (Treibs and Kreuzer, 1969), the
BODIPY fluorophore possesses a number of advantageous qualities including high
photostability, strong and narrow wavelength emission in the visible spectrum, and
an overall uncharged state (Monsma et al., 1989; Pagano et al., 1991).
To administer BODIPY FA analogs to live zebrafish larvae, we developed a
feeding assay that utilizes liposomes to create a suspension of relatively hydrophobic
FA analogs in embryo media (Carten, unpublished). Following a short liposome feed,
digestive organ structure and metabolic function can be assessed, as the fluorescent
FAs accumulate readily throughout numerous larval organs and tissues. With this
assay, we have observed that different chain length FAs (short, medium, and long)
accumulate in distinct patterns throughout digestive organs and tissues, with each
chain length suited to visualize particular larval organs and cellular structures. Long
126 Jennifer L. Anderson et al.

chain fatty acids (LCFA) appear in cytoplasmic LD in enterocytes and hepatocytes.


Short chain fatty acids (SCFA) accumulate primarily in the hepatic and pancreatic
ducts and are particularly suited to illuminate ductal networks. Medium chain fatty
acids (MCFA) accumulate in LDs throughout a wide range of cell types, as well as
ductal and arterial networks, and reveal the subcellular structures of multiple cell
types. Because this feeding assay enables the rapid assessment of digestive function
and FA metabolism in live zebrafish larvae, it has the potential for use in genetic and
pharmacologic screens to identify genes involved in FA metabolism and potential
drug targets.

C. BODIPY Cholesterol
In addition to FA analogs, a number of sterol analogs are available for use with
cultured cells and zebrafish larvae (e.g., NBD-cholesterol, BODIPY-cholesterol).
Initially created to visualize cholesterol partitioning into membranes, these analogs
were found to preferentially enter into liquid-disordered domains, making them less
useful for studies of sterol trafficking in cells (Li et al., 2006). To address these
limitations a new BODIPY-tagged cholesterol analog was synthesized with a mod-
ified fluorophore linker (Li and Bittman, 2007). Recent studies utilizing the
improved BODIPY-cholesterol analog found it to partition into the cholesterol-rich
liquid-ordered membrane domain (Ariola et al., 2009) and interact with membranes
in ways similar to native sterols, making it a powerful new tool for imaging sterol
trafficking in live cells (Marks et al., 2008).
Studies done in the zebrafish have found that BODIPY-cholesterol localizes to the
yolk of developing zebrafish larvae (Holtta-Vuori et al., 2008). Ongoing work in the
Farber lab is examining the localization of BODIPY-cholesterol in zebrafish larval
intestinal enterocytes after a high-fat meal and comparing its localization to that of
LD (revealed by BODIPY-labeled FA). Recent data suggest that the initial uptake of
sterol and of FA segregate into nonoverlapping compartments (Walters, unpub-
lished). These types of experiments will ultimately enable the development of a
clearer model for the uptake and trafficking of dietary lipid in intestinal enterocytes.

D. Fluorescent Reporters of Lipid Metabolism


The ability to perform forward genetic studies in zebrafish by mutagenizing the
entire genome and screening for particular phenotypes has made this vertebrate
model popular (Driever et al., 1996; Haffter et al., 1996). Mutagenesis methods
commonly utilized by the zebrafish community include gamma-ray irradiation to
generate large deletions and translocations (Fisher et al., 1997), soaking founder fish
in mutagenic chemicals such as ethylnitrosourea (ENU) to generate point mutations,
(Driever et al., 1996; Haffter et al., 1996), and retrovirus- or transposon-mediated
gene insertions (Chen and Farese, 2002; Ivics et al., 2004). We perform an ongoing
5. Zebrafish Lipid Metabolism 127

ENU-mutagenesis screen to search for mutations that perturb lipid processing. To


screen for mutants defective in lipid processing, we soak zebrafish larvae in fluo-
rescent lipid reporters that are swallowed and allow lipid processing to be visualized
in vivo (Farber et al., 2001).
One fluorescent reporter we routinely use in our screen is the phosphoethanola-
mine analog PED6, [N-((6-(2,4-dinitro-phenyl)amino)hexanoyl)-1-palmitoyl-2-
BODIPY-FL-pentanoyl-sn-glycerol-3-phosphoethanolamine] (Fig. 8A). Following
ingestion, PED6 is cleaved by phospholipase A2 (PLA2), resulting in the release of
the fluorescent labeled acyl chain (Farber et al., 1999) (Fig. 8C). When zebrafish
larvae (5 dpf) are immersed in media containing PED6, bright green fluorescence is
observed in the intestine, gall bladder, and liver (Fig. 8E). We also utilize the sterol
analog 22-NBD-cholesterol (22-[N-(7-nitronbenz-2-oxa-1,3-diazol-4-yl) amino]-
23,24-bisnor-5-cholen-3-ol) (Fig. 8B) to visualize cholesterol absorption in live
larvae and screen for mutants (Fig. 8F). This reagent is different from PED6 in that
it continuously fluoresces and due to its hydrophobicity it is slightly more difficult to
administer via feeding.
Because PED6 and NBD-cholesterol provide rapid readouts for digestive organ
morphology and lipid processing, we used them to perform the first forward genetic
physiologic screen in zebrafish to identify new genes that regulate lipid metabolism.
One mutation identified with these lipid reporters, fat-free (ffr), was a recessive
lethal mutation. Although ffr mutants appeared morphologically normal, they exhib-
ited significantly diminished fluorescence in their intestine and the gall bladder
following PED6 treatment (Fig. 9) (Farber et al., 2001). ffr larvae were further
characterized using NBD-cholesterol to determine if the uptake and trafficking of
sterol-like molecules was also impaired. In contrast to wild-type larvae, which
accumulated NBD-cholesterol in their gall bladders within a few hours of feeding,
ffr mutants were unable to concentrate NBD-cholesterol in their gall bladders. This
observation suggests that ffr larvae have a significant defect in bile secretion and/or
transport. When ffr mutants were incubated with BODIPY FL-C5, a medium chain
FA analog, they had nearly normal digestive organ fluorescence. Because this
medium chain length FA is less hydrophobic and is not dependent on emulsifiers,
such as bile, for absorption, this further suggests that the ffr mutation attenuates
biliary synthesis or secretion (Ho et al., 2003).
To better characterize the metabolic defects of ffr larvae, we immersed mutants in
media containing radioactive oleic acid (250 mCi/mmol, 3 h), followed by whole
embryo lipid extraction and TLC analysis. We found that ffr larvae have significantly
reduced radioactivity incorporated into phosphatidylcholine (PC) fraction (p<0.05)
in comparison to wild-type larvae (Fig. 10). PC is a major component of bile and its
overall reduction in ffr mutants is consistent with impaired liver lipid synthesis.
The positional cloning of ffr indicated that this gene encodes a protein with no
known function. Further sequence analysis revealed ffr to be well conserved from
invertebrates to mammals. Further studies found ffr to regulate a number of cellular
processes including Golgi structure/maintenance, protein sorting, vesicle
128 Jennifer L. Anderson et al.

[(Fig._8)TD$IG]

Fig. 8 PED6 and NBD-cholesterol visualize lipid uptake in larval zebrafish. (A) The chemical
structures of PED6. The BODIPY-labeled acyl chain of PED6 is normally quenched by the dinitrophenyl
group at the sn-3 position. Upon PLA2 cleavage at the sn-2 position, the BODIPY-labeled acyl chain is
unquenched and can fluoresce. Bright field (B) and fluorescent (C) images of 5 dpf larva following
soaking in PED6 for 6 h. PED6 labeling reveals lipid processing in the gall bladder (arrowhead) and
intestine (arrow). (D) The chemical structure of NBD-cholesterol. The NBD-cholesterol analog
contains a NDB fluorophore where the alkyl tail at the terminal end of cholesterol would normally
reside. (E) Soaking zebrafish larvae (5 dpf) in NBD-cholesterol (3 mg/ml, solubilized with fish bile)
for 2 h visualizes cholesterol uptake in the gall bladder (arrowhead) and intestine (arrow).
5. Zebrafish Lipid Metabolism 129
[(Fig._9)TD$IG]

Fig. 9 PED6 labeling of fat-free(ffr) larvae reveals defective lipid processing. (A) ffr larvae appear
morphologically normal in comparison to wild-type siblings (6 dpf). (B). ffr mutants have diminished
intestinal and gall bladder fluorescence when labeled with PED6, indicating abnormal lipid processing.

trafficking, and intestinal lipid absorption and processing (Ho et al., 2006). Recent in
vivo and in vitro work suggests that ffr is a novel Arf GTPase family effector that
regulates the phospholipase D (PLD) activity. ffr mutants have excess PLD, leading
to higher levels of phosphatidic acid (Wang et al., 2006), which is known to increase
membrane curvature (Begle et al., 2009). The excessive membrane curvature in ffr
mutants may cause abnormal Golgi-vesicle trafficking of nutrient transporters to the
cell surface, leading to higher extracellular blood glucose levels and impaired lipid
metabolism (Liu et al., 2010).
130 Jennifer L. Anderson et al.

[(Fig._0)TD$IG]

Fig. 10 ffr larvae have abnormal phospholipid metabolism as shown by thin layer chromatography
(TLC). To identify the metabolic defects in ffr larvae, mutants and wild-type larvae (4 dpf) were incubated
5. Zebrafish Lipid Metabolism 131

After identifying the molecular nature of the zebrafish ffr mutation, the human
ortholog was located by BLAST searches of public databases. While the human gene
was present in these databases, there was no data on its possible function. This example
demonstrates the power of forward genetic methods in zebrafish to assign functions to
mammalian genes. Other reports of forward genetic screens demonstrate the utility of
PED6 and similar synthetic analogs to identify genes involved in digestive processes.
For example, Wantanabe et al. used PED6 to identify genes that regulate liver mor-
phogenesis and bile synthesis in Medaka fish (Watanabe et al., 2004).

VI. Triple Screen: Phospholipase, Protease and Swallowing


Function Assays
Although PED6 has proven successful in identifying abnormal lipase activity in
zebrafish mutants, there can be significant variability in the digestive tract fluores-
cence observed between wild-type siblings (Fig. 11A). Such variable labeling can
make the use of PED6 in genetic screens difficult, as an increase or decrease in
fluorescence cannot be attributed solely to mutations and may reflect interindividual
differences in reporter ingestion. To address this issue, it was reasoned that PED6
could be used in conjunction with other reporters of digestive function to create a
physiologically relevant readout of in vivo digestive processes.
Hama et al. (Hama et al., 2009) recently demonstrated that concurrent feeding of
PED6, EnzChek protease reporter (Invitrogen Inc.), and nonhydrolyzable micro-
spheres allows one to monitor lipase, protease, and swallowing activities, respec-
tively, in larval zebrafish. Since the three reporters fluoresce at distinct wavelengths,
simultaneous viewing of all three signals is possible (Figs. 11B and 11C). The
EnzChek protease reporter consists of the phosphoprotein casein labeled with mul-
tiple red or green BODIPY fluorophores. The proteolytic cleavage of the quenched
reporter generates highly fluorescent casein fragments, with total fluorescence
proportional to enzyme activity (Jones et al., 1997). EnzChek has been previously
used to detect the activity of metallo, serine, acid and thiol proteases in a number of
biological systems (Haugland and Kang, 1988; Jones et al., 1997; Menges et al.,
1997; Sarment et al., 1999). Unlike PED6 and EnzChek, which report enzymatic

with radioactive oleic acid (C18:1) for 20 h, followed by total lipid extraction and radioactive TLC
analysis. (A, B) Chromatograms generated from scans of TLC plates run with total lipids extracted from
wild-type (A) and ffr (B) larvae. The y-axis reflects radioactivity counts (cpm; 2–3% of actual activity
detected for 3H); the x-axis shows various oleic acid metabolites determined by the migration distance
from the start of the TLC plate measured in centimeters (cm). The major metabolites derived from oleic
acid (here FA) are PC, phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylethanolamine
(PE), phosphatidylglycerol (PG), and TAG. (C) Comparison of relative FA metabolites between ffr and
wild-type larvae reveals that ffr mutants have significantly decreased PC production (n = 9, mean  SD).
Reprinted from Methods in Enzymology with permission from Elsevier (Ho et al., 2003).
132 Jennifer L. Anderson et al.

[(Fig._1)TD$IG]

Fig. 11 Concurrent feeding of PED6, EnzChek, and fluorescent microspheres enables the assessment
of larval digestive function. To correct for interindividual variability observed in wild-type siblings
(6 dpf) labeled with PED6 (A), multiple reporters of the digestive function can be used. (B) PED6,
EnzChek and microspheres each fluoresce at distinct wavelengths, allowing simultaneous screening for
lipase and protease activities, and swallowing function, respectively. (C) PED6 and EnzChek signal in the
gall bladder (arrowhead) and intestine (arrow) of wild-type zebrafish. Microspheres in the intestine
indicate normal ingestion. (D) Intestinal protease and phospholipase activity correlate, validating the
use of the ratio of PED6 to EnzChek signal as readout of the digestive function. Figure from Hama et al.
(2008), Am. J. Physiol. Gastrointest. Liver Physiol., used with permission from Am. Physiol. Soc.
5. Zebrafish Lipid Metabolism 133

function, nonhydrolyzable microspheres assess swallowing and intestinal lumen


integrity.
Administering this screening cocktail to larval zebrafish revealed a correlation
between the intestinal protease and phospholipase activity, consistent with the
hypothesis that the variance observed in PED6 fluorescence was partly due to
differing amounts of PED6 consumed by each larva (Fig. 11D). This work demon-
strated that the ratio of PED6 to EnzChek fluorescence can serve as a readout of the
digestive function, since the ratio in individual larvae is unaffected by differences in
reporter ingestion.
After validating the triple screening method, Hama et al. (2009) demonstrated the
utility of this assay in evaluating the role of the exocrine pancreas in the digestive
function. The exocrine pancreas secretes many of the gastric lipases and proteases
needed for the breakdown and subsequent uptake of nutrients (Layer and Keller,
1999). Previous work has shown that morpholino knockdown of the ptf1a transcrip-
tion factor can selectively prevent exocrine pancreas development (Lin et al., 2004).
Analysis of ptf1a mutants (5 dpf) using the triple screen found that these larvae retain
normal levels of lipase activity yet have reduced protease activity. Further investiga-
tion revealed that there is a marked increase in pancreatic phospholipase A2 (groups
IB and III) expression between 5 and 6 dpf, supporting the notion that the exocrine
pancreas is not the main source of phospholipase activity in 5 dpf fish. However,
6 dpf ptf1a mutant larvae exhibit decreased amounts of both protease and lipase
activity, suggesting the exocrine pancreas begins providing gastric lipases after 5 dpf.
The regulation of phospholipase and protease activity was also examined using the
triple screening method. The peptide hormone Cholecystokinin (CCK) facilitates
digestion by causing the secretion of gastric enzymes into the small intestine after
food consumption (Raybould, 2007). Release of CCK into the circulatory system
activates the CCK receptor A (CCK-RA) in the exocrine pancreas. Larvae (5 dpf)
treated with CCK-RA antagonist showed a reduction in protease activity but had
unaffected lipase activity. Much like the ptf1a mutants, 6 dpf larvae had lower levels
of both protease and lipase activity. Not surprisingly, the effect of CCK-RA antag-
onist was abolished in ptf1a morphants. This work suggests that CCK signaling
regulates zebrafish secretion of exocrine pancreas-derived intestinal proteases ear-
lier in development (5 dpf) and phospholipases later (6 dpf).
The utility of employing multiple reagents to screen for the digestive function
using larval zebrafish has been demonstrated by others in the field. Recent work
from Clifton et al. utilized a multi-tiered approach to identify and describe novel
compounds that inhibit dietary lipid absorption (Clifton et al., 2010). In the initial
screen, larvae (5 pdf) were incubated in PED6 after compound exposure and assayed
for a reduction in gall bladder fluorescence. Results were validated in adult fish and
inactive or toxic compounds were removed from the study. Further narrowing was
done through the use of fluorescent microspheres to confirm swallowing, as previ-
ously described. Absorption of fluorescent cholesterol (NBD-cholesterol) and FA
analogs (SCFA BODIPY-C5, LCFA BODIPY-C16) was also assayed to better under-
stand the mechanism of action for seven potentially novel compounds and two
134 Jennifer L. Anderson et al.

known lipid absorption inhibitors, orlistat and ezetimibe. Interestingly, the authors
found that ezetimibe was found to reduce metabolism of both PED6 and BODIPY-
C16 in addition to NBD-cholesterol. Five of the seven compounds identified in the
initial PED6 screen had comparable results. AM1–43, a dye commonly used to
visualize endocytosis, and the EnzChk protease assay were used to screen for
physiological disruptions, while the lipophilic dye ORO was used to determine
whether reduced gall bladder fluorescence was due to reduced lipid processing.
AM1–43 staining after ezetimibe treatment was significantly reduced, supporting
a disruptive role in endocytosis, perhaps more pronounced than previously
described. Those with reduced AM1–43 staining underwent further testing to deter-
mine whether cholesterol synthesis was normal: larvae were pretreated with a
reagent that extracts membrane cholesterol (methyl-ß-cyclodextran;MßC) and
observed for recovery. By using a series of well-established assays, three compounds
were identified for testing in mammals and ezetimibe was implicated as playing a
larger role in intestinal endocytic dynamics than previously thought. These studies
further establish the zebrafish system as a useful and translatable model for testing
and prioritizing new compounds for follow-up studies in mammals.
As with any in vivo system, there is inherent variability (developmental timing,
intestinal microenvironment), which will result in signal variation. Therefore, cau-
tion should be taken to minimize this variability by carefully scoring digestive organ
morphology of larvae prior to and after reporter ingestion, discarding sickly larvae
that lack swim bladders or exhibit developmental delay, and using properly staged
larvae.

VII. Zebrafish Models of Human Dyslipidemias

Recently, an adult zebrafish model for early atheroschlerosis has been developed
through administration of a high-cholesterol diet (HCD) (Stoletov et al., 2009). After
receiving a 4% cholesterol diet from 5 weeks postfertilization (wpf) to 13–17 wpf,
fish developed hypercholesterolemia, as demonstrated by their four-fold plasma
total cholesterol levels. No weight gain or increase in TAG was observed.
Lipoprotein profiles of HCD-fed fish showed, in addition to the wild-type control
HDL fraction, strong fractions for LDL and VLDL. Antibody studies showed that the
fish had oxidized plasma lipoproteins, correlating to the development of atheroscle-
rotic lesions. Staining of sections indicated early lesions of atherosclerosis (fatty
streaks) while antibody (human L-plastin) staining implicated macrophage infiltra-
tion. Live larvae were used to visualize myeloid cell and lipid accumulation, endo-
thelial cell defects, and increased PLA2 activity, signs of early atherosclerosis. This
novel model has been used in transplant studies to demonstrate in vivo the require-
ment of toll-like receptor-4 in macrophage lipid uptake.
The zebrafish has also emerged as a model of glucose metabolism and thus,
diabetes, allowing the role lipids play in the onset and pathologies of both subtypes
of this disease to be examined. Diabetes was once studied primarily in the context
5. Zebrafish Lipid Metabolism 135

glucose uptake and metabolism; however, the causal effects of dietary lipids on type
2 diabetes are now being examined more closely. Larval zebrafish contain insulin-
secreting beta cells that are triggered to release insulin in response to elevated blood
glucose and FA levels, much like in humans. Work done by Elo et al. has shown that
the exposure of larval and adult zebrafish to high glucose levels results in hyper-
glycemia (Elo et al., 2007). Furthermore, treatment with the antidiabetic com-
pounds Glipizide and Metformin reduces blood glucose levels in adult zebrafish
and lowers the expression of phosphoenolpyruvate carboxykinase, which is regu-
lated by insulin and catalyzes the rate-limiting step of gluconeogenesis, in larval
zebrafish (Elo et al., 2007).

VIII. Summary
In this, chapter we have presented novel approaches undertaken using the zebra-
fish to investigate lipid metabolism and signaling. The optical transparency of
zebrafish embryos and larvae can be fully exploited by using transgenic lines,
fluorescent reporters, and lipid analogs to visualize metabolic events.
Furthermore, the high genetic conservation across lipid signaling and metabolic
pathways allows pharmacological regents to be utilized to study the importance of
lipids during early development. The high fecundity, small size, and genetic tracta-
bility of these organisms make them ideal for high-throughput screening efforts to
identify genes involved in lipid metabolism and signaling and thus identify potential
therapeutic targets for human diseases.
Despite the immense potential of the zebrafish as a genetic and therapeutic
screening tool, these organisms are currently underutilized by the academic and
pharmaceutical research communities. While we have discussed some of the excep-
tions here, many studies use the zebrafish to investigate developmental events such
as pattern formation and early neurogenesis, with few taking advantage of the system
to study digestive physiology and lipids. We advocate the increased use of zebrafish
in metabolic studies and fully expect a continued upward trend in the use of this
organism as its contributions to our understanding of human disease become more
apparent.

Acknowledgments
The authors would like James Walters and Mike Sepanski of the Carnegie Institution for Science for the
EM image of the zebrafish enterocyte. Sections of this chapter contain modified text from our review in
Clinical Lipidology: Carten JD, Farber SA: A new model system swims into focus: using the zebrafish to
136 Jennifer L. Anderson et al.

visualize intestinal lipid metabolism in vivo. Clin. Lipidol. 4(4): 501–515 (2009). The article is available at
http://www.futuremedicine.com/doi/full/10.2217/clp.09.40.

Financial Disclosure
The authors have no affiliations or financial arrangement with any organization that has a financial
interest or stake in the material discussed in this manuscript. The Carnegie Institution does hold a patent
together with the University of Penn. on the invention of the author (S.A.F.) that describes the use of
fluorescent lipids in zebrafish for high-throughput screening (Pub. No.: US 2009/0136428 A1). The
authors have no current consultancies, honoraria, stock ownership or options, expert testimony or royalties
regarding the material described. However, the Carnegie Institution and/or U. Penn. can license this
technology in the future, potentially providing royalties to the author (S.A.F). Research performed by
the authors and described in this manuscript was supported by the Carnegie Institution Endowment and
with grants from the US National Institutes of Health (RO1 GM63904 and RO1 DK060369).

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CHAPTER 6

Development of the Zebrafish Enteric


Nervous System
Iain Shepherd* and Judith Eiseny
*
Department of Biology, Emory University Rollins Research Building, Atlanta Georgia
y
Institute of Neuroscience, 1254 University of Oregon, Eugene, Oregon

Abstract
I. Introduction
II. Organization of the Zebrafish Intestinal Tract
III. Early Development of the ENS
IV. Genetic Approaches to Studying ENS Development
V. Molecular Mechanisms of ENS Development
VI. ENS Differentiation
VII. Regulation of Gut Motility
VIII. Zebrafish ENS as a Model for Understanding Human Diseases
IX. Future Prospects
Acknowledgments
References

Abstract
The enteric nervous system (ENS) is composed of neurons and glia that mod-
ulate many aspects of intestinal function. The ability to use both forward and
reverse genetic approaches and to visualize development in living embryos and
larvae has made zebrafish an attractive model in which to study mechanisms
underlying ENS development. In this chapter, we review the recent work describ-
ing the development and organization of the zebrafish ENS and how this relates to
intestinal motility. We also discuss the cellular, molecular, and genetic mechan-
isms that have been revealed by these studies and how they are providing new
insights into human ENS diseases.

METHODS IN CELL BIOLOGY, VOL 101 0091-679X/10 $35.00


Copyright 2011. Elsevier Inc. All rights reserved. 143 DOI 10.1016/B978-0-12-387036-0.00006-2
144 Iain Shepherd and Judith Eisen

I. Introduction
The enteric nervous system (ENS) is composed of neurons and glia that provide
intrinsic innervation to the intestinal tract and modulate functions such as motility,
homeostasis, and secretion (Burns and Pachnis, 2009; Furness, 2006). The ENS is
the largest division of the peripheral nervous system and is the only division able to
function independent of innervation from the central nervous system (Furness,
2006). Considering that as many as 40% of patients who visit medical practitioners
do so because they have intestinal malfunctions (Gershon, 1998), learning how the
ENS develops and functions is crucial for understanding human health and disease,
as well for understanding basic mechanisms of neural development.
The ENS is derived entirely from the neural crest (Le Douarin and Kalcheim, 1999).
Studies from many species have led to an understanding of the basic mechanisms of
neural crest formation (Sauka-Spengler and Bronner-Fraser, 2008), although it remains
unclear when enteric neural crest becomes specified. Many signaling pathways includ-
ing the BMP, FGF, WNT, and Notch signaling pathways are required for induction of
neural crest and specification of distinct subtypes of neural crest derivatives (Sauka-
Spengler and Bronner-Fraser, 2008). However, what specifies and guides ENS pre-
cursors to the intestinal tract is still unknown. Defined roles though have been estab-
lished for specific signaling pathways necessary for migration, proliferation, and
differentiation of enteric neural crest precursors after these cells have reached the
gut. These signaling pathways include the GDNF, Endothelin, BMP, Hedgehog (Hh),
Retinoic Acid, Notch, Semaphorin, and Netrin pathways (reviewed in Burzynski et al.,
2009). Several transcription factors have also been implicated in ENS development
including Mash1, Pax3, Phox2b, Hand2, Hox11a, and Hoxb5 (reviewed in
Burzynski et al., 2009). Although considerable work has been carried out on ENS
development, there are still many outstanding questions that remain unresolved. For
example, we still do not have a good understanding of how the lineages of specific
types of enteric neurons are specified, how enteric circuitry is established, or the extent
of ENS abnormalities that may result in human intestinal disorders (Gershon, 2010).
The ability to use both forward and reverse genetic approaches and to visualize
development in living embryos and larvae has made zebrafish an excellent model in
which to study mechanisms underlying ENS development and to gain insights into
diseases affecting the human ENS (Burzynski et al., 2009). Here we review studies
using zebrafish that have provided a new understanding of the cellular, molecular,
and genetic mechanisms underlying ENS development and the establishment of
intestinal motility. Many of these studies are also providing new insights into human
digestive tract diseases.

II. Organization of the Zebrafish Intestinal Tract


The intestinal tract, often referred to as the gut, is a complex organ composed of
several different cell types, including the gut epithelium, gut musculature,
6. Development of the Zebrafish Enteric Nervous System 145
[(Fig._1)TD$IG]

Fig. 1 Comparison of mammalian (A) and zebrafish (B) intestinal architecture. Reprinted from
Wallace et al. (2005) with permission from Elsevier.

vasculature, immune cells, and the ENS. The teleost gut is generally similar to that of
mammals, although it is somewhat simpler in overall architecture (Wallace et al.,
2005) (see Fig. 1). For example, in zebrafish, the esophagus connects directly to the
intestine without the presence of a stomach (Wallace et al., 2005). The beginning of
the intestine is defined by the insertion of the common hepatic–pancreatic duct and
by digestive enzymes characteristic of the intestine (Wallace and Pack, 2003). Even
though there is no stomach, the anterior embryonic intestine is enlarged and may
function as a food reservoir. This anterior intestinal region, known as the intestinal
bulb, also has retrograde motility that differs from the predominantly anterograde
propulsive motility observed in the posterior intestine (Holmberg et al., 2007). The
intestinal bulb also contains a few scattered goblet cells that produce both acid and
neutral mucins (Ng et al., 2005). Furthermore, the patterns of sox2, barx1, gata5, and
gata6 expression in the anterior zebrafish intestine resemble those of the developing
146 Iain Shepherd and Judith Eisen

mammalian stomach (Muncan et al., 2007) and may allow this section of the gut to
develop additional functions of storage and mixing of luminal contents. In addition
to lacking a stomach, the structure of the zebrafish gut epithelium is simpler than that
of amniotes. The zebrafish gut epithelium lacks crypts and is arranged in broad,
irregular folds rather than forming villi (Ng et al., 2005; Wallace et al., 2005).
Zebrafish also lack a submucosa layer found in the guts of amniotes
(Wallace et al., 2005) that contains a large amount of connective tissue and large
blood vessels and lymphatics. The vascular tissue in the zebrafish intestine is found
in the mucosa and the muscularis.
In addition to host cells, the vertebrate gut is home to a vast consortium of
microbial cells typically referred to as the gut microbiota (Cheesman and
Guillemin, 2007; Kanther and Rawls, 2010). The collective genome of the gut
microbiota exceeds that of the host by several orders of magnitude and encodes
critical digestive capacities absent from the host genome (Gill et al., 2006), suggest-
ing a crucial role for this coevolved community in intestinal development and
function. The role of the microbiota in gut development has been investigated by
rearing animals in a sterile environment and comparing them with conventionally
reared animals. The importance of the microbiota in gut development has been
revealed by studies in both mouse and zebrafish showing that in the absence of
gut microbiota, programs of gut epithelial cell type specification and maturation are
altered (Backhed et al., 2005; Bates et al., 2006). Interestingly, one aspect of zebra-
fish gut function that is affected by the absence of the microbiota is motility
(Bates et al., 2006), raising the possibility that the gut microbiota influence the
development of the ENS, although this has not yet been tested.

III. Early Development of the ENS


The zebrafish ENS, like that of all other vertebrates, is derived from the neural
crest (Kelsh and Eisen, 2000). In amniote vertebrates, both vagal and sacral neural
crest contribute to the ENS (Furness, 2006). In contrast, lineage studies in zebrafish
suggest that the entire ENS is derived from vagal neural crest (Shepherd, unpub-
lished data). Consistent with the simpler structure of the zebrafish gut compared
with amniotes, migration of neural crest cells along the zebrafish gut also appears
simpler than that in amniotes. For example, in amniotes, neural crest-derived ENS
precursors migrate caudally along the developing gut in multiple chains that follow
complex and unpredictable trajectories (Druckenbrod and Epstein, 2005; Young
et al., 2004). In zebrafish, neural crest-derived ENS precursors migrate from the
vagal region to the anterior end of the gut primordium, associate with it, and then
migrate as two parallel chains of cells along the length of the developing gut (Fig. 2)
(Elworthy et al., 2005; Olden et al., 2008; Shepherd et al., 2004). Subsequently, ENS
precursors migrate circumferentially around the gut and differentiate into enteric
neurons and glia. The final organization of the zebrafish ENS is also simpler than
that of amniotes, which have two distinct layers of enteric ganglia, the submucosal
6. Development of the Zebrafish Enteric Nervous System 147
[(Fig._2)TD$IG]

Fig. 2 Migration of neural crest cells into the zebrafish gut. (A-C) Ventral views of embryos with yolk
removed and anterior to left. Arrows indicate migrating enteric precursors. (A) Vagal region showing
crestin expression at 36 hpf. (B) Vagal region showing persistence of phox2b-expressing cells at the
anterior end of the gut at 48 hpf. (C) Somites 3–10 of 48 hpf embryo hybridized showing phox2b
expression. (D) Transverse section of phox2b expression in 48 hpf embryo at level of somite 8; broken
outline indicates border of gut endoderm. Modified from Shepherd et al. (2004) with permission of the
Company of Biologists.
148 Iain Shepherd and Judith Eisen

and the myenteric (Fig. 1). Zebrafish lack submucosal ganglia, and myenteric
neurons are typically arranged as single neurons or neuron pairs (Wallace et al.,
2005). How the pattern of ENS precursor migration is determined is currently
unknown, although recent studies in avians and zebrafish suggest that vascular
endothelial cells may be involved in this process (Nagy et al., 2009).

IV. Genetic Approaches to Studying ENS Development

Unbiased forward mutant screens provide an important methodology for identi-


fying genes involved in a process of interest (Grunwald and Eisen, 2002). Several
such screens have now revealed many genes involved in zebrafish ENS develop-
ment. colourless, one of the first ENS mutants to be isolated, results from a lesion in
the sox10 gene (Dutton et al., 2001; Kelsh and Eisen, 2000), which was already
known to be important in ENS development in mammals (Southard Smith et al.,
1998). Zebrafish screens have also identified other mutations in genes not previously
associated with ENS development (Chen et al., 1996; Schilling et al., 1996), for
example, the DNA polymerase delta 1 mutant flathead (pold1), the elys nucleopore
assembly protein mutant flotte lotte (ahctfi) and the rpc2, and RNA polymerase III
subunit mutant slim jim (polr3b) (Davuluri et al., 2008; de Jong-Curtain et al., 2008;
Plaster et al., 2006; Wallace et al., 2005; Yee et al., 2007). All of these mutants have
pleiotropic phenotypes and thus they were not identified based on ENS defects.
Nonetheless, these studies contribute to our knowledge about gut development and
offer the potential to help dissect the genetic basis of human intestinal disorders.
Two screens have specifically focused on identifying mutations affecting the ENS
(Kuhlman and Eisen, 2007; Pietsch et al., 2006). In both cases, mutants were
identified by a change in the number or distribution of enteric neurons as revealed
by labeling with an antibody to Elavl3 (previously known as HuC/D), a pan-neuronal
protein expressed in differentiated neurons. The screen conducted by Pietsch and
colleagues isolated 6 mutations and the screen conducted by Kuhlman and
Eisen isolated 13 mutations. The majority of mutants isolated in both screens
(15 of 19 mutants) have pleiotropic phenotypes; however, Kuhlman and Eisen
identified four mutants that appear to affect the ENS specifically. The majority
of mutants isolated in both screens (16 of 19 mutants) have a decrease in the number of
enteric neurons along the entire length of the intestine; however, Kuhlman and Eisen
identified three mutants in which enteric neurons are restricted to the anterior region
of the gut. Identifying the genes that underlie these mutant phenotypes should provide
important new insights into the molecular mechanisms involved in ENS development.
The genes responsible for two of the mutations identified in the screen conducted
by Pietsch and colleagues have now been determined. One of the mutations is an
allele of the previously identified DNA polymerase delta 1 mutant flathead
(Plaster et al., 2006). The other mutant, lessen, is a null mutation in the med24
(previously known as trap100) gene (Pietsch et al., 2006). Med24 is a component of
the mediator cotranscriptional activation complex previously shown to have an
6. Development of the Zebrafish Enteric Nervous System 149

essential role in mouse embryogenesis (Ito et al., 2002). The med24 null mutant
mouse has a placental defect that causes early embryonic lethality, thus no ENS
phenotype has been reported. The zebrafish med24 mutant ENS phenotype results
from a defect in ENS precursor proliferation after these cells reach the intestine.
Surprisingly, Med24 is not expressed in ENS precursors but instead is expressed in
the intestinal endoderm. Genetic chimera analysis revealed that wild-type endoderm
could rescue the mutant phenotype when transplanted into med24 mutants, suggest-
ing that an endoderm-derived or endoderm-regulated factor has an altered expres-
sion in med24 mutants, and that the change in expression of this factor is responsible
for the ENS phenotype (Pietsch et al., 2006).

V. Molecular Mechanisms of ENS Development


Despite developmental and architectural differences between the ENS of zebra-
fish and that of amniote vertebrates, most of the molecular mechanisms underlying
ENS development are conserved among species. For example, signaling through the
RET receptor tyrosine kinase is critical for ENS development in both amniotes and
zebrafish. ret mRNA is expressed in ENS precursors and differentiating neurons in
the developing zebrafish gut (Bisgrove et al., 1997; Marcos-Gutierrez et al., 1997;
Shepherd et al., 2004). As in other vertebrate species, zebrafish have two ret iso-
forms (ret9 and ret51), of which ret51 is unnecessary for complete colonization of
the gut by ENS precursors (Heanue and Pachnis, 2008). RET acts together with
GFRa, a member of the family of GPI-anchored cell surface receptors, to form a
receptor complex that mediates signals of the GDNF neurotrophic factor family
(Airaksinen and Saarma, 2002). The role Gfra in development of the zebrafish ENS
was tested using morpholino antisense oligonucleotides to create morphants lacking
one or more Gfra orthologues. Morpholino-mediated knockdown of the two ortho-
logues of Gfra1 results in complete loss of ENS neurons and their precursors
(Shepherd et al., 2004). However, morpholino-mediated knockdown of Gfra2 has
no apparent effect on ENS development, at least through the first few days of
development (Shepherd, unpublished data). Whether this gene functions later in
ENS development has not been addressed. Similar to knockdown of Ret and
Gfra1, knockdown of GDNF also results in complete loss of zebrafish ENS neurons
and their precursors (Shepherd et al., 2001). Two other GDNF family members,
Neurturin and Artemin, are reported to be present in zebrafish by immunoreactivity,
although whether they function in zebrafish ENS development is unknown (Lucini
et al., 2004, 2005). Interestingly, the Endothelin signaling pathway, which interacts
with the RET signaling pathway during the development of the ENS in mammals and
avians (Landman et al., 2007), may not be required for zebrafish ENS development
because a mutation that perturbs the function of the Endothelin receptor, Ednrb1,
does not result in an ENS phenotype in zebrafish (Parichy et al., 2000).
In addition to Ret signaling, the function of several transcription factors iden-
tified in mouse and avian as potential regulators of Ret expression has also been
150 Iain Shepherd and Judith Eisen

tested in zebrafish ENS development (Burzynski et al., 2009). Mutants lacking


Sox10 (Dutton et al., 2001; Kelsh and Eisen, 2000) or Foxd3 (Montero-Balaguer
et al., 2006; Stewart et al., 2006) lack an ENS, as do zebrafish in which Phox2b
(Elworthy et al., 2005) or Pax3 (Minchin and Hughes, 2008) has been knocked
down with morpholino antisense oligonucleotides. In addition to characterizing
animals in which genes have been mutated or transiently knocked down, the
regulatory regions of the ret, phox2b, and sox10 genes have been analyzed
(Antonellis et al., 2008; Dutton et al., 2008; Fisher et al., 2006; McGaughey
et al., 2008).

VI. ENS Differentiation


There are many different types of enteric neurons, including motor neurons,
interneurons, and intrinsic primary afferent (sensory) neurons. Each of these
classes can be further subdivided based on neuronal morphology, physiology,
and biochemistry. Extensive immunohistochemical analysis in amniotes has
revealed that each ENS neuron expresses several different neurotransmitters,
and the chemical coding hypothesis posits that the combinatorial expression of
these neurotransmitters can be used to functionally define each neuronal class
(Furness, 2006). Studies from a number of different laboratories indicate that,
similar to amniotes, both excitatory and inhibitory neurotransmitters are
expressed by neurons in the zebrafish ENS. Thus, enteric neurons expressing
pituitary adenylate cyclase-activating polypeptide (PACAP), vasoactive intestinal
polypeptide (VIP), calcitonin gene-related polypeptide (CGRP), nitric oxide
(NO), neurokinin-A (NKA), substance P, acetylcholine, and serotonin have all
been reported (Holmberg et al., 2003, 2004, 2006; Kuhlman and Eisen, 2007;
Olden et al., 2008; Olsson et al., 2008; Pietsch et al., 2006; Poon et al., 2003).
Knowledge of the precise chemical coding of the embryonic, larval, and adult
zebrafish ENS does not currently exist, nor is there a detailed description of the
morphologies of the different types of zebrafish enteric neurons. However, a
recent study has significantly advanced our understanding of chemical coding
in the zebrafish ENS by carefully detailing the proportions of neurons expressing
specific neurotransmitters, neurotransmitter synthetic enzymes, and downstream
signaling components and how these proportions change over time
(Uyttebroek et al., 2010). In addition, this study examined coexpression of several
different neurotransmitters, neurotransmitter synthetic enzymes, and downstream
signaling components (Fig. 3) including VIP, PACAP, neuronal nitric oxide
synthase (nNOS), choline acetyltransferase (ChAT), calbindin (CB), calretinin
(CR), and serotonin (Uyttebroek et al., 2010). The distribution and timing of the
appearance of these markers in the developing zebrafish ENS is comparable to
that seen in amniotes. This information, combined with a detailed analysis of
neuronal morphology, will shed considerable light on ENS circuitry in the
zebrafish.
6. Development of the Zebrafish Enteric Nervous System 151
[(Fig._3)TD$IG]

Fig. 3 Confocal images showing coexpression of neurochemical markers in adult zebrafish ENS.
(a–c) Colocalization of CB (a, green) and ChAT (b, magenta; c, merged). Arrowheads, neurons expressing
only CB. (d–f) Colocalization of CR (d, green) and nNOS (e, magenta; f, merged). (g–i) Colocalization of
nNOS (g, green) and ChAT (h, magenta; i, merged). O!A: From oral to aboral side of the intestine.
Modified from Uyttebroek et al. (2010) with permission of John Wiley and Sons, Inc. (For interpretation
of the references to color in this figure legend, the reader is referred to the web version of this book.)

VII. Regulation of Gut Motility

Intestinal motility arises from coordinated activity of three different cell popula-
tions, the ENS, interstitial cells of Cajal (ICCs), and gut smooth muscle (Furness,
2006; Rich et al., 2007). The ability to image motility directly in living zebrafish
larvae (Holmberg et al., 2003, 2004, 2006, 2007; Kuhlman and Eisen, 2007) has
revealed the complexity of gut motility patterns in vivo. The first spontaneous
contractions appear in the zebrafish gut at about 3.5 days postfertilization (dpf)
(Kuhlman and Eisen, 2007), well before the onset of feeding at 5–6 dpf
152 Iain Shepherd and Judith Eisen

(Olsson et al., 2008). The earliest contractions are focal, but begin to propagate along
the intestine by 4 dpf and the regularity of contractions increases over the next
several days (Holmberg et al., 2006, 2007; Kuhlman and Eisen, 2007). Before larvae
begin to feed, regular anterograde and retrograde contractions spread from a com-
mon point in the middle intestine. Retrograde contractions in the anterior intestine
may mix food, as a compensation for the absence of a stomach, whereas posterior
contractions appear to be primarily propulsive. The posterior most portion of the gut
also propagates short contraction waves in both directions (Holmberg et al., 2004).
ENS cells expressing neuronal markers are first detected at approximately 2 dpf in
the zebrafish intestine (Holmberg et al., 2003, 2006; Holmberg, 2003; Kelsh and
Eisen, 2000), which is prior to the onset of gut motility. The number of enteric
neurons increases rapidly over the next 3 days, a time during which gut smooth
muscle is also differentiating (Holmberg, 2003; Olden et al., 2008; Olsson et al.,
2008; Seiler et al., 2010; Wallace et al., 2005). Elegant work from the Holmberg lab
has shown that although excitatory cholinergic tonus and inhibitory nitergic tonus
are present in the zebrafish gut from the onset of feeding (Holmberg et al., 2004,
2006), the spontaneous gut contractions seen at 4 dpf are not initiated by intrinsic or
extrinsic neuronal innervation (Holmberg et al., 2007). Instead, the ENS has been
suggested to have an increasingly important role in modulating intestinal activity
late in development, after the onset of feeding behavior (Holmberg et al., 2007).
Consistent with this idea, recent work from the Pack lab provides evidence for
zebrafish intestinal motility in the absence of innervation (Davuluri et al., 2010).
Motility of the zebrafish gut in the absence of ENS function parallels the situation in
mouse (Anderson et al., 2004; Ward et al., 1999) and human (Huizinga et al., 2001)
in which gut motility can develop in the absence of the ENS. In fact, recent studies in
mouse also reveal that the earliest gut contractions are not mediated by the ENS
(Roberts et al., 2010). Despite this, and consistent with the idea that the ENS
modulates intestinal activity, the regularity of gut contractions in zebrafish appears
to roughly correlate with the number of neurons in gutwrencher mutants. These
mutants have a decreased number of ENS neurons along the entire length of the
intestine and there is an associated decrease in the regularity of gut contractions
(Kuhlman and Eisen, 2007) (Fig. 4). However, whether ICCs or gut smooth muscle
are also affected in this mutant has not been determined.
The initiation of spontaneous gut contractions in zebrafish could be mediated by
ICCs. ICCs prominently express the Kit receptor tyrosine kinase and are mesodermal
in origin (Lecoin et al., 1996), being derived from the same precursors as intestinal
smooth muscle cells (Young, 1999). ICCs generate pacing signals that drive contraction
of gut smooth muscle and coordinate neuronal input (Furness, 2006). This cell type is
present in zebrafish, although ICCs have not been detected at stages prior to 7 dpf using
Kit antibody staining (Rich et al., 2007), thus it is currently unknown precisely when
these cells develop in zebrafish. However, like the ENS, ICCs are dispensable for the
earliest gut motility in mouse, thus Young and her colleagues suggest that the earliest
gut contractions are likely to be myogenic (Roberts et al., 2010). Neither ICC activity
nor myogenic activity of the gut musculature has been studied in zebrafish, leaving
open the question of the origin of the earliest contractions. A more complete analysis of
6. Development of the Zebrafish Enteric Nervous System 153
[(Fig._4)TD$IG]

Fig. 4 gutwrencher mutants have fewer ENS neurons and less regular gut motility than wild types.
(A-B) Elavl staining of mid-intestine of 5.5 dpf wild type (A) and gutwrencher mutant (B) larvae reveals
that gutwrencher mutants have fewer enteric neurons. (C) Schematic showing correlation between the
number of enteric neurons and GI motility. Hatched arrows show spontaneous, focal contractions. Long
arrows show direction of contraction waves. Green dots represent enteric neurons. Modified from
Kuhlman and Eisen (2007) with permission of John Wiley and Sons, Inc. (For interpretation of the
references to color in this figure legend, the reader is referred to the web version of this book.)

ENS function will require developing techniques to record electrophysiologically from


ENS neurons, ICC cells, and intestinal muscles.

VIII. Zebrafish ENS as a Model for Understanding Human


Diseases
Functional disorders of the human intestine have an enormous impact on the
quality of life and extract a high societal cost (Gershon, 2010). Most prevalent
154 Iain Shepherd and Judith Eisen

among these and the focus of most research is Hirschsprung’s disease (HSCR), the
major human congenital disorder affecting the intestinal tract (Burzynski et al.,
2009; Gershon, 2010). HSCR pathology is characterized by the absence of enteric
ganglia in a distal segment of the gut, resulting in tonic contraction of the aganglionic
segment and intestinal obstruction. Only about 30% of HSCR cases are familial,
whereas about 70% of HSCR cases occur as an isolated trait. In the majority of
families exhibiting such nonsyndromic HSCR, disease transmission displays com-
plex inheritance patterns, thus nonsyndromic HSCR is assumed to be a multifacto-
rial disorder (Amiel et al., 2008; Badner et al., 1990). Studies from a number of
different groups have shown that mutations in the RET coding sequence account for
about 50% of familial and 15–20% of sporadic cases of HSCR (Burzynski et al.,
2009). Mutational analysis shows that genes that interact with RET, such as sox10,
gdnf, and phox2b, are also HSCR loci. Thus, studies of these genes in zebrafish, as
well as new genes identified in mutant screens, should provide important insights
into the mechanisms underlying this devastating human disease.
HSCR is also manifested as one phenotype of other diseases. For example,
patients with Mowat-Wilson syndrome exhibit HSCR as well as craniofacial dys-
morphology and other defects. Mutations in the gene encoding the Sip1 transcription
factor are implicated in Mowat-Wilson syndrome in humans. Recent work has
shown that morpholino-mediated knockdown of the two zebrafish orthologues of
human SIP1 results in loss of enteric precursors in the intestine and a HSCR-like
phenotype (Delalande et al., 2008), paving the way for future studies that will shed
more light on the mechanisms underlying this disease.
HSCR is also a part of the clinical phenotype of Goldberg–Shprintzen Syndrome
(GOSHS), another rare genetic disorder that is characterized by central nervous
system and ENS defects and craniofacial abnormalities. This syndrome is caused by
null mutations in Kif1-binding protein (KBP; also known as KIAA1279) – a protein
whose precise biological function is unknown (Brooks et al., 2006). Surprisingly, a
zebrafish kbp null mutant does not exhibit any of the clinical phenotypes associated
with the disease condition (Lyons et al., 2008). However, a recent study revealed that
KBP interacts with the stathmin-like protein Stmn2 (previously known as Scg10)
both in a yeast two-hybrid assay and in vivo. The yeast two-hybrid study used mouse
KBP to screen a mouse cDNA library; subsequently, the interaction between KBP
and Stmn2 was confirmed by immunoprecipitation. To determine whether kbp and
stmn2 interact genetically, an epistasis experiment was undertaken in zebrafish. This
experiment revealed an interaction between these two genes by morpholino-medi-
ated knockdown (Alves et al., 2010); aspects of the double morphant phenotype are
similar to the GOSHS clinical phenotype. This study in zebrafish provides new
insights into the cellular mechanisms underlying GOSHS by suggesting that the
central nervous system and ENS phenotypes of this disease result from disruption of
the normal microtubule dynamics in neurons requiring Stmn2.
In addition to enhancing our understanding of the known HSCR-related genes,
recent studies in zebrafish have provided novel insights into another disease, Bardet–
Biedl syndrome (BBS). BBS is a pleiotropic syndrome that combines facial
6. Development of the Zebrafish Enteric Nervous System 155

dysmorphogenesis and HSCR. Perturbing the function of zebrafish bbs8, one of the
genes associated with this syndrome results in craniofacial and ENS defects that
mirror those in human patients and mouse models and result from aberrant migration
of cranial neural crest cells (Tobin et al., 2008). Because noncanonical Wnt signaling
is known to regulate cranial neural crest migration (De Calisto et al., 2005), the
authors of this study knocked down Wnt signaling in zebrafish and found a neural
crest migration defect similar to that seen in bbs8 morphants (Tobin et al., 2008).
They also tested the potential involvement of Hh signaling, which is known to be
important for patterning neural crest cells during craniofacial development
(Tapadia et al., 2005). They found that Hh signaling was perturbed and they were
able to place bbs8 within the Hh signaling pathway (Tobin et al., 2008). In inde-
pendent studies, Hh signaling was also shown to be essential for ENS development
by acting as a potent mitogen for zebrafish ENS precursors (Reichenbach et al.,
2008) and to control neural crest cell proliferation and differentiation by modulating
responsiveness of cultured mouse neural crest cells to GDNF (Fu et al., 2004).
Together these studies provide important new insights into BBS by revealing a
potential link between the HSCR phenotype of BBS patients, Hh signaling, and
RET signaling.

IX. Future Prospects

ENS development is a complex process involving specification, proliferation,


migration, survival, and differentiation of neural crest cells and their derivatives,
which must finally form circuitry that regulates motility and other intestinal func-
tions. Studies in a variety of models including mammals, avians, and zebrafish have
provided an excellent base of knowledge for understanding these processes; how-
ever, many of the cellular and molecular mechanisms are as yet unresolved. The
conservation of mechanisms across species coupled with the ability to perform both
forward and reverse genetic screens, to follow development of individual cells or cell
populations in living embryos and larvae, and to characterize intestinal motility in
living animals have made zebrafish an attractive model in which to study basic
mechanisms of ENS development. These studies will certainly be enhanced by
learning more about zebrafish ENS circuitry.
Although HSCR has been the primary focus of research on human ENS-related
disorders, there are likely to be other, as yet unrecognized, disorders that cause
abnormal ENS development and result in abnormal ENS function (Gershon,
2010). At least some of these disorders may arise late in development as a result
of failure to elaborate the appropriate lineages of enteric neurons or glia (Gershon,
2010). The experimental tractability of zebrafish including the ability to follow cell
lineages in real time in living embryos and larvae has the potential to reveal cellular
and genetic interactions involved in these later processes.
As well as providing new information about the molecular underpinnings of
diseases affecting the human ENS, zebrafish may be useful to study physiological
156 Iain Shepherd and Judith Eisen

processes in real time. For example, a recently devised screen for intestinal motility
based on ingestion and clearing of fluorescent food (Field et al., 2008) should
enhance the ability to identify and characterize genes affecting gut motility.
Learning to record electrophysiologically from zebrafish ENS neurons in living
embryos and larvae will also provide a clear advance to understanding the mechan-
isms by which the ENS regulates intestinal function, and will provide an important
new methodology for characterizing ENS mutants. Motility and other aspects of
ENS and gut function are regulated by the neurotransmitter, serotonin. In addition to
being present in some ENS neurons, serotonin is produced by enterochromaffin cells
in the gut epithelium, and drugs that alter 5HT-4 and 5HT-3 serotonin receptors have
become major therapeutic agents for a variety of intestinal tract malfunctions,
including irritable bowel syndrome (Gershon and Tack, 2007). A recent study using
differential pulse voltammetry with implantable carbon-fiber microelectrodes to
monitor changes in serotonin levels in the zebrafish intestine in vivo (Njagi et al.,
2010) opens the door to studying serotonin actions in relation to location and
numbers of particular receptors in specific intestinal regions of living animals.
Finally, intestinal diseases such as irritable bowel syndrome and inflammatory bowel
disease alter gut motility and can also affect enteric neuron function (Furness, 2006;
Lomax et al., 2005). These diseases may result from dysregulation of the intestinal
microbiota (Baker et al., 2009; Cheesman and Guillemin, 2007; Furness, 2006;
Salonen et al., 2010); thus, it is crucial to understand how the microbiota influence
ENS function, and whether they also play a role in the later aspects of normal ENS
development or homeostasis.

Acknowledgments
Thanks to Robert Kelsh, David Raible and Kenneth Wallace for critical reading of manuscript drafts.
We thank our many colleagues for discussions of ENS development and apologize to any of them whose
work was inadvertantly left out of this review. Our original research on the ENS is supported by NIH
HD22486 (JE) and DK067285 (IS).

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CHAPTER 7

A Guide to Analysis of Cardiac Phenotypes


in the Zebrafish Embryo
Grant I. Miura and Deborah Yelon
Division of Biological Sciences, University of California, San Diego, La Jolla, California

Abstract
I. Introduction
II. Defects in Heart Size
III. Defects in Heart Shape
IV. Defects in Cardiac Function
V. Summary
References

Abstract
The zebrafish is an ideal model organism for investigating the molecular mechan-
isms underlying cardiogenesis, due to the powerful combination of optical access to
the embryonic heart and plentiful opportunities for genetic analysis. A continually
increasing number of studies are uncovering mutations, morpholinos, and small
molecules that cause striking cardiac defects and disrupt blood circulation in the
zebrafish embryo. Such defects can result from a wide variety of origins including
defects in the specification or differentiation of cardiac progenitor cells; errors in the
morphogenesis of the heart tube, the cardiac chambers, or the atrioventricular canal
or problems with establishing proper cardiac function. An extensive arsenal of
techniques is available to distinguish between these possibilities and thereby deci-
pher the roots of cardiac defects. In this chapter, we provide a guide to the exper-
imental strategies that are particularly effective for the characterization of cardiac
phenotypes in the zebrafish embryo.

I. Introduction
Cardiac birth defects are found in as many as 1 in 100 infants (Hoffman and
Kaplan, 2002). The prevalence of these defects provides a strong motivation for
METHODS IN CELL BIOLOGY, VOL 101 0091-679X/10 $35.00
Copyright 2011. Elsevier Inc. All rights reserved. 161 DOI 10.1016/B978-0-12-387036-0.00007-4
162 Grant I. Miura and Deborah Yelon

studies of the molecular mechanisms responsible for normal cardiac form and
function. Although it is suspected that many congenital heart diseases have a genetic
basis (Pierpont et al., 2007), only a modest number of genes have been identified as
disease loci (Bruneau, 2008; Ransom and Srivastava, 2007). Therefore, an increas-
ingly growing community of investigators is eager to employ effective methods for
determining the precise roles of essential genes during cardiogenesis.
In this regard, the zebrafish is an ideal model organism for studying the genetic
regulation of heart development (Schoenebeck and Yelon, 2007). The zebrafish
embryo is externally fertilized and transparent, allowing easy visualization of the
heart and its contractility. The simple architecture of the zebrafish heart also aids
analysis: it is composed of two major chambers, a ventricle and an atrium, each of
which contains an inner layer of endocardium and an outer layer of myocardium. The
particular characteristics of each cardiac tissue can be readily visualized with cel-
lular resolution, facilitating distinctions between normal and aberrant phenotypes. In
addition, since the zebrafish embryo does not require a functional cardiovascular
system for its survival (Pelster and Burggren, 1996), cardiac defects can be analyzed
in detail throughout embryogenesis.
To date, hundreds of studies have taken advantage of these benefits of the zebra-
fish for identifying crucial cardiac genes. Classical genetic screens have unearthed a
prodigious number of mutations causing a variety of defects in the embryonic
zebrafish heart (Alexander et al., 1998; Beis et al., 2005; Chen et al., 1996;
Stainier et al., 1996; Warren et al., 2000). Notably, genetic screens have been
particularly effective at identifying two large categories of mutant phenotypes:
mutations that disrupt the initial formation of the midline heart tube (Alexander
et al., 1998; Chen et al., 1996; Stainier et al., 1996) and mutations that disrupt
cardiac function (Chen et al., 1996; Stainier et al., 1996; Warren et al., 2000).
Additionally, knockdown of gene function via injection of gene-specific morpholi-
nos has been instrumental in revealing the functions of numerous cardiac genes (Bill
et al., 2009; Eisen and Smith, 2008). Small molecule screens have also contributed to
the identification of essential cardiac pathways (Kaufman et al., 2009; Peal et al.,
2010): for instance, several compounds have been shown to disrupt cardiac valve
morphogenesis (Scherz et al., 2008), and others have been shown to influence
cardiac action potential (Milan et al., 2003, 2009).
Whether caused by mutations, morpholinos, or small molecules, cardiac defects
typically manifest as pericardial edema and faulty blood circulation that are evident
by 48 h postfertilization (hpf), if not earlier. This common phenotype can result from
a wide variety of origins including defects in the specification or the differentiation
of cardiac progenitors (Fig. 1A,B); errors in the morphogenesis of the heart tube, the
cardiac chambers, or the atrioventricular canal (AVC) (Fig. 1C–E); or problems with
establishing proper cardiac function. Recent studies have pioneered a variety of
experimental strategies for distinguishing between these possibilities and thereby
deciphering the roots of cardiac defects. In this chapter, we provide a guide for
investigators who aspire to use state-of-the-art techniques to characterize cardiac
phenotypes in zebrafish embryos.
7. A Guide to Analysis of Cardiac Phenotypes in the Zebrafish Embryo 163

II. Defects in Heart Size


Heart size defects can readily be assessed in the zebrafish embryo: an enlarged
heart, a shrunken heart, or an apparent absence of cardiac tissue can easily be
detected even on a dissecting microscope (Chen et al., 1996; Stainier et al.,
1996). The perceptible size of the organ is a product of the number of cardio-
myocytes and the morphology and arrangement of those individual cells. This
section focuses on the possible causes of production of an inappropriate number
of cardiac cells.
To determine whether heart size defects are a function of cell number defects, it is
useful to employ a reporter transgene, Tg(cmlc2:DsRed2-nuc) (Mably et al., 2003),
that expresses DsRed in the nuclei of all differentiated cardiomyocytes. Nuclear
localization of the fluorescent signal facilitates counting of cardiomyocytes in indi-
vidual embryos; combining this technique with the use of an antibody recognizing an
atrium-specific myosin heavy chain (S46; anti-atrial myosin heavy chain (Amhc)
(Berdougo et al., 2003)) allows quantitative analysis of the number of cells in each
chamber (Schoenebeck et al., 2007). However, nuclear DsRed expression is most
robust and resolvable in embryos that are 48 hpf or older, since the DsRed protein
requires approximately 24 h to properly fold and localize after initial cardiac myosin
light chain 2 (cmlc2) promoter activation (Baird et al., 2000; Bevis and Glick, 2002;
Lepilina et al., 2006). To count cells at earlier stages, an alternate approach visua-
lizes cardiomyocyte nuclei with an antibody recognizing the transcription factor
Mef2, which is detectable in cardiomyocytes as early as 24 hpf (Targoff et al., 2008).
Increased or decreased numbers of cardiomyocytes could be the result of defects
in the specification of cardiac progenitor cells. Fate maps of the late blastula have
demonstrated that cardiac progenitors arise from bilateral multipotential zones
within the embryonic margin (Fig. 1A) (Keegan et al., 2004). During gastrulation,

[(Fig._1)TD$IG]

Fig. 1 Phases of heart formation in the zebrafish embryo. (A) Schematic lateral view, dorsal to the
right, of the late blastula. Ventricular (red) and atrial (yellow) myocardial progenitor cells are spatially
organized within bilateral marginal zones at this stage. (B–D) Schematic dorsal views, anterior up,
depicting the locations of ventricular (red) and atrial (yellow) cardiomyocytes during cardiac morpho-
genesis. (B) Myocardial progenitors migrate to form bilateral heart fields within the ALPM. (C) The
process of cardiac fusion recruits cardiomyocytes to the embryonic midline, where they form a cardiac
cone. (D) Continued migration of cardiomyocytes elongates the cardiac cone to create the heart tube. (E)
Schematic frontal view, anterior up, depicting the morphologically distinct ventricle (red) and atrium
(yellow) that are separated by a constriction at the atrioventricular canal following chamber emergence.
Adapted from Schoenebeck and Yelon (2007). (See Plate no. 9 in the Color Plate Section.)
164 Grant I. Miura and Deborah Yelon

the cardiac progenitors migrate to reside in bilateral fields in the anterior lateral plate
mesoderm (ALPM) (Fig. 1B) (Keegan et al., 2004; Schoenebeck et al., 2007).
Gastrula fate maps have shown that the dimensions of these heart fields correspond
well with the expression pattern of hand2 in the ALPM (Schoenebeck et al., 2007).
Differentiation of progenitors into cardiomyocytes begins around the 14 somite
stage, as indicated by the expression of myocardial markers such as cmlc2
(Yelon et al., 1999). Ventricular and atrial progenitors are spatially organized both
within the lateral margin and the ALPM (Fig. 1A,B) (Keegan et al., 2004;
Schoenebeck et al., 2007). Once they differentiate, ventricular and atrial cardiomyo-
cytes can be distinguished by molecular markers, such as ventricular myosin heavy
chain (vmhc) and amhc (Fig. 1C) (Berdougo et al., 2003; Yelon et al., 1999). Thus, a
failure to produce the right numbers of differentiated ventricular or atrial cardio-
myocytes could reflect an alteration in the number of cardiac progenitors selected
from within a multipotential zone, a change in the size of the heart fields within the
ALPM, or a modification of the effective production of differentiated cells by the
heart fields.
Fate mapping techniques can be employed to distinguish between these possibil-
ities. Construction of a fate map in mutant, morphant, or drug-treated embryos can
reveal alterations in the size of the cardiac progenitor pool, the organization of the
progenitors, and their productivity in terms of the number of cardiomyocytes gen-
erated (Keegan et al., 2004, 2005; Schoenebeck et al., 2007; Thomas et al., 2008;
Waxman et al., 2008). In these experiments, embryos are injected at the one-cell
stage with a caged fluorescein dextran lineage tracer that can subsequently be
photoactivated in selected cells using a laser (Keegan et al., 2004; Schoenebeck
et al., 2007). The locations of the labeled cells are noted, and their contributions to
the myocardium are assessed at later stages. In addition to determining whether the
progeny of selected cells become ventricular and/or atrial cardiomyocytes, it is
possible to count the number of cardiomyocytes derived from the labeled cells,
particularly when using anti-fluorescein immunohistochemistry to detect the lineage
tracer. This sensitive technique can resolve differences from the wild-type fate map
and thereby indicate the origin of a defect in heart size: cardiac progenitors might be
missing from their usual locations or might be found in atypical locations, ventric-
ular and atrial progenitors might be disorganized, and individual progenitors might
give rise to fewer or more cardiomyocytes than usual.
Several studies have utilized cell counting and fate mapping techniques to char-
acterize heart size defects and their origins in zebrafish embryos (Schoenebeck
et al., 2007; Thomas et al., 2008; Waxman et al., 2008). In one example, this
combination of experimental strategies was used to show that hedgehog (Hh) sig-
naling promotes the specification of cardiac progenitor cells (Thomas et al., 2008).
In zebrafish smoothened mutants, Hh signaling is disrupted by the loss of function of
the Smoothened receptor (Chen et al., 2001; Varga et al., 2001). These mutant
embryos exhibit misshapen hearts that appear smaller than their wild-type counter-
parts (Fig. 2A,B) (Thomas et al., 2008). Cell-counting experiments demonstrated
that the shrunken appearance of the smoothened mutant heart is a consequence of a
7. A Guide to Analysis of Cardiac Phenotypes in the Zebrafish Embryo 165
[(Fig._2)TD$IG]

Fig. 2 Hedgehog signaling promotes the specification of myocardial progenitor cells. (A, B) Lateral
views of wild-type (A) and smoothened mutant (B) hearts at 48 hpf. Immunofluorescence with MF20 and
S46 antibodies allows visualization of the ventricle (red) and atrium (yellow), both of which are misshapen
in smoothened mutants. (C) Quantification of cardiomyocyte number at 52 hpf in wild-type and smooth-
ened mutant hearts. Values represent the mean cell number ( standard deviation) for each category, and
asterisks indicate statistically significant differences from wild type. (D) Fate maps of myocardial pro-
genitors at 40% epiboly in wild-type and cyclopamine-treated embryos. Longitude coordinates of the first
tier of the embryonic margin are depicted with dorsal as the origin (0 ) and ventral as 180 . Each dot
represents an individual labeling experiment. Red dots represent embryos in which labeled blastomeres
contributed to ventricular myocardium, and black dots represent embryos in which the labeled cells did not
become cardiomyocytes. Adapted from Thomas et al. (2008). (See Plate no. 10 in the Color Plate Section.)

reduced number of cardiomyocytes in both the ventricle and atrium (Fig. 2C)
(Thomas et al., 2008). These results suggested that Hh signaling could modulate
chamber size either through regulating specification of cardiac progenitors or pro-
liferation of cardiomyocytes. Fate mapping in embryos treated with cyclopamine, a
166 Grant I. Miura and Deborah Yelon

Smoothened antagonist, detected cardiac progenitor cells in the lateral margin only
half as often as in wild-type control embryos (Fig. 2D), indicating inadequate
specification of the cardiac progenitor population (Thomas et al., 2008). In contrast,
the productivity of successfully specified progenitor cells remained normal in
cyclopamine-treated (CyA) embryos (4.6-labeled cardiomyocytes per experiment
in wild-type embryos and 4.5-labeled cardiomyocytes per experiment in cyclopa-
mine-treated embryos), indicating that Hh signaling is not essential for cardiomyo-
cyte proliferation at the stages examined (Thomas et al., 2008). These data, together
with additional results, led to the conclusion that Hh signaling plays an important
role in directing multipotential cells into the myocardial lineage.
In addition to resulting from altered progenitor specification or cardiomyocyte
production, defects in heart size could be a consequence of inappropriate timing of
myocardial differentiation. A recent study has shown that the zebrafish myocardium
forms during two distinct phases of myocardial differentiation (de Pater et al., 2009).
The first phase creates the primitive heart tube, beginning with the differentiation of
the ventricle and progressing to the atrium. During the second phase, a separate
population of late-differentiating cardiomyocytes forms the outflow tract at the
arterial pole of the ventricle (Fig. 3). Therefore, defects in the number of cardio-
myocytes could reflect ineffective execution of the timing of myocardial differen-
tiation; for example, loss of Fgf signaling has been shown to inhibit the second phase
of differentiation and thereby lead to an inadequate number of ventricular cardio-
myocytes (de Pater et al., 2009).
Two types of transgenic assays have been developed to monitor the timing of
myocardial differentiation in zebrafish embryos, and both can be effective for detect-
ing defects in timing. One strategy uses a pair of independent reporter transgenes
(de Pater et al., 2009): a transgene that expresses GFP in differentiated cardiomyo-
cytes under the control of the cmlc2 promoter, Tg(cmlc2:egfp) (Huang et al., 2003),
and a transgene that expresses DsRed under the control of the same promoter, either
Tg(cmlc2:DsRed2-nuc) (Mably et al., 2003) or Tg(cmlc2:dsRed) (Kikuchi et al.,
2010). This assay takes advantage of the difference in GFP and DsRed protein-folding
kinetics to distinguish early-differentiating and late-differentiating populations of
cells (de Pater et al., 2009; Lepilina et al., 2006). Since DsRed requires more time
to mature and fluoresce than does GFP, early-differentiating cardiomyocytes express
both GFP and DsRed at timepoints when the late-differentiating cells express only
GFP (de Pater et al., 2009). Thus, confocal imaging of DAPI-stained double trans-
genic embryos facilitates counting of both early-differentiated and late-differentiated
cardiomyocyte nuclei; for example, analysis of double transgenic embryos at 48 hpf
has been used to examine whether heart size defects are due to an altered number of
late-differentiating cells at the arterial pole of the ventricle (de Pater et al., 2009).
A second, complementary strategy uses a reporter transgene, Tg(cmlc2:kaede)
(de Pater et al., 2009), that expresses the green-to-red photoconvertible fluorescent
protein Kaede under the control of the cmlc2 promoter. Prior to photoconversion,
differentiated cardiomyocytes exhibit green fluorescence but not red fluorescence
(Fig. 3A,B). Upon exposure to UV light, the Kaede protein is cleaved and converts
7. A Guide to Analysis of Cardiac Phenotypes in the Zebrafish Embryo 167
[(Fig._3)TD$IG]

Fig. 3 Photoconversion of Kaede provides an assay for the timing of cardiomyocyte differentiation.
Lateral views of Tg(cmlc2:kaede) embryos, ventricle to the left. (A, B) Prior to photoconversion, the heart
exhibits green, but not red, Kaede fluorescence. (C, D) After photoconversion, green Kaede fluorescence
has been converted into red Kaede fluorescence. (E, F) Sixteen hours later, red Kaede fluorescence persists
in the cells that were expressing Tg(cmlc2:kaede) at 32 hpf. These cells also contain newly generated green
Kaede fluorescence. In contrast, newly differentiated cells in the outflow tract at the arterial pole (arrow)
exhibit only green, and not red, Kaede fluorescence. Adapted from de Pater et al. (2009).
168 Grant I. Miura and Deborah Yelon

from its green form to its red form (Fig. 3C,D). At later stages, any cells exhibiting
green fluorescence, but not red fluorescence, are interpreted as having initiated
differentiation after the time of photoconversion. For example, photoconversion of
embryos at 32 hpf, followed by the examination of fluorescence at 48 hpf, reveals
green fluorescent cardiomyocytes at the arterial pole of the ventricle (Fig. 3E,F).
Thus, this assay can be used to identify embryos with defects in the late differenti-
ation of arterial pole cardiomyocytes.

III. Defects in Heart Shape


In order for the heart to be an effective pump, the cardiac chambers and the
atrioventricular valve (AVV) need to acquire specific morphologies that are crucial
for their function. Because of the dynamic nature of cardiac morphogenesis, defects
in heart shape can have a variety of origins, ranging from complete failure of
cardiomyocyte migration at early stages to subtle displacement of atrioventricular
cushions at later stages. In this section, we discuss a series of experimental strategies
for determining the possible causes of a misshapen heart in a zebrafish embryo.
A number of zebrafish mutations have been shown to cause dramatic defects in
cardiomyocyte migration during early steps of heart morphogenesis (Kikuchi et al.,
2000, 2001; Kupperman et al., 2000; Reiter et al., 1999). Instead of exhibiting a single
heart at the midline, these embryos have a pair of separate hearts in bilateral positions,
a condition known as cardia bifida. During wild-type development, differentiated
cardiomyocytes move from their lateral positions in the ALPM toward the embryonic
midline, where they merge to assemble the heart tube through a process called cardiac
fusion (Fig. 1C) (Glickman and Yelon, 2002). In embryos with cardia bifida, cardiac
fusion fails because cardiomyocytes fail to migrate to the midline; this phenotype is
typically evident in the aberrant expression patterns of myocardial markers, such as
cmlc2, between 18 and 22 hpf. Analyses of cardia bifida mutants have highlighted two
different requirements for medial cardiomyocyte movement. One group of cardia
bifida mutations (e.g., casanova, bonnie and clyde, faust, miles apart) disrupt the
formation of the anterior endoderm that is adjacent to the migrating myocardium
(Alexander et al., 1999; Kikuchi et al., 2000, 2001; Kupperman et al., 2000; Reiter
et al., 1999), and a second group of mutations (e.g., natter, hands off) disrupt the
organization of the extracellular matrix (ECM) that surrounds the cardiomyocytes
(Garavito-Aguilar et al., 2010; Trinh and Stainier, 2004). Together, these phenotypes
indicate the importance of myocardium–endoderm and myocardium–ECM interac-
tions during cardiac fusion. Therefore, when investigating new cardia bifida pheno-
types, it is valuable to examine the specification and morphogenesis of the anterior
endoderm, using appropriate markers (e.g., Tg(-0.7her5:EGFP), axial, sox17
(Kawahara et al., 2009; Kupperman et al., 2000; Osborne et al., 2008; Tallafuss and
Bally-Cuif, 2003)), as well as the deposition and composition of the ECM, using
immunofluorescent detection of relevant components (e.g., fibronectin and laminin
(Arrington and Yost, 2009; Garavito-Aguilar et al., 2010; Trinh and Stainier, 2004)).
7. A Guide to Analysis of Cardiac Phenotypes in the Zebrafish Embryo 169
[(Fig._4)TD$IG]

Fig. 4 Tracking the paths of individual cardiomyocytes during cardiac fusion. Selected images from a
time-lapse of cardiac fusion in a wild-type embryo expressing Tg(cmlc2:egfp) between the 16 and 20
somite stages; dorsal views, anterior to the top. Arrows indicate paths traveled by individual cells from
their starting positions (A, C) to their ending positions (B, D). Red arrows indicate medial movement, and
yellow arrows indicate angular movement. (A, B) Cardiac fusion initiates with medial movement toward
the midline. (C, D) Cardiomyocytes in anterior and posterior regions and then transition into angular
patterns of movement that create the cardiac cone. Adapted from Holtzman et al. (2007). (See Plate no. 11
in the Color Plate Section.)

Subtle errors in cardiomyocyte movement do not necessarily result in a cardia


bifida phenotype but can still interfere with the shape of the heart tube. Time-
lapse confocal microscopy has been instrumental in elucidating the wild-type
patterns of directed cardiomyocyte movements that underlie heart tube dimen-
sions (Baker et al., 2008; de Campos-Baptista et al., 2008; Holtzman et al., 2007;
Smith et al., 2008). Wild-type cardiac fusion begins with medially directed move-
ments of the cardiomyocytes that recruit the bilateral populations toward each
other (Fig. 4A,B) (Holtzman et al., 2007). Then, anterior and posterior subsets of
cardiomyocytes change to an angular direction of movement in order to construct
a ring-like configuration of myocardium at the midline, referred to as the cardiac
cone (Figs. 1C and 4C,D) (Holtzman et al., 2007). Finally, the cardiac cone tilts to
the left, due to the differential leftward migration of cardiomyocytes: cells in the
posterior region of the cone exhibit a greater overall leftward displacement com-
pared with cells in the anterior region of the cone (Fig. 5) (Smith et al., 2008).
170 Grant I. Miura and Deborah Yelon

[(Fig._5)TD$IG]

Fig. 5 Patterns of cardiomyocyte movement during heart tube elongation. (A) Selected images from a
time-lapse of heart tube elongation in a wild-type embryo expressing Tg(cmlc2:egfp) beginning at the 23-
somite stage; dorsal views, anterior to the top. The cardiac cone was divided into four regions (I–IV), and
the movements of individual cardiomyocytes from each region were tracked. (B) Bar graphs indicate the
mean displacement rate and meandering index for tracked cardiomyocytes; error bars indicate standard
errors. Comparisons of the displacement rate (displacement/time) and meandering index (displacement/
track length) for each quadrant indicate that posterior cardiomyocytes exhibit higher rates of displacement
than anterior cardiomyocytes, whereas the meandering index is comparable for each subset of cardio-
myocytes. Adapted from Smith et al. (2008).

Continued leftward and anterior migration of cardiomyocytes completes the elon-


gation of the heart tube (Fig. 1D). Thus, heart tube morphology depends on the
coordination of carefully choreographed patterns of cell movement, and disrup-
tion of these patterns can result in defects in heart shape.
To distinguish whether a dysmorphic heart originates with problems during cardiac
fusion or tube elongation, it is sufficient to use myocardial markers, such as cmlc2,
vmhc, and amhc, at a series of timepoints between 16 and 30 hpf to identify the first
signs of abnormal cardiac morphology. Additional information about the cellular
mechanisms underlying morphological defects can come from examination of mar-
kers that exhibit apicobasal polarity in cardiomyocytes, such as aPKC, b-catenin, and
ZO-1 (Rohr et al., 2006; Trinh and Stainier, 2004); apicobasal polarity is disrupted by
mutations in heart and soul (has; prcki) and nagie oko (nok; mpp5) (Horne-
Badovinac et al., 2001; Peterson et al., 2001; Rohr et al., 2006), both of which block
heart tube elongation. Finally, to determine the specific type of cell movement defects
disrupting cardiac cone or heart tube formation, it is necessary to conduct appropriate
time-lapse analyses. Several studies have utilized the transgene Tg(cmlc2:egfp)
(Huang et al., 2003) to track individual cardiomyocyte movements through time-
lapse confocal microscopy (Baker et al., 2008; de Campos-Baptista et al., 2008;
Holtzman et al., 2007; Rohr et al., 2008; Smith et al., 2008). Such analyses can
ascertain multiple parameters related to cell movement including direction of cell
displacement (medial, angular, leftward, anterior, etc.), rate of cell displacement, and
degree of meandering (Figs. 4 and 5). For example, time-lapse analysis of
7. A Guide to Analysis of Cardiac Phenotypes in the Zebrafish Embryo 171

cardiomyocyte movements during cardiac fusion in cloche mutant embryos revealed


normal medial movement toward the midline but failure to execute angular move-
ments, resulting in a dysmorphic cardiac cone (Holtzman et al., 2007). Since cloche
mutants lack endocardium, these data suggested that myocardium–endocardium
interactions play an important role in directing cardiomyocyte behavior. In other
examples, reduction of BMP signaling and reduction of Nodal signaling were shown
to alter the direction, speed, and coherence of cardiomyocyte displacement during
tube elongation, demonstrating the importance of these signaling pathways for driv-
ing leftward cell movement (de Campos-Baptista et al., 2008; Smith et al., 2008).
Although many types of heart shape defects originate during heart tube assembly,
dysmorphic phenotypes can also appear during the stages of cardiac chamber emer-
gence. Between 24 and 48 hpf, the heart tube transforms into morphologically evident
cardiac chambers that exhibit distinct curvatures: a bulging concave curvature desig-
nated as the outer curvature (OC) and a recessed convex curvature called the inner
curvature (IC) (Fig. 1D,E) (Auman et al., 2007). Regional changes in cardiomyocyte
size and shape create the differences between the chamber curvatures. In the linear heart
tube (LHT), ventricular cardiomyocytes appear relatively small and round. During

[(Fig._6)TD$IG]

Fig. 6 Regionally confined cell shape changes underlie the emergence of chamber curvatures. (A–C)
Confocal projections of live hearts expressing Tg(cmlc2:egfp) with mosaic expression of Tg(cmlc2:
dsRed). Arrows point to representative cells expressing both dsRed and egfp. Ventricular cells in the
LHT at 28 hpf (A) and in the IC at 52 hpf (C) are relatively cuboidal, whereas cells in the OC at 52 hpf (B)
are flattened and elongated. (D, E) Bar graphs indicate the mean surface area and circularity index of cells
in the LHT, IC, and OC. Error bars indicate standard error, and asterisks indicate statistically significant
differences from LHT values. OC cells exhibit an increase in surface area with an accompanying decrease
in circularity. Adapted from Auman et al. (2007).
172 Grant I. Miura and Deborah Yelon

chamber emergence, cells in the ventricular OC become enlarged and elongated,


creating a bulging morphology, whereas cells in the ventricular IC retain a rounded
morphology and exhibit a smaller increase in size (Fig. 6) (Auman et al., 2007). Errors
in the execution of these cell shape changes can result in abnormal ventricular shape: a
compact and narrow ventricle if cells fail to expand normally or a dilated and round
ventricle if expansion is excessive (Auman et al., 2007).
To determine if a dysmorphic chamber phenotype is caused by defects in cardi-
omyocyte morphology, it is necessary to visualize and assess cardiomyocyte size and
shape using a marker that outlines cell boundaries (e.g., phalloidin or an anti-Dm-
grasp antibody) together with a myocardial reporter transgene like Tg(cmlc2:egfp)
(Auman et al., 2007; Deacon et al., 2010). Size is typically expressed in terms of
cardiomyocyte surface area, and shape is typically evaluated using a circularity
index that quantifies divergence from a perfectly circular morphology (Fig. 6D,
E). For example, morphometric analysis has shown that knockdown of the
microRNA mir-143 results in ventricular OC cells that are small and circular, having
failed to undergo the expansion and elongation seen in wild-type embryos; these
results indicate an important role of this microRNA in modulating the expression of
determinants of cardiomyocyte morphology (Deacon et al., 2010). When evaluating
the origins of chamber emergence defects, it is also important to keep in mind that
defects in cardiac function, such as reduced blood flow and/or reduced contractility,
can have a significant impact on cardiomyocyte morphology (Auman et al., 2007).
In this regard, it is valuable to consider whether an observed dysmorphic chamber
could be a secondary consequence of a primary defect in heart function.
In addition to being caused by defects in heart tube assembly and chamber
emergence, dysmorphic hearts can result from specific errors in the development
of the AVC, a constriction of the heart tube found at the boundary between the atrium
and the ventricle (Fig. 1E). In wild-type embryos, specification of the AVC is made
evident by the restricted expression of markers such as bmp4 and versican in the
myocardium and notch1b and has2 in the endocardium (Hurlstone et al., 2003;
Walsh and Stainier, 2001). As AVC differentiation proceeds, the adhesion molecule
Dm-grasp is detectable in the AVC endocardial cells, but not in the remainder of the
endocardium (Beis et al., 2005). This is accompanied by distinct cell morphology
changes in both the endocardium and myocardium: endocardial cells undergo tran-
sition from a squamous shape to a cuboidal shape, while myocardial cells undergo
apical constriction and extend their basolateral surfaces (Beis et al., 2005; Chi et al.,
2008a). The AVC endocardial cushions will subsequently be remodeled into the
leaflets of the AVV (Scherz et al., 2008).
Errors in AVC formation frequently lead to a failure of constriction at the atrio-
ventricular boundary that is morphologically apparent by 48 hpf. Additionally, AVC
defects often result in retrograde blood flow, visible as blood vacillating in between
the chambers (Beis et al., 2005; Stainier et al., 1996). To determine whether these
phenotypes indicate an error in AVC specification or differentiation, it is appropriate
to use the AVC molecular markers described above, as well as fluorescent reporter
transgenes (such as Tg(cmlc2:ras-gfp) and Tg(flk1:ras-cherry)) to mark myocardial
7. A Guide to Analysis of Cardiac Phenotypes in the Zebrafish Embryo 173

and endocardial cell borders and thereby visualize AVC cell shapes (Chi et al.,
2008a). For example, one group of mutations (e.g., jekyll, slipjig) have been shown
to disrupt the initial specification of the AVC; these mutants fail to exhibit restricted
expression of AVC molecular markers and fail to undergo AVC-specific myocardial
and endocardial cell shape changes (Chi et al., 2008a; Walsh and Stainier, 2001).
Conversely, adenomatous polyposis coli (apc) mutants fail to restrict AVC differ-
entiation to the proper location: the cardiac chambers in apc mutant embryos exhibit
expanded expression of AVC markers and excessive endocardial cushions that
extend beyond their normal boundaries (Hurlstone et al., 2003).
Subtle errors in valve function can cause retrograde blood flow even when initial
AVC specification and differentiation occur normally. The use of specialized fluo-
rescent microscopy techniques such as selective plane illumination microscopy
(SPIM), which can capture optical sections in thick samples rapidly and with
minimal photobleaching (Huisken and Stainier, 2009; Huisken et al., 2004), has
been instrumental in visualizing valve function defects in live embryos. SPIM
analysis of wild-type embryos expressing Tg(flk1:egfp) (Jin et al., 2005) in endo-
cardial cells and Tg(gata1:dsRed) (Traver et al., 2003) in erythrocytes has revealed
key features of AVV maturation and function (Fig. 7) (Scherz et al., 2008). At 55 hpf,
the AVV undergoes three phases of motion: myocardial contractions bring the sides
of the AVC together (Fig. 7A), then the juxtaposed sides of the endocardium engage

[(Fig._7)TD$IG]

Fig. 7 Time-lapse imaging of atrioventricular valve function during development. Selected images
from SPIM analysis of embryos expressing Tg(flk1:egfp) and Tg(gata1:dsRed) at 55 hpf (A–C) and
72 hpf (D–F), with accompanying schematics. (A–C) At 55 hpf, the AVC initially closes as a consequence
of myocardial contraction (A), then closes further through a rolling motion (B), and finally relaxes in
order to reopen (C). (D–F) Similar motions take place at 72 hpf, but the increased thickness of the AVC
endocardium occludes the lumen and prevents retrograde blood flow during the steps of rolling and
relaxation. Adapted from Scherz et al. (2008).
174 Grant I. Miura and Deborah Yelon

in a rolling motion (Fig. 7B), and, finally, the AVC relaxes (Fig. 7C). At this stage,
the relatively immature AVV is unable to prevent retrograde blood flow. Over time,
the increasing thickness of the AVV begins to occlude the AVC lumen, preventing
retrograde blood flow upon relaxation (Fig. 7D–F). Thus, examination via SPIM can
reveal regulators of specific aspects of AVV morphology and behavior. For example,
treatment of embryos with Cox2 inhibitors has been shown to displace the AVV
toward the ventricle and thereby interfere with AVV rolling and lumen occlusion,
indicating the importance of the Cox2 signaling pathway for the prevention of
retrograde blood flow (Scherz et al., 2008).

IV. Defects in Cardiac Function


The heart must regulate circulation efficiently with appropriately robust contrac-
tions triggered by propagated electrical impulses. Defects in cardiac function can
range from absence of heartbeat to subtle arrhythmia, and these problems can be
caused by abnormalities in the contractile apparatus or the conduction system. In this
section, we discuss several experimental techniques that are suitable for determining
the causes of defective heart function.
It is convenient to monitor heart function in the zebrafish embryo, since the con-
tracting heart and flowing blood are readily visible on a dissecting microscope. The
heart tube begins to contract by 24 hpf, and robust, serial contractions of the atrium
and ventricle are apparent by 48 hpf. Correspondingly, sarcomere assembly begins
within the primitive heart tube, and mature sarcomeres are organized by the time of
chamber emergence (Huang et al., 2009). The cardiac conduction system matures over
the same timeframe. Within the heart tube, electrical activity travels unidirectionally
and smoothly from the venous pole to the arterial pole (Chi et al., 2008b; Milan et al.,
2006). Once the cardiac chambers form, a cardiac conduction delay separates the atrial
and ventricular rhythms; this delay is a consequence of the formation of specialized
conduction tissue at the AVC (Fig. 8) (Chi et al., 2008b; Milan et al., 2006).
Qualitative assessment of heart function can rapidly discern the presence of severe
defects such as failure of chamber contraction or lack of blood flow through the
dorsal aorta. Quantitative methods can provide a more refined understanding of less
dramatic phenotypes. Assessment of heart rate is straightforward and can elucidate
even subtle alterations in the speed and rhythm of contraction. Additionally, degree
of contractility can be quantified using high-speed video microscopy to determine
ventricular fractional shortening, a measurement of systolic contractile function
normalized to the diameter of the heart (Fink et al., 2009; Rottbauer et al., 2005,
2006). Finally, dynamics of blood flow can be measured by tracking movement of
erythrocytes or fluorescent molecules introduced into circulation (Hove et al., 2003;
Schwerte and Pelster, 2000); alterations in the pace or pattern of blood flow can
provide a complementary indication of subtle aberrations in cardiac function.
To determine whether functional defects originate with contractile apparatus
abnormalities, it is effective to use transmission electron microscopy to inspect
7. A Guide to Analysis of Cardiac Phenotypes in the Zebrafish Embryo 175
[(Fig._8)TD$IG]

Fig. 8 Optical mapping of cardiac conduction. (A) Sequence of images depicts calcium activation
initiating in the sinus venosus (top) and concluding in the ventricle (bottom) of a live heart expressing Tg
(cmlc2:gCaMP) at 48 hpf. (B) Optical map of the pattern of calcium excitation shown in (A); isochronal
lines represent every 60 ms. Images 5–10 (A) and isochronal lines 5–10 (B) indicate conduction delay at
the AVC. Adapted from Chi et al. (2008).

sarcomere structure directly. Examination of sarcomere formation between 24 and


48 hpf can distinguish between errors in myofibril assembly and maintenance. For
example, in silent heart mutants, which lack the cardiac troponin T gene tnnt2,
myofibrils fail to assemble, as the thick filaments are disorganized in the absence of
176 Grant I. Miura and Deborah Yelon

normal thin filaments (Sehnert et al., 2002). In contrast, in pickwick mutants, which
lack titin, nascent myofibrils form normally but higher-order sarcomeric structures
are absent, suggesting a key role of Titin in sarcomere organization and maintenance
(Xu et al., 2002).
For heart function phenotypes that are not associated with defects in the contrac-
tile apparatus, it is logical to investigate whether cardiac conduction is aberrant.
Electrical currents can be assayed in vivo by electrocardiography, and similar patch
clamp techniques can be used to stimulate the heart to test its excitability
(Rottbauer et al., 2001). For optical mapping of cardiac conduction, calcium flux
can be monitored using fluorescent dyes (Ebert et al., 2005; Langenbacher et al.,
2005; Milan et al., 2006) or with a fluorescent calcium indicator transgene
(Tg(cmlc2:gCaMP)) (Chi et al., 2008b), and transmembrane action potential can
be evaluated using voltage-sensitive dyes (Pana´kova´ et al., 2010). This combination
of techniques can detect a wide variety of conduction abnormalities, ranging from
cell-intrinsic defects in calcium handling to failure to develop specific types of
conduction tissue. For example, the arrhythmia observed in tremblor (ncx1h)
mutants is caused by defects in calcium extrusion that result in elevated intracellular
calcium in cardiomyocytes (Ebert et al., 2005; Langenbacher et al., 2005). In a
different case, slipjig (foxn4) mutants fail to specify the AVC and therefore do not
develop specialized AVC conduction tissue; as a consequence, they exhibit an
absence of atrioventricular conduction delay (Chi et al., 2008a).

V. Summary
A wide variety of techniques are available to investigators seeking to determine the
origins of defects in heart size, shape, and function in the zebrafish embryo. Whereas
some of the applicable techniques require specific reporter transgenes or specialized
microscopes, most are readily accessible and should facilitate characterization of
cardiac phenotypes in a broad range of laboratories. Experimental strategies will
continue to evolve as more molecular markers, new transgenes, and advances in
microscopy enhance the resolution of our inspection of cardiac development. These
future prospects, together with the always increasing number of relevant mutations,
morpholinos, and small molecules, promise a strong continuation of the utility of the
zebrafish for illuminating the molecular mechanisms responsible for cardiogenesis.

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CHAPTER 8

Chemical Approaches to Angiogenesis


in Development and Regeneration
Sean Hasso and Joanne Chan
Vascular Biology Program and Department of Surgery, Children’s Hospital Boston, Harvard Medical School,
Boston, Massachusetts, USA

Abstract
I. Introduction
II. Chemical Approaches and Zebrafish Vascular Development
A. Conserved Vascular Construction Provides the Basis for Chemical Approaches
B. Analysis of Vascular Function in Larval and Adult Zebrafish
C. Chemical Library Screening and Analysis of Vascular Mutations
D. Chemical Treatments and Chemical Screening
E. Microangiography
F. Fluorescent Bead Assay for Vascular Permeability and Extravasation
G. Adult Angiography
III. Novel Chemical Strategies to Investigate Mechanisms of Angiogenesis
A. Chemical and Genetic Sensitization of Vascular Phenotypes
B. Effects of Hemodynamic Forces on Vascular Development
C. Role of Mural Cell Recruitment in Vessel Maturation
D. Novel Use of the Zebrafish Model to Address Pathogen–Host Vascular Effects
IV. Summary
Acknowledgments
References

Abstract
Research on blood vessel formation has provided a great deal of information
regarding both normal and pathological forms of angiogenesis during develop-
ment and in different disease states. Central to these studies is the role of the
vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs).
VEGF stimulation promotes the division, survival, and migration of endothelial
cells, and is necessary for the formation of blood and lymphatic vessels. The
conserved functions of the VEGF ligands and receptors from fish to mammals

METHODS IN CELL BIOLOGY, VOL 101 0091-679X/10 $35.00


Copyright 2011. Elsevier Inc. All rights reserved. 181 DOI 10.1016/B978-0-12-387036-0.00008-6
182 Sean Hasso and Joanne Chan

have allowed a near-seamless translation of a cellular and molecular mechanism


of vascular assembly between vertebrate models. An added advantage to the
conserved gene function is the ability to apply chemical approaches to modulate
zebrafish angiogenesis. In this chapter we will discuss current and potential future
uses of chemical strategies in the zebrafish model to further our understanding of
angiogenesis, lymphangiogenesis, and regenerative biology.

I. Introduction

Research on blood vessel formation over the last 30 years has generated a
wealth of information about both physiological and pathological forms of angio-
genesis (Folkman, 2007). The vascular endothelial growth factor (VEGF, referring
to VEGF-A) and its receptors (VEGFRs) play crucial roles in promoting endo-
thelial cell division, survival, and migration. These processes are essential to all
forms of blood vessel formation, including embryonic development, neovascular-
ization in regeneration and wound healing, as well as in pathological angiogenesis.
In the case of hyperactive vessels, such as those found in tumors or macular
degeneration, attacking blood vessels through blocking the VEGF signaling path-
way has proven successful (Chung et al., 2010). The first FDA-approved drug, an
antibody targeting the VEGF ligand (Bevacizumab/Avastin), has been used in
combination with chemotherapy for the treatment of colorectal cancer since 2004
(approved later for treatment of lung, brain and liver cancers). A similar anti-
VEGF antibody drug (Ranibizumab/Lucentis) is used to inhibit the aberrant
choroidal neovascularization that occludes central vision in the vascularized form
of macular degeneration. An alternate strategy to block this pathway is to use
small molecule inhibitors that block VEGF receptor (VEGFR) function. Of these
compounds, two have been approved for cancer treatment (Sorafenib/Nexavar;
Sunitinib/Sutent), while many others are under development (Zhang et al., 2009).
The fact that these small molecules inhibit the same targets in the zebrafish has
accelerated the use of chemical approaches in developmental studies. Although
the zebrafish is a relatively new animal model for angiogenesis research, the
conservation of cellular processes, angiogenic genes, and signaling pathways
across vertebrate biology has demonstrated its usefulness in defining the intricate
mechanisms involved in building a blood vessel. Added advantages of this con-
servation include the ability to use small molecules to investigate distinct events
during vascular development and the reciprocal use of zebrafish angiogenesis in
chemical library screening to identify active compounds. Like the terms for
forward and reverse genetics, chemical biology can be applied in a similar man-
ner: either in an unbiased chemical library screen or as a selective chemical probe
to dissect a particular pathway. Here we will review current studies and suggest
novel uses of chemical biology and genetics in the zebrafish model to investigate
blood and lymphatic vessel formation.
8. Chemical Approaches to Angiogenesis in Development and Regeneration 183

In our laboratory, we have used a reverse chemical approach to investigate the


VEGF signaling pathway in vivo, using a chemical VEGFR inhibitor, PTK787/
ZK222584 (PTK/ZK (Bayliss et al., 2006; Chan et al., 2002; Wood et al., 2000)).
In a study testing 20 kinase inhibitors profiled against 119 kinases, PTK/ZK was
found to be one of the most selective (Fabian et al., 2005). This small molecule
demonstrated robust effectiveness in blocking blood vessel formation in zebrafish
embryos (Chan et al., 2002), as well as in adults during regenerative angiogenesis
(Bayliss et al., 2006). Using this selective compound, we induced a receptor block-
ade to demonstrate that the downstream components of the VEGFR signaling
pathway such as PI3K-AKT and PLCg 1-MAPK are required for proper angiogen-
esis. In addition, we established the use of the adult zebrafish as a highly sensitive
model for chemical modulation of regenerative angiogenesis that can reveal genetic
sensitivities (Bayliss et al., 2006).

II. Chemical Approaches and Zebrafish Vascular Development


A. Conserved Vascular Construction Provides the Basis for Chemical Approaches
As we learn more about the development of the blood and lymphatic vessels in the
zebrafish, similarities to the mammalian system in endothelial cell processes,
genetic programs, and signaling pathways have been recognized (Chung et al.,
2010; Isogai et al., 2001, 2003; Kuchler et al., 2006; Swift and Weinstein, 2009;
Yaniv et al., 2006). These observations serve to illustrate the translational value of
data generated in the fish model and led to significantly increased use of the
zebrafish system for vascular biology studies (Alders et al., 2009; Hogan et al.,
2009a; Lieschke and Currie, 2007; Nicoli et al., 2010; Siekmann and Lawson, 2007).
Since detailed descriptions of blood and lymphatic vessel development have been
presented elsewhere (Ellertsdottir et al., 2010; Hogan et al., 2009a; Isogai et al.,
2001, 2003; Kuchler et al., 2006; Yaniv et al., 2006), a brief outline of the major steps
will be provided here to illustrate the potential future uses of a chemical approach.
The development of the zebrafish vasculature over the first 5 days postfertiliza-
tion (dpf) shares a number of common steps with other vertebrates: (1) ventral
mesodermal progenitors give rise to both blood and endothelial lineages; (2) angio-
blasts migrate from the lateral plate mesoderm to form the major axial vessels, the
aorta, and cardinal vein, through the process of vasculogenesis; blood flow is
established in these vessels by 24–26 h postfertilization (hpf); (3) angiogenic sprout-
ing from the dorsal aorta and posterior cardinal vein forms intersegmental or inter-
somitic vessels along the trunk that are mostly patent by 48 hpf; (4) differentiation of
venous endothelial cells initiates lymphangiogenesis, leading to the formation of the
thoracic duct by 5–6 dpf. While early developmental events are crucial to establish-
ment of the basal vascular network in zebrafish, they represent the first and simplest
forms of vasculogenesis, angiogenesis, and lymphangiogenesis. In adults, deregu-
lation of the key molecules governing these basic processes results in increased
vascular permeability, irregular vessel diameter, poor pericyte coverage, and
184 Sean Hasso and Joanne Chan

deregulated angiogenesis; all contribute to the poor perfusion and function of path-
ological vessels found in angiogenesis-dependent diseases such as cancer, diabetes,
and ischemic heart disease (Carmeliet, 2005; Chung et al., 2010).

B. Analysis of Vascular Function in Larval and Adult Zebrafish


In addition to embryonic angiogenesis, the zebrafish model can also be used to
investigate later vascular events during larval, juvenile, and adult stages. The avail-
ability of transgenic zebrafish lines with fluorescently labeled endothelial or blood
cells has provided a major advantage in the analysis of vascular function over the past
few years (e.g. endothelial-EGFP, endothelial-RFP; erythrocyte-RFP; platelet-
EGFP; myeloid-EGFP (Choi et al., 2007; Flores et al., 2010; Geudens et al.,
2010; Hall et al., 2007; Hogan et al., 2009b; Hsu et al., 2004; Jin et al., 2005;
Lawson and Weinstein, 2002; Traver et al., 2003); also see Fig. 1A, B). These lines
allow direct visualization of blood vessel formation and of hematopoietic and
myeloid cell movement throughout the first few days of development. Currently,
blood and lymphatic vessel development has been carefully defined from 1 to 7 dpf
by a number of groups (Geudens et al., 2010; Hogan et al., 2009b; Isogai et al., 2001,
2003; Kuchler et al., 2006; Yaniv et al., 2006). Recent focus has been placed on the

[(Fig._1)TD$IG]

Fig. 1 Zebrafish blood vessels in larval development and regenerated adult caudal fin. Lateral views
shown with anterior to the left in all panels. (A) Brightfield view of zebrafish at 3 dpf, HB, hindbrain;
heart and ear are indicated. (B) Vasculature of larval zebrafish at 4 dpf labeled by EGFP (Tg(fli1:EGFP)y1
(Lawson and Weinstein, 2002), PAV (parachordal vessel); PL (parachordal lymphangioblast), aorta or vein
are indicated (B0 ). (C) Zebrafish caudal fin at 5 day postamputation (dpa), in brightfield, endothelial-
EGFP, and angiography. Orange line indicates clip site, distal region is newly regenerated tissue, indicat-
ing vascular plexus formation and blood flow is shown by angiography with red fluorescent dye (boxed,
C0 , C00 ). D. High magnification view of fin rays showing that arteries are found within bony rays (D0 ) and
microvasculature within interray mesenchyme (D00 ). (For interpretation of the references to color in this
figure legend, the reader is referred to the web version of this book.)
8. Chemical Approaches to Angiogenesis in Development and Regeneration 185

development of lymphatic vessels from 2 to 7 dpf. In order to distinguish lymphatic


endothelial cells from patent blood vessels, the use of microangiography, lymphan-
giography, and transgenic lines has provided an efficient means to evaluate both the
development and function of the thoracic duct at the same time (Geudens et al.,
2010; Hogan et al., 2009b; Kuchler et al., 2006; Yaniv et al., 2006).
In adults, most of the endothelial specific transgenes remain active, and can be
used to observe a nascent vascular network during regeneration or repair, which
contain endothelial cells at various stages of vessel formation and lumenization.
Microangiography can also be performed to facilitate visualization of functional
blood flow (discussed below). The semitransparent caudal fin of the adult zebrafish
provides an accessible model to visualize regenerative and injury-induced angio-
genic processes. To observe pathological angiogenesis, within the adult tissues and
organs, one may use a transparent mutant line such as casper (roy–/–; nacre–/–
(White et al., 2008)) in an endothelial-specific transgenic background.

C. Chemical Library Screening and Analysis of Vascular Mutations


Chemical biology using the zebrafish model began with a simple visual analysis of
phenotypic changes after addition of chemical from a library to zebrafish embryos
(Peterson et al., 2000). In this historical screen, three embryos were placed into each
well of a 96-well plate, and individual compounds at 1 mM were added from a
chemical library of 1100 small molecules. This forward chemical genetic approach
was applied to identify chemical suppressors of the cardiovascular mutation, grid-
lock, which revealed a connection with the VEGF signaling pathway (Peterson et al.,
2004).
The recessive zebrafish gridlock mutation (grlm145/m145) was identified owing to
the lack of blood flow to the trunk of the embryo by 2 days of development
(Weinstein et al., 1995). Using a microangiography method, the blockade was
localized to the anterior trunk, where the paired lateral dorsal aortae normally merge
to form the single midline aorta. The gridlock phenotype resembles coarctation of
the aorta, a form of human congenital cardiovascular defect (Weinstein et al., 1995).
Positional cloning revealed that grl encodes a basic helix-loop-helix (bHLH) protein
belonging to the Hairy/Enhancer of the Split family. A point mutation in the zebra-
fish, grlm145, bypasses the stop codon and extends the predicted mutant protein
sequence by 44 amino acids (Zhong et al., 2000). These data are consistent with
the observation that some of the recessive mutants established collateral blood flow
and survive to adulthood (Peterson et al., 2004; Weinstein et al., 1995). Expression
of the grl gene in the lateral plate mesoderm is followed by arterial-specific gene
expression. Using Notch pathway reagents, a role for the Notch-grl/hry2 interaction
in formation of the dorsal aorta was demonstrated. Compromised grl function in the
grlm145/m145 hypomorph reduced the expression of arterial genes and enhanced
venous marker expression (Zhong et al., 2001, 2000).
In a suppressor screening strategy, Peterson et al. (2004) took advantage of the
survival of grlm145/m145 adult zebrafish to generate large numbers of recessive
186 Sean Hasso and Joanne Chan

embryos. Visual scoring for a suppressor effect depended on the chemical’s


ability to overcome the genetic defect and provide blood flow to the trunk and
tail. After screening 5000 compounds at 2 mg/ml, two structurally related
chemicals, GS4012 and GS3999, were shown to rescue the grlm145/m145 pheno-
type (Peterson et al., 2004). In particular, treatment time points from 12 to 24 hpf
were most effective, while chemical addition beyond the 24 hpf time point did
not result in rescue. To investigate the mechanism, several angioblast specifica-
tion genes (shh, grl, flt4, ephrinB2, and VEGF-A) were examined for changes in
mRNA expression level by quantitative RT-PCR (qPCR). This analysis revealed
that both compounds induce the expression of VEGF-A mRNA in a dose-depen-
dent manner in either wild-type or grl embryos. In addition, the authors dem-
onstrated that grlm145/m145 mutants expressed lower levels of VEGF-A mRNA, as
compared with wild-type controls. This study provided the first connection
between grl and the VEGF signaling pathway using an unbiased, forward chem-
ical screening strategy. Later, this group expanded their screen by another 7000
compounds to identify one that is structurally related to known PI3K inhibitors
(Hong et al., 2006). In the past few years, refined versions of chemical screening
strategies have provided insights into vertebrate biology, such as the role of
prostaglandins in hematopoietic stem cell renewal (North et al., 2007).
Chemical modification of the rest versus awake states has been examined using
automated video monitoring across wells of zebrafish larvae (Rihel et al., 2010).
The diverse application of this simple, unbiased strategy has revealed a number
of novel biological interactions.

D. Chemical Treatments and Chemical Screening


Key to the identification of compounds that reversed the gridlock phenotype was
the time and dose of chemical administration. Small molecule inhibitors are typi-
cally added to embryo or fish medium at 100 nM to 10 mM in our laboratory,
depending on the solubility of the inhibitor. For poorly soluble inhibitors, we gen-
erally refresh the drug treatment every 24 h. Additionally, we have found that adult
zebrafish regenerative angiogenesis is highly sensitive to the VEGFR inhibitor
PTK787/ZK222584 (PTK/ZK), which gives partial inhibition at 100 nM;
(Bayliss et al., 2006). By contrast, the embryonic vascular response is less sensitive
to these compounds (Chan et al., 2002). It is important to note that adult zebrafish
require a larger volume of solution to minimize distress. Typically, we administer
compounds to adults using at least 1 liter of fish water (Bayliss et al., 2006).
Furthermore, we have found pulsed administration of a chemical is an effective
method to target a narrow window of vascular development for analysis. In these
experiments, chemical inhibitors are added to embryo medium (in E3 medium
(5 mM NaCl, 0.17 mM KCl, 0.4 mM CaCl2, 0.16 mM MgSO4)) for a pulse of 6 h
and then washed out three times with embryo medium, followed by a recovery period
before examination of vascular function (Bayliss et al., 2006; Bolcome and Chan,
2010; Bolcome et al., 2008; Chan et al., 2002).
8. Chemical Approaches to Angiogenesis in Development and Regeneration 187

Chemical library screening is an unbiased approach based on the treatment of


whole embryos or larval zebrafish arrayed in wells and exposed to chemical com-
pounds. Recent reports have described how to perform chemical library screening on
zebrafish embryos or larvae in different arrays, with an increased ability for auto-
mation (e.g. Clifton et al., 2010; North et al., 2007; Paik et al., 2010; Peal et al.,
2010; Rihel et al., 2010; Stern et al., 2005; Zon and Peterson, 2005). However, they
share these essential components: a fish assay for a specific readout and a small
molecule chemical library. The fish assay has ranged from visual inspection of wild-
type fish, to suppression of a particular genetic mutation or cell type (visualized by
GFP, by in situ hybridization, or by antibody staining). The use of automated video
capture or staining equipment has helped zebrafish investigators to screen more
compounds in a shorter time. The wells where embryonic phenotypes are altered
by the presence of chemical(s) are scored as positive. Positive and negative controls
(e.g., known chemical effector, no chemical treatment, DMSO carrier) are distrib-
uted randomly in wells on the plate so that the person scoring the phenotype is blind
to treatment identity. A number of chemical compound libraries are available com-
mercially or through the NIH. Typically, a compound is added to a well of 3–5
embryos at 1–30 mM, or 2.5–10 mg/ml, in embryo medium (E3 medium) in a 96-
well or 384-well plate format. It is also possible to add a number of compounds to the
same well, identify the combination that induced a phenotypic change, then refine
the secondary screening to determine the active compound, the concentration
required and the timing of chemical effectiveness (Clifton et al., 2010; North
et al., 2007; Paik et al., 2010; Peal et al., 2010; Rihel et al., 2010; Stern et al.,
2005; Zon and Peterson, 2005).
In the adult zebrafish, the caudal or tail fin is routinely clipped for genotype
analysis and for regeneration studies. The amputated fin regenerates all of its cell
types (bone precursors, blood vessels, nerves, pigment and skin) while continuing to
function. We took advantage of this regenerative capacity to examine adult angio-
genesis under chemical modulation (Bayliss et al., 2006). We noted that chemical
treatment with the VEGFR inhibitor, PTK/ZK, blocked regenerative angiogenesis at
submicromolar levels, demonstrating that this adult tissue is more sensitive than the
embryo to VEGF pathway inhibition. Nanomolar concentrations of the inhibitor
(100 nM for partial or 500 nM for complete blockade of regenerative angiogenesis)
are effective, which approaches the sensitivity observed in cell culture experiments
(Chan et al., 2002). Using this inhibitor, we demonstrated an angiogenic limit to
tissue regeneration (1 mm) without an appreciable effect on the cellular differen-
tiation events required for the regenerative process. We then investigated whether the
effects of chemical inhibition would be enhanced in three embryonic vascular
mutants: PLCg 1y10 (Lawson et al., 2003), grlm145 (Weinstein et al., 1995), and
cloche (clom39 (Stainier et al., 1995)). In these experiments, the tail fin is clipped
and allowed to regenerate over the next 3 days in the absence or the presence of
partial VEGFR inhibition. Using an adult angiography technique, we determined the
amount of vessel function in the newly regenerated fin tissue. We found that both
PLCg 1y10/+ and grlm145/m145 adults displayed an increased sensitivity to the VEGFR
188 Sean Hasso and Joanne Chan

inhibitor, while the clom39/+ heterozygous adults behave in a manner similar to wild-
type controls. Therefore, this assay shows that genetic differences can be revealed in
an adult zebrafish, either in the heterozygous or homozygous states, facilitating
future studies to examine the genetic component of the angiogenic response
(Bayliss et al., 2006).

E. Microangiography
Blood flow during the first week of zebrafish vascular development can easily be
visualized using a dissecting microscope. The microangiography technique intro-
duces an inert fluorescent reagent into the circulation to visualize areas of functional
blood flow for recording and analysis. This method was originally described by
Weinstein et al. (1995), who injected a fluorescent agent into the zebrafish vascu-
lature ventrally through the sinus venosus. Our laboratory has since modified this
technique to increase throughput, allowing the injection of 100–200 embryos
(48 hpf) in 1 h (Bolcome et al., 2008). Using an endothelially labeled transgenic
zebrafish background, endothelial cell biology and blood flow can be evaluated at
the same time. In addition to inert fluorescent dyes, sized microspheres and large
proteins may also be delivered in this manner (Bayliss et al., 2006; Bolcome and
Chan, 2010; Bolcome et al., 2008). Typically, injected reagents are mixed with
phenol red (0.05%) to monitor fluid delivery during the microinjection. Zebrafish
embryos at 48 hpf are anesthetized with 0.02% tricaine (Tricaine Methane Sulfonate,
MS-222, Sigma, A5040) and arranged dorsal side-up in rows on agarose ramps.
Volumes of 40 nl are microinjected directly into the duct of Cuvier (also known as
the common cardinal vein) using a gas driven microinjector (Medical Systems
Corp.). Following injection, embryos are transferred into fresh embryo medium
for recovery. Beyond the first 3 days of development, and throughout adulthood,
microangiography should be performed through direct injection into the heart. For
these stages, we have observed high reproducibility and fish survival.

F. Fluorescent Bead Assay for Vascular Permeability and Extravasation


The use of sized fluorescent microspheres in microangiography can provide
information about vessel size and extravasation. We have developed these techni-
ques to examine changes in vascular leakage (Bolcome and Chan, 2010; Bolcome
et al., 2008). Microsphere mixtures are prepared before microinjection using the
microangiography technique. Typically, we inject a mixture of two different bead
diameters, for example, 100 nm (diameter) blue and 500 nm red fluorescent bead
suspensions (Duke Scientific Corp.) to determine whether disruptions of endothelial
cell junctions are large enough for bead extravasation. We have mostly performed
these injections at 48–72 hpf. For fluorescence microscopy of bead extravasation,
embryos were anesthetized with tricaine (Sigma), and then mounted in 4% methyl-
cellulose for photography.
8. Chemical Approaches to Angiogenesis in Development and Regeneration 189

G. Adult Angiography
To evaluate regenerative angiogenesis in adult zebrafish, we have developed an
angiography assay in a transgenic zebrafish line Tg(fli1:EGFP)y1 (Lawson and
Weinstein, 2002), so that endothelial cell growth and migration, as well as blood
flow, can be assessed at the same time (Bayliss et al., 2006). By 3 days postcaudal fin
amputation, a network of endothelial cells, or a vascular plexus, is formed in the
newly regenerated fin tissue (Fig. 1C, D). At this point, new blood vessels are being
formed but not all endothelial cells labeled by EGFP contribute to patent vessels
(Fig. 1C; (Bayliss et al., 2006)). The use of angiography facilitates this distinction.
For these experiments, we typically use needles of the same size as those used for
one-cell stage embryo injection. However, the needle is broken at a higher point to
create an opening of 30 nm. Adult zebrafish are anesthetized with 0.02% tricaine
for 2–4 min. The fish are gently positioned ventral side up on a slotted sponge using a
plastic disposable spoon. A small incision is made at the midline, just above the heart
and ventral to the gills. Approximately 5 ml of a fluorescent agent is delivered
directly into the heart using standard microinjection equipment. Fluorescent agents
we have used include lectins or a 70 kD dextran, conjugated to Texas Red. The fish
are then placed under a fluorescent microscope to assess functional blood vessels,
and then immediately placed into a recovery tank. Ideally, the entire procedure
should take 5–10 min with all zebrafish recovering within 2–3 min.

III. Novel Chemical Strategies to Investigate Mechanisms of


Angiogenesis

The zebrafish is a versatile vertebrate model organism amenable to both forward


genetic and chemical library screens (Lieschke and Currie, 2007). In this section, we
will discuss possible next steps in applying a chemical approach in the zebrafish
model to investigate vascular biology using intact blood vessels.

A. Chemical and Genetic Sensitization of Vascular Phenotypes


Classical forward genetic screens rely on phenotypic identification of homozy-
gous mutants in the F3 generation. Recently, the use of chemical sensitization in
combination with genetics has been reported for a myocardial repolarization screen
(Milan et al., 2009) as well as a senescence screen (Kishi et al., 2008), where the use
of insertional zebrafish mutants provided rapid identification of the genetic lesions
that validated the screening strategy. For an angiogenic screen, we propose that the
use of selective chemical agents, such as a VEGFR inhibitor, can facilitate the
identification of suppressor or enhancer mutations in heterozygotes of the F2 gen-
eration. The utility of this chemically assisted enhancer or suppressor screening is
that it can be a tool: (1) to identify polymorphisms or other mutations which alter
drug effects such as loss of binding to its target or (2) to identify other factors acting
190 Sean Hasso and Joanne Chan

either upstream or downstream of the drug target or in its signaling pathway. Using
selective inhibitors to enhance a vascular effect, subtle changes in protein function
may be revealed. For example, the role of PLCg 1 downstream of VEGFR signaling
has been demonstrated in zebrafish mutants (Lawson et al., 2003; Rottbauer et al.,
2005), correlating with its function in the mouse model, as an essential effector for
VEGFR2/flk-1 signaling (Sakurai et al., 2005; Takahashi et al., 2001). However,
since increased sensitivity to chemical VEGFR inhibition can be revealed in het-
erozygous adult zebrafish (Bayliss et al., 2006), it is likely that this type of mutation
can be revealed through a combined chemical and genetic screening strategy.
A chemically assisted suppressor/enhancer genetic screen is performed as an alter-
native to the traditional ENU mutagenesis screen. Protocols for ENU treatment and
establishment of the F1 generation can be found elsewhere (Haffter et al., 1996).
Following these initial steps, mutagenized F1 fish are outcrossed to wild-type fish
generating the heterozygous F2 population. To circumvent problems arising from a
maternal effect, only males from the F1 generation should be used. Pairwise matings of
mutagenized F1 males to wild-type females can be performed. Each clutch is treated
with a chemical agent of choice. The exact timing and concentration of the chemical
agent should be determined in advance of screening to determine the appropriate
concentration in which the phenotype is most distinct but causes the least lethality.
Embryos should be monitored both for the appearance of the normal phenotype
induced by drug treatment and embryos which show an enhancement/suppression of
the phenotype. Each F1 adult is screened three times, and the embryos from the third
clutch would be raised to adulthood. Photographic documentation should be generated
for each specimen as a reference for comparison when following the phenotype over
successive generations. Once an acceptable number of genomes have been screened,
and a worthwhile number of potential mutants have been identified, phenotypic
characterization and standard positional cloning techniques can begin for each line.

B. Effects of Hemodynamic Forces on Vascular Development


The role of blood flow in vascular remodeling has been observed for many years;
however, the precise molecular mechanisms are largely unknown. A recent study
demonstrated a synergistic role for mechanical forces and VEGF signaling in the
remodeling of aortic arch blood vessels (Nicoli et al., 2010). In this study, the authors
demonstrated that blood flow induced the expression of the mechanosensitive tran-
scription factor, klf2a, which acts upstream of an endothelial-specific microRNA,
mir-126, to stimulate VEGF-A mRNA expression (Nicoli et al., 2010). To investigate
this connection further, we suggest that the adult zebrafish tail fin model could
provide an additional model that is responsive to chemical biology and accessible to
qPCR analysis (Bayliss et al., 2006). We have determined that angiogenesis in this
newly regenerated tissue proceeds through blastemal and fin tip VEGF ligand
expression and the recruitment of endothelial cells. Revascularization of this tissue
occurs under active blood flow, is sensitive to VEGFR inhibitors, and is reduced in
genetic lines with mutations in VEGF pathway genes (Bayliss et al., 2006). Thus, by
8. Chemical Approaches to Angiogenesis in Development and Regeneration 191

combining chemical biology with expression analysis, it may be possible to identify


candidate genes or microRNAs that play important roles in the remodeling of the
nascent vascular plexus into functional blood vessels.

C. Role of Mural Cell Recruitment in Vessel Maturation


An emerging area of vascular biology research is the role of perivascular support
cells (mural cells) in blood vessel function. These include both vascular smooth
muscle cells and pericytes; both have contractile ability and function to support
needs of the endothelial cell within a blood vessel. Vascular smooth muscle cells
wrap around large arteries, while pericytes are found adjacent to smaller diameter
vessels (Cleaver and Melton, 2003). In mammals, these cells have been difficult to
define due to the requirement for coexpression for a number of markers (Gerhardt
and Betsholtz, 2003). The search for mural cells in the zebrafish model began with
identification of a vascular smooth muscle actin gene, sm22a. In situ RNA staining
using this marker showed that it is highly expressed in intestinal smooth muscle
(Georgijevic et al., 2007). In addition, vascular smooth muscle cells have been
shown to express sm22a in the adult zebrafish tail fin, with higher levels around
the artery than vein (Bayliss et al., 2006) as has been reported for mammalian vessels
(Cleaver and Melton, 2003).
Recently, a detailed expression study suggests that sm22a (also known as trans-
gelin) is expressed in the developing embryonic vessels of the zebrafish embryo
(Santoro et al., 2009). Using ultrastructural analysis of adult and larval zebrafish,
Santoro et al. (2009) tracked the progression of perivascular cells. They also devel-
oped a zebrafish-specific antibody against Transgelin to localize the protein in the
cellular environment near blood vessels. With these tools, we suggest that a chemical
approach could be developed to examine the roles of PDGF (platelet-derived growth
factor) and VEGF signaling in mural cell development using both larval and adult
zebrafish and selective chemical inhibitors for PDGFR (e.g. Murphy et al. (2010))
and VEGFR (e.g. PTK/ZK). This chemical approach should provide significant
insights into the role of endothelial-pericyte interaction, as proposed in a previous
study where endothelial cells restrict pericyte proliferation (Greenberg et al., 2008).

D. Novel Use of the Zebrafish Model to Address Pathogen–Host Vascular Effects


Our laboratory has begun to investigate the potential use of the zebrafish as a
surrogate host to dissect the signaling pathways affected by the lethal anthrax toxin.
Anthrax infections lead to cold-like symptoms that can be easily dismissed. Within a
week of these symptoms, anthrax toxin proteins are released into the host blood
stream, leading to widespread damage in a number of organ systems (Young and
Collier, 2007). Vascular delivery of purified anthrax toxin induces rapid death in the
Fischer rat model accompanied by pulmonary edema and pleural effusions, symp-
toms typical of the human disease (Beall et al., 1962). We have begun to develop a
192 Sean Hasso and Joanne Chan

zebrafish model to examine the host pathways disrupted by this toxin based upon
studies showing that host entry receptors are expressed during blood vessel forma-
tion, ANTXR1/TEM8 (tumor endothelial factor 8) and ANTXR2/CMG2 (capillary
morphogenesis gene 2) (Young and Collier, 2007). Thus, we designed a vascular
delivery method for the toxin proteins by modification of the microangiography
technique in the zebrafish at 48 hpf (Bolcome et al., 2008).
Interestingly, the zebrafish vascular model for anthrax lethal toxin entry demon-
strated conserved host cell components. Using mutant or fusion toxins, we demon-
strated that the enzymatic activity of lethal toxin is responsible for the induction of
vascular leakage (Bolcome et al., 2008). Since lethal toxin has the ability to cleave
the MEK/MKK family of related kinases (Duesbery et al., 1998), we used chemical
biology to demonstrate that inactivation of the MEK1/2 pathway was sufficient to
induce vascular leakage in the zebrafish model. We have also shown increased
vascular permeability through the use of a sized fluorescent microsphere extrava-
sation assay (Bolcome et al., 2008). Recently, we have developed transgenic zebra-
fish models of regulated MEK pathway activation and confirmed these findings
(Bolcome and Chan, 2010). We showed that anthrax lethal toxin-induced vascular
leakage can be prevented by using a transgene that activates the MEK1/2 signaling
pathway (Bolcome and Chan, 2010). These studies demonstrate another novel use of
the zebrafish model to interrogate a human disease-relevant question.

IV. Summary
The zebrafish is an ideal model for vascular biology studies owing to the acces-
sibility of chemical and genetic analyses on intact blood and lymphatic vessels. For
the past 15 years, zebrafish researchers have demonstrated conservation in gene
usage and cellular mechanisms in building blood vessels. The creative use of chem-
ical biology in the zebrafish model will continue to contribute to the basic biology of
angiogenesis and reveal common mechanisms utilized in mammalian models and in
human diseases. Thus, the chemical approach to angiogenesis in the zebrafish model
provides a unique system to examine fundamental questions in vascular biology.

Acknowledgments
We thank Daniel Reed and Kim Bellavance for help in the preparation of figure panels. We acknowl-
edge support from the Department of Defense, Grant #TS093079 and the Manton Foundation for Orphan
Disease Research (JC).

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CHAPTER 9

Laser-Induced Thrombosis in Zebrafish


Pudur Jagadeeswaran, Maira Carrillo, Uvaraj P. Radhakrishnan,
Surendra K. Rajpurohit and Seongcheol Kim
Department of Biological Sciences, University of North Texas, Denton, Texas, USA

Abstract
I. Introduction
II. Vascular Occlusion
III. Methods
A. Laser setup
B. Preparation of larvae for laser thrombosis
C. Laser ablations
D. Normal values and larval stages in laser thrombosis
IV. Future Perspectives
References

Abstract
In the event of injury to the vasculature in vertebrate organisms bleeding is stopped
by a defense mechanism called hemostasis. Even though biochemical studies charac-
terized a number of factors, classical genetic methods have not been applied to study
hemostasis. We introduced zebrafish as an animal model to study genetics of hemo-
stasis. To conduct genetic studies of hemostasis, we required a global screening
method to address all the factors of hemostasis such as those present in plasma, in
platelets or those present in the endothelium. Therefore, we developed a global laser
induced thrombosis method which can assay all these components. In this paper, we
describe the principle of this method as well as provide the detailed protocol so this
could be used as a screening tool to measure hemostasis in any laboratory.

I. Introduction
Hemostasis is a well-orchestrated defense mechanism that comes to the rescue in
the event of injury to the vasculature in vertebrate organisms (Jagadeeswaran et al.,

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Copyright 2011. Elsevier Inc. All rights reserved. 197 DOI 10.1016/B978-0-12-385464-3.00002-9
198 Pudur Jagadeeswaran et al.

2005, 2007). In mammals, when a vessel wall is ruptured, platelets are anchored to
the subendothelial matrix via collagen and von Willebrand factor (vWF). These
proteins activate the platelets, which initiate the secretion of three agonists ADP,
thromboxane, and serotonin, and these agonists further activate and amplify the
aggregation of platelets. These agonists act via their respective receptors and initiate
a variety of signaling pathways that ultimately result in the activation of aIIbb3,
which causes platelet aggregation along with fibrinogen as a bridging molecule.
aIIbb3 activation subsequently leads to ‘‘outside in signaling’’ and initiates the
platelet retraction. Along with these events tissue factor on the surface of the cells
in the subendothelial matrix binds to the preexisting factor VIIa and initiates coag-
ulation by cleaving factor X to Xa, which then cleaves prothrombin to generate
minuscule amount of thrombin. This then converts factor XI to XIa; XIa then cleaves
factor IX to IXa; factor IXa, along with cofactor VIIIa, then cleaves larger amounts
of factor X to Xa, thus generating explosive amounts of thrombin from prothrombin.
The thrombin generated will cleave fibrinogen to fibrin forming a clot that firmly
plugs the ruptured vessel and stops bleeding. Interestingly, these reactions occur on
the surface of platelets. The newly generated thrombin also enhances platelet acti-
vation. The fibrin formed will be stabilized by the crosslinking of fibrin monomers
by factor XIIIa, generated by thrombin cleavage of factor XIII. The clots generated
are lysed subsequently by the plasmin enzyme. The clotting cascade and the plasmin
system are regulated by inhibitor pathways, which maintain balance. The vessel wall
itself is involved in inhibiting the adverse coagulation and platelet aggregation
inside the vessel by providing a layer of endothelial cells that secrete anticoagulant,
fibrinolytic, and platelet inhibitory factors such as thrombomodulin, tissue plasmin-
ogen activator, and nitric oxide, respectively.
When this mechanism of hemostasis and the proper balance of reactions go awry,
it is pathological resulting in either bleeding disease or thrombosis. Despite extensive
investigations several pathways remain elusive, such as the mechanisms involved in
the initiation of hemostasis. Therefore, we introduced zebrafish as a model to study
this process because this fish has been used as a genetic model to study development.
Furthermore, in the human hemostasis community there was a notion that fish
hemostasis may be primitive and that fish do not carry cell fragments called platelets
as in mammals and therefore zebrafish may not be a suitable model to study
mammalian hemostasis (Jagadeeswaran et al., 2007). For these reasons, we charac-
terized zebrafish hemostasis and provided evidence that these fish carry most of the
factors governing hemostasis that are found in mammals. Furthermore, despite the
fact that fish thrombocytes carry nucleus, we showed that thrombocytes are mam-
malian equivalents of platelets and also carry nuclear factors similar to megakar-
yocytes. Although the thrombocytes are nucleated, the signaling pathways are well
conserved, and therefore there is no reason why these thrombocytes cannot be used
to model platelet activation; after all the functional molecules of the platelet signal-
ing reside in the cytoplasm and the membrane surface all of which are present in
thrombocytes. Therefore, we have been using zebrafish to model mammalian hemo-
stasis and to identify novel factors governing hemostasis using the power of genetics.
9. Laser-Induced Thrombosis in Zebrafish 199

In order to do this, we need genetic screening methods. Even though we developed


several assays to measure hemostatic function, none of these methods will address all
the factors of hemostasis such as those present in plasma, in platelets or those present
in the endothelium. Therefore, we developed a global method that can assay all these
components. We describe below the principle of the method and one of the screening
tools we developed called laser-induced thrombosis (Gregory et al., 2002).

II. Vascular Occlusion


The main initiating event in hemostasis is the injury to the vessel wall. Even
though fish can be injured by a variety of methods such as crushing and needle
poking, uniform injury is important to quantify hemostasis. Therefore, we intro-
duced a laser injury to the zebrafish larvae. In our work, we used pulsed nitrogen
laser light to cause a lesion in the caudal vein or artery leading to vascular occlusion.
We have demonstrated that it is possible to wound the larval vessel uniformly at a
defined location both at the arterial side and at the venous side. Furthermore, we have
shown that the vascular occlusion is not due to thermal agglutination of the cells but
due to actual formation of fibrin in the venous occlusive mass by injecting FITC-
labeled fibrinogen. We also demonstrated that arterial occlusion is due to thrombo-
cytes. Due to the fact that thrombocytes and other blood cells are large, it is easy to
visualize directly the thrombus formation as well as vascular occlusion. Therefore, it
is possible to measure the time taken to occlude (TTO) the vessel from the time of
injury by the laser beam for both arterial and venous occlusions. Also, we noted that
it is possible to measure the time taken to adherence (TTA) of the first cell from the
time of laser injury. Furthermore, we observed that if we keep the larvae for some-
time after the complete vascular occlusion the thrombus begins to dissolve and it is
also possible to measure the time to dissolution (TTD) of the thrombus. The validity
of the TTO assay has been verified by using the antisense morpholino knockdown
technology to knockdown proteins such as prothrombin and factor VII and demon-
strating that venous TTO was prolonged. We will present the detailed procedure of
vascular occlusion as performed in our laboratory.

III. Methods
A. Laser setup
The laser setup required for this is the micropoint laser unit supplied by Photonics
Inc. This setup has a pulsed nitrogen laser light attached to a fluorescence micro-
scope (Fig. 1). In our laboratory we have two units, one unit attached to Nikon
Optiphot microscope and a second one attached to a confocal microscope. In both
these units a nitrogen laser pumped through coumarin dye is configured according to
the manufacturers’ procedures. For recording the assay, a camera and a videocassette
recorder or a computer are connected to a monitor.
200 Pudur Jagadeeswaran et al.

[(Fig._1)TD$IG]

Fig. 1 Laser setup. C, camera; L, thermoline laser; M, fluorescence microscope; V, videocassette


recorder attached to a monitor.

B. Preparation of larvae for laser thrombosis


1. On day 1, the male and female zebrafish, which have been kept separately, are
placed in the breeding tank supplied by Aquaneering Inc.
2. On day 2, the embryos are retrieved from the bottom of the breeding tank and
allowed to hatch as described in the zebrafish manual.
3. On day 4, 1.6% low-melt agarose is prepared by boiling and refluxing in a
microwave, and then maintained at 35  C. A small rectangular chamber is created
on a glass microscopic slide by lightly coating one side of a rectangular gasket
with petroleum jelly and pressing it onto the slide (Fig. 2).
4. For each assay, several larvae (at least one dozen larvae) are transferred to a 1.5-
mL Eppendorf tube along with fish water and the volume of this water is adjusted
to 0.5 mL.
5. Six microliters of a 10 mM tricaine solution are added to this 0.5 mL to anes-
thetize the larvae.
9. Laser-Induced Thrombosis in Zebrafish 201
[(Fig._2)TD$IG]

Fig. 2 Preparation of zebrafish larvae for laser injury. Rectangular chamber (RC) constitutes a black
gasket placed on the microscopic slide (MS) which holds agarose in which larvae (shown by arrow) are
embedded.

6. Then 0.5 mL of the 1.6% agarose solution is added, and gently the larvae are
mixed by inversion and immediately poured onto the rectangular chamber on
the microscope slide. (Note: The larvae usually position themselves flat on
their side so the vessels are clearly visible, but some larvae require gentle
reorientation under the dissection scope with a pipette tip as the agarose
solidifies.)
7. At this time, the pattern of the larvae is roughly drawn on a paper and numbered
so the larvae can be targeted according to a numbered scheme.

C. Laser ablations
1. The accuracy of the laser is tested by placing a slide-shaped mirror on the stage
and triggering the laser through the 10 objective. (Note: The laser damages the
mirror and allows a small beam of light through, which can be adjusted to be
visible at the intersection of the crosshairs embedded in the eyepiece.)
2. The nitrogen laser repetition rate is set at 10 pulses per second and the pulse
generator rate is set at 10 Hz.
3. The coumarin laser attenuator plate is adjusted to power level 10 for the younger
larvae and up to 14 for older larvae.
4. The prepared slide is placed under the microscope and viewed with a 2 objec-
tive lens for orientation, and then individual larval vessels are located with the
20 objective lens. The area for laser ablation is selected by counting five to six
somites toward the caudal end from the anal pore.
5. Using a hand switch for the laser, the artery or vein is targeted and damaged for
5 s, with the subsequent thrombus formation clearly recorded onto videotape.
6. At the start of an ablation, a hand is waived across the microscope’s light source to
mark the starting point of the laser pulse. Approximate TTO is recorded. Arterial
202 Pudur Jagadeeswaran et al.

[(Fig._3)TD$IG]

Fig. 3 Venous thrombosis. Arrows show the site of laser injury (left panel) and the venous thrombus
(right panel).

occlusion assay is performed on day 6 (the image of thrombus in vein is shown in


Fig. 3).
7. After completion of ablations, the larvae continue to be monitored for the amount
of time taken for thrombus dissolution and restoration of local circulation.
8. The video is then reviewed to count the precise times taken for adherence and
occlusion.

D. Normal values and larval stages in laser thrombosis


The normal times for TTA, TTO, and TTD are approximately 4 s, 21 s, and 90 min,
respectively, for the vein; and 11 s, 75 s, and 5 min, respectively, for the artery.
Zebrafish embryos hatch from the chorion 3 days postfertilization (dpf). For several
days after hatching, the blood vessels in these larvae are visible, although melanin
granules mask them as the development continues. In our method, we target two
blood vessels, namely the caudal artery and caudal vein. We can focus the laser beam
on these vessels at exact locations through the use of muscle segments called
myotomes acting as reliable markers for repeatedly selecting an exact location of
the vessel for injury. In the mouse model due to anatomic variations and the need for
invasive exposure of the vessels, it is very difficult to consistently target the same
vessel locations. In zebrafish, venous thrombosis can be induced about 2 dpf and is
especially effective after hatching. However, arterial thrombosis cannot be induced
effectively until 5–6 dpf, when a sufficient amount of thrombocytes are in circulation.

IV. Future Perspectives

This assay has been applied in our laboratory to characterize mutants of hemo-
stasis such as Victoria (Gregory et al., 2002) in addition to finding a novel factor
9. Laser-Induced Thrombosis in Zebrafish 203

affecting hemostasis (manuscripts in preparation). Since the implementation of this


tool, many laboratories have used this method to either characterize zebrafish
mutants or knockdown platelet factors. We have previously used transgenic zebra-
fish thrombocytes labeled by green fluorescent protein (GFP) for visualizing throm-
bus formation and also introduced thrombocyte labeling methods by intravital
staining for quantification of thrombus formation (Gregory and Jagadeeswaran,
2002; Jagadeeswaran, 2005). Even without using the above fluorescence methods
the density of accumulating cells in normal larvae can be quantified by measuring
the loss of transparency. Recently, another transgenic line where the blood vessels as
well as thrombocytes are labeled with GFP has been used to detect the accumulation
of young and mature thrombocytes (Jagadeeswaran et al., 2010; Thattaliyath et al.,
2005). The advantage of this over the fish with only thrombocytes labeled with GFP
is that the vasculature is also visible; thus giving the real-time visualization of
thrombus along with the vessel. With the vascular occlusion method described here,
it is possible to conduct larval screens globally for the entire hemostatic pathways.
We have hitherto performed screens for venous occlusion, and in the future, other
screens for arterial occlusion should be feasible. The dissolution of thrombus assay
in both arteries and veins should provide additional screens for the future. Since the
methods are now available to measure global hemostasis in larvae, it should be easy
to screen for antithrombotic compounds by treating the zebrafish larvae with these
compounds and performing TTO assays. Thus, the larval vascular occlusion assay
presents a powerful tool not only to discover factors affecting hemostasis but also to
screen for novel antithrombotic drugs.

References
Gregory, M., Hanumanthaiah, R., and Jagadeeswaran, P. (2002). Genetic analysis of hemostasis and
thrombosis using vascular occlusion. Blood Cells Mol. Dis. 29, 286–295.
Gregory, M., and Jagadeeswaran, P. (2002). Selective labeling of zebrafish thrombocytes: quantitation of
thrombocyte function and developmental detection. Blood Cells Mol. Dis. 28, 417–427.
Jagadeeswaran, P. (2005). A green light for the thrombopoietic program. Blood 106, 3684.
Jagadeeswaran, P., Gregory, M., Day, K., Cykowski, M., and Thattaliyath, B. (2005). Zebrafish as a
genetic model for hemostasis and thrombosis. J. Throm. Haemost. 3(1), 46–53.
Jagadeeswaran, P., Kukarni, V., Carrillo, M., and Kim, S. (2007). Zebrafish: from hematology to hydrol-
ogy. J. Throm. Haemost. 300–304.
Jagadeeswaran, P., Lin, S., Weinstein, B., and Kim, S. (2010). Loss of GATA1 and gain of FLI1 during
thrombocyte maturation. Blood Cells Mol. Dis. 44(3), 175–180.
Thattaliyath, B., Cykowski, M., and Jagadeeswaran, P. (2005). Young thrombocytes initiate thrombus
formation in zebrafish. Blood 106(1), 118–124.
CHAPTER 10

Endoderm Specification, Liver


Development, and Regeneration
Trista E. North*,z and Wolfram Goesslingy,z
*
Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston,
Massachusetts, USA
y
Genetics Division, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA
z
Harvard Stem Cell Institute, Cambridge, Massachusetts, USA

Abstract
I. Review of the Literature
A. Endoderm Progenitor Differentiation and Specification
B. Liver Specification and Growth
C. Biliary Differentiation
II. Embryonic and Larval Protocols to Analyze Liver Formation
A. Chemical Screens
B. Histology
C. Fluorescence-Activated Cell Sorting
III. Liver Injury and Regeneration Protocols
A. Acetaminophen Injury
B. Mechanical Injury
C. Genetic Ablation
D. Transient Genetically Induced Apoptosis
IV. Assessment of Liver Function
A. Liver Enzymes
B. Lipid Metabolism
V. Summary
References

Abstract
The endoderm is the innermost germ layer that gives rise to the lining of the gut,
the gills, liver, pancreas, gallbladder, and derivatives of the pharyngeal pouch. These
organs form the gastrointestinal tract and are involved with the absorption, delivery,

METHODS IN CELL BIOLOGY, VOL 101 0091-679X/10 $35.00


Copyright 2011. Elsevier Inc. All rights reserved. 205 DOI 10.1016/B978-0-12-387036-0.00010-4
206 Trista E. North and Wolfram Goessling

and metabolism of nutrients. The liver has a central role in regulating these processes
because it controls lipid metabolism, protein synthesis, and breakdown of endoge-
nous and xenobiotic products. Liver dysfunction frequently leads to significant
morbidity and mortality; however, in most settings of organ injury, the liver exhibits
remarkable regenerative capacity.

I. Review of the Literature


A. Endoderm Progenitor Differentiation and Specification
The specification and development of the primitive endoderm are governed by a
well-orchestrated successive interaction of signaling pathways and transcription
factors. As shown by fate mapping experiments, endodermal progenitors arise from
the marginal cell layers of the blastula. Their dorsoventral orientation reflects the
organization of the endodermal structures after gastrulation, with the cells most
dorsally located giving rise to the anterior endoderm, and the most ventral cells
developing into the posterior portions of the gastrointestinal tract (Bally-Cuif et al.,
2000; Warga and Nusslein-Volhard, 1999).
Several pathways are involved in the earliest steps of endodermal specification.
Nodal signaling is essential during gastrulation as it induces both mesoderm and
endoderm. Additionally, as some Nodal genes are expressed asymmetrically, it has
been implicated in left–right development (Burdine and Schier, 2000). Ligands for
the nodal pathway are members of the TGFb growth factor family. In the zebrafish,
these are squint (sqt/nodal-related 1 (ndr1)) and cyclops (cyc/ndr2), which bind to
the activin A receptor 1b (acvr1b) and to the EGF-CFC co-receptor one-eyed pin-
head (oep). Mutations in sqt, cyc, and oep, or overexpression of the nodal inhibitor
antivin, lead to a failure of endoderm development. The zinc finger transcription
factor gata5 (also called faust) and the mix-family homeodomain transcription
factor bonnie and clyde (bon) (Kikuchi et al., 2000) act downstream of nodal to
activate sox32 (also called casanova).
Sox32 is required for endoderm development, and its overexpression converts
mesoderm to endoderm (Alexander et al., 1999; Aoki et al., 2002; Dickmeis et al.,
2001; Kikuchi et al., 2001); it has been successfully used to direct the fate of
transplanted cells in blastula transplantation experiments. The fibroblast growth
factor (FGF)/extracellular signal-regulated kinase (ERK) pathway directly inhibits
endoderm formation by sox32 phosphorylation (Poulain et al., 2006). Sox32 func-
tions to positively regulate sox17 expression in a cell-autonomous and nodal-inde-
pendent manner (Kikuchi et al., 2001; Poulain et al., 2006; Warga and Nusslein-
Volhard, 1999). The zebrafish analog of Oct4, pou5f1, is required to maintain sox32
expression and also collaborates with sox32 to induce sox17 expression (Alexander
et al., 1999; Lunde et al., 2004; Reim et al., 2004).
Another group of genes strongly expressed in endodermal precursors are the fox or
forkhead box transcription factors: foxa1, foxa2 (previously called hnf3b), and foxa3.
Murine studies have identified this family as pioneer factors that bind to promoter
10. Endoderm Specification, Liver Development, and Regeneration 207

regions and enable binding of other transcription factors to chromatin. Their expres-
sion and function are required for the normal development of endodermal organs.
foxa3 is the first to be expressed, appearing at the dorsal blastula margin to mark
endodermal precursors, followed by induction of foxa2 during the shield stage. foxa1
is expressed in the primitive endoderm toward the end of gastrulation, and expres-
sion of all three genes persists in the endoderm through the later stages of organo-
genesis. For these reasons, they have been widely used as general markers for
endodermal patterning; the transgenic reporter line gut:GFP (Tg(XlEef1a1:GFP))
is expressed in a pattern resembling that of foxa3 and has proven a highly useful
marker for these pan-endodermal progenitor cells.

B. Liver Specification and Growth


Our understanding of the morphological aspects and genetic factors involved in
zebrafish liver development has substantially increased over the past decade. The
liver develops from anterior endodermal progenitor cells. Progenitors fated to
become liver are identifiable between the 18-somite stage (16 h postfertilization
(hpf)) and 24 hpf. At 24 to 28 hpf, hepatocyte precursors aggregate in the anterior
endoderm, leading to a thickening and leftward looping of the endodermal rod,
combined with a restriction of previously pan-endodermal gene expression
(Field et al., 2003). This so-called gut-looping is currently thought to occur as a
result of asymmetric migration of the epithelial lateral plate mesoderm (LPM);
failure of gut-looping leads to laterality abnormalities of endodermal organs, typi-
cally bilateral livers and pancreata (Chen et al., 2001; Horne-Badovinac et al., 2003).
Recent data reveal that the bHLH transcription factor hand2 is involved in the
process of gut-looping. It functions to remodel the extracellular matrix through
matrix metalloproteinase-mediated reduction of laminin deposition along the
boundary of LPM and gut, leading to LPM asymmetry (Yin et al., 2010).

1. Genetic Markers of Hepatogenesis


The liver primordium appears as a prominent bud extending to the left from the
midline over the yolk sac between 24 and 28 hpf (Field et al., 2003), when markers of
hepatic precursors, such as hematopoietically expressed homeobox (hhex) and pros-
pero-related homoebox 1 (prox1), as well as GATA-binding protein 6 (gata6),
become restricted in their expression in this region (Ober et al., 2003; Yee et al.,
2005); hhex morphant studies have revealed its central role in liver development
(Her et al., 2003), while studies in other vertebrates have postulated a function for
prox1 in hepatoblast proliferation (Kamiya et al., 2008) and hepatocyte migration
(Sosa-Pineda et al., 2000). As hepatic development progresses, markers of hepato-
blast maturation are sequentially expressed, such as the copper-binding protein gene,
ceruloplasmin (cp), at 34 hpf (Korzh et al., 2001), followed by the antioxidant and
transport protein gene, selenoprotein P, plasma, 1b (sepp1b) (Thisse et al., 2003;
Tujebajeva et al., 2000). Differentiated hepatocytes, however, are only recognizable
208 Trista E. North and Wolfram Goessling

by 44–48 hpf, when the liver expresses genes indicative of hepatocyte function such
as liver fatty acid binding protein (fabp1a or fabp10a) (Her et al., 2003), transferrin-
a (tfa) (Mudumana et al., 2004), transferrin receptor 2 (tfr2) (Fraenkel et al., 2009),
and the recently described group specific component (gc) member of the albumin/
a-fetoprotein/afamin family (Noel et al., 2010). Between 55 and 60 hpf, endothelial
cells previously surrounding the liver primordium begin to invade the liver to
establish a vascular network; this is coincident with the apical–basal polarization
of the hepatocytes, suggesting that the endothelium delivers instructive signals for
hepatocyte polarity and structure during the process of angiogenesis
(Sakaguchi et al., 2008).

2. Specific Signals Involved in Liver Formation

2.1 WNT Signaling


WNT signaling is involved in many critical processes during early development,
including axis formation and the specification and growth of several organs. The
mutant prometheus (prt), with defective wnt2bb signaling, demonstrated markedly
reduced expression of the liver markers hhex at 24 hpf, and of cp and sepp1b at
52 hpf (Ober et al., 2006). However, much as the name implies the homozygous
mutants eventually recover their livers and survive into adulthood, indicating an
important, but replaceable role for wnt signaling during liver specification and
growth. Studies using adenomatous polyposis coli (apc) mutant zebrafish
(Hurlstone et al., 2003), with impaired b-catenin degradation, and heat-shock
inducible transgenic fish to regulate wnt activity revealed two phases of wnt signal-
ing during hepatogenesis (Goessling et al., 2008): repression is required during early
somitogenesis for anterior endoderm and liver specification, consistent with find-
ings in Xenopus (McLin et al., 2007; Zorn et al., 1999). In contrast after mid-
somitogenesis, wnt activity enhances hepatic progenitor proliferation and liver size
at maturation and beyond, explaining the prometheus mutant phenotype.

3. FGF and BMP Signaling


In addition to their essential roles in early endoderm specification, FGF and bone
morphogenetic protein (BMP) signaling are also important for liver development.
Heat shock-mediated expression of a dominant-negative bmp receptor at 18 hpf or a
mutation in the type I BMP receptor gene, alk8, resulted in decreased expression of
hhex, prox1, gata4, gata6, foxa3, and ceruloplasmin, indicating the requirement of
BMP signaling for hepatoblast specification (Shin et al., 2007). BMP signaling also
remains involved in the later steps of hepatogenesis. Similarly, induction of a
dominant-negative form of the FGF receptor gene, fgfr1a, diminished hepatoblast
specification, which could in part be overcome by overexpression of bmp2. BMP
signaling not only affects liver formation, but may also impact the fate decisions of
endodermal progenitors: bmp2 signals through alk8 to enhance liver differentiation
10. Endoderm Specification, Liver Development, and Regeneration 209

and decreases the size of the pancreas (Chung et al., 2008). A similar effect is
observed after WNT activation, where wnt8 overexpression during later somitogen-
esis increased liver size at the expense of exocrine pancreas (Goessling et al., 2008).
All these data provide in vivo evidence for the existence of bipotential hepatopan-
creatic progenitors, originally postulated from studies in the mouse (Deutsch et al.,
2001).

4. Retinoic Acid
Another recently recognized pathway affecting liver development is retinoic acid
(RA) signaling, which is a well-characterized regulator of pancreas formation
(Stafford et al., 2006; Stafford and Prince, 2002). The RA synthesis enzyme gene,
aldehyde dehydrogenase 1a2 (aldh1a2; previously called retinal dehydrogenase 2,
raldh2) is expressed in the developing endoderm, and aldh8a1 (previously called
raldh4) in the growing liver bud (Liang et al., 2008). Impaired RA synthesis in
aldh1a2 mutants and in embryos treated with the aldh1a2 inhibitor DEAB decreases
prox1 and hhex expression at 30 hpf, indicating an important role for RA in liver
formation (Alexa et al., 2009). In medaka, the aldh1a2 mutant hio exhibits low
expression of wnt2bb in the LPM and delayed hepatic budding with decreased gata6,
prox1, and foxa3 expression (Negishi et al., 2010), implying that RA may act through
modulation of the WNT pathway.

5. Epigenetic Factors Regulating Liver Development


Recent studies have highlighted a role for epigenetic regulation in liver develop-
ment. Zebrafish mutants for histone deacetylase 1 (hdac1) demonstrate impaired
liver and pancreas formation (Noel et al., 2008). Similarly, morpholino knockdown
or chemical inhibition of hdac3 between 6 and 16 hpf delayed liver and exocrine
pancreas formation, possibly by acting through growth differentiation factor 11
(gdf11) (Farooq et al., 2008). Mutations in another epigenetic factor gene, DNA
methyltransferase 1 (dnmt1, also called dandelion), also caused embryos to exhibit
smaller liver size at 100 hpf, in this case through apparent induction of apoptotic cell
death (Anderson et al., 2009).

C. Biliary Differentiation
The mammalian liver contains a heterogeneous group of cell types, dominated by
hepatocytes. Another major subtype is biliary cells that form the bile ducts, conduits
to transport the bile secreted by the hepatocytes. Bile is a mixture of bile acids,
phospholipids, and cholesterol that are important for lipid digestion. As in humans,
zebrafish have a gallbladder that stores the bile (produced in the liver) between
meals, which is then secreted into the gut lumen. The hepatic architecture in zebra-
fish differs somewhat from that of mammals. Rather than forming the rosette pattern
of portal fields seen in mammalian livers, zebrafish liver consists of tubules of
210 Trista E. North and Wolfram Goessling

hepatocytes that are located around biliary cells. Hepatic maturation and growth
continues through the embryonic and larval stages as hepatobiliary differentiation
occurs (Wallace and Pack, 2003), which is governed by conserved factors.
Cytokeratin staining identifies the first biliary cells in the liver between 50 and
60 hpf, forming biliary ducts by 70 hpf (Lorent et al., 2004). As in the mammalian
liver, biliary differentiation depends on Onecut factors, which include many hepatic
nuclear factors. In zebrafish onecut3 is the functional homolog of the mammalian
Onecut factor Onecut1 (previously called Hnf6), and onecut3 morphants exhibit a
decreased number of shorter, poorly organized biliary ducts and impaired biliary
lipid secretion (Matthews et al., 2008). Onecut3 acts upstream of zebrafish onecut1,
which is equally important for biliary differentiation and signaling through down-
stream targets hnf1ba (previously called vhnf1) and the vacuolar sorting protein
vps33b (Matthews et al., 2004, 2005). Knockdown of onecut1 and vps33b also
results in impaired biliary architecture and reduced biliary lipid secretion. In addi-
tion, the vps33b morphants exhibit other defects consistent with the human disease
phenotype for the rare arthrogryposis-renal dysfunction-cholestasis syndrome
(Matthews et al., 2005).
As in mammals, notch signaling is also involved in zebrafish biliary specification
and growth. Compound morphants of notch 2 or 5 combined with knockdown of the
notch ligand jagged 2, or combined knockdown of jagged 2 and jagged 3 demon-
strate severely impaired biliary architecture (Lorent et al., 2004). Chemical inhibition
of Notch signaling by the g -secretase inhibitor DAPT inhibits further biliary growth
and remodeling (Lorent et al., 2010). In addition, epigenetic factors have been
recently recognized to be important for biliary development. S-adenosylhomocys-
teine hydrolase (ahcy) mutants with reduced DNA methylation due to accumulation
of S-adenosyl homocysteine as well as embryos treated with 5-azacytidine, a chem-
ical inhibitor of DNA methyl transferase I (dnmt1), demonstrated defects in bile duct
formation and impaired biliary physiology (Matthews et al., 2010). These findings
provide a zebrafish model for the clinical disease of biliary atresia, a rare defect in
children where the bile ducts are absent, obstructing bile flow within the liver.

II. Embryonic and Larval Protocols to Analyze Liver Formation


A. Chemical Screens
Chemical screens have become an increasingly popular and successful approach
to identify novel modulators and pathways that affect zebrafish development and
organogenesis (North et al., 2007; Zon and Peterson, 2010) and may have applica-
bility to human disease. The impact of this approach depends on the library of
chemicals used, with the utilization of libraries consisting of known biological
compounds enabling the direct identification of regulatory pathways. Examples of
these libraries are the Prestwick 1 collection (1120 compounds), NIH Clinical
Collection 1 (450 compounds), NINDS Custom Collection (1,040 compounds),
Biomol 3 ICCB Known Bioactives library (480 compounds), Biomol 4 FDA
10. Endoderm Specification, Liver Development, and Regeneration 211

approved library (640 compounds), and Sigma LOPAC1280 (library of pharmaco-


logically active compounds, 1280). We have recently performed a chemical screen
for modifiers of liver formation, exposing fish through all stages of liver develop-
ment from 18 somites to 72 h, covering the period of hepatoblast specification,
budding, and hepatocyte growth (North TE, Goessling W, manuscript in prepara-
tion). Use of increasingly available organ-specific transgenic reporter lines, such as
Tg(-2.8fabp10:EGFP)as3 (Her et al., 2003), enable real-time assessment of organ
size, enhancing the speed of assessment. Both fluorescent microscopy of transgenic
reporter fish and in situ hybridization for hepatocyte markers can be used to assess
the effects of chemical treatment. Fluorescence-based evaluation has the advantage
that no processing is required, and effects can be analyzed in vivo. Recently, an
automated method using confocal microscopy was used to study axonal growth and
regeneration, serving as a proof-of-principle that high-throughput approaches are
feasible (Pardo-Martin et al., 2010). In contrast, in situ hybridization enables anal-
ysis in batches and by different observers as well as direct comparisons across plates
processed on different days. The use of automated in situ hybridization decreases
mechanical workload and enhances throughput. This approach enables a qualitative
assessment that includes liver size, position, and laterality, and allows observations
of other global developmental defects.
Protocol – Chemical Screen for Liver Formation:
1. Harvest and pool stage-matched embryos; about 250–500 embryos are required
for each 48-well plate.
2. Keep embryos in incubator at 28.5  C until 18 somite stage.
3. Prepare multi-well plates. We have found that 48-well plates (such as Greiner)
allow better embryo survival than 96-well plates when filled with 5–10 embryos.
Fill each well with 1 mL E3 embryo buffer.
4. Prepare chemical compounds from library. Most premade libraries will have
stocks of 10–20 mM compounds, dissolved in DMSO. Transfer 1 ml of each
compound into each well. (Note: in our experience, combinatorial exposure to
two or more bioactive compounds at the same time results in excessive toxicity.)
5. Using a small spatula, aliquot 5–10 embryos/well using the ‘‘Caviar’’ method to
minimize transfer of additional water. This can be achieved most easily by
removing excess embryo buffer from the embryo plate before aliquotting the
embryos manually.
6. Incubate embryos in drug at 28.5  C until 72 hpf.
7. Process for in situ hybridization. We use fabp10a as a robust standard hepatocyte
marker. Using the BioLane HTI 16V in situ robot by Intavis (H€ olle&H€ uttner,
Germany) will enhance the ease and speed of processing. Alternatively, when
fluorescent reporter fish are being used, these will be assessed with a fluorescent
stereoscope, while the fish are anesthetized with tricaine.
8. Score embryos for liver size and position, using untreated and/or DMSO-treated
embryos as reference. Liver size is most easily assessed in embryos in the lateral
position, with the left side facing up.
212 Trista E. North and Wolfram Goessling

9. Identify compound wells that affect organ formation and correlate with the
chemical library; group related hits according to mechanism of action.

B. Histology
Histological assessment has increasingly been used to examine tumor formation in
adult zebrafish. However, it is also an effective method to determine cell morphology
and tissue architecture in zebrafish embryos and larvae. Sections can be prepared as
cryosections or embedded in plastic or paraffin. Hematoxylin and eosin (H&E)
staining enables the rapid identification of basic anatomical structures. Available
online resources, such as the Zebrafish Atlas (http://www.zfatlas.psu.edu/reference.
php), can serve as an excellent reference for normal organ structure and cell appear-
ance. Serial sectioning through the liver or other organs of interest enables the
objective assessment of cell number in response to chemical or genetic modulation.
Basic H&E staining also facilitates the selection of specific sections for advanced
staining procedures. Apoptosis can be demonstrated by TUNEL staining (Millipore
Apoptag Kit), cell proliferation by BrdU incorporation (see protocol), or immuno-
histochemistry for phosphorylated histone 3 (pH3, Santa Cruz Biotechnology) or
proliferating cell nuclear antigen (PCNA). Oil-red-O staining can be performed to
detect neutral lipids and to assess lipid accumulation in genetic or environmental
models of hepatic steatosis (Matthews et al., 2009; Passeri et al., 2009; Sadler et al.,
2005). Periodic acid-Schiff staining reveals glycogen, which can similarly be altered
in the liver in response to environmental toxins (Lam et al., 2006; Ung et al., 2010).

C. Fluorescence-Activated Cell Sorting


The qualitative assessment of organ development in the zebrafish has been well
established, mainly through the use of in situ hybridization and organ-specific
reporter fish lines. However, there are few methods that provide a quantitative and
objective measure of cell number and morphology in response to genetic or
chemical modulation. Fluorescence-activated cell sorting (FACS) in combination
with transgenic reporter lines or fluorescent dyes has been increasingly used in
recent years to assess both cell number and the size and shape of organ-specific
cell types. This method can easily be applied to a variety of endodermal organs
and supporting cell types (such as the vasculature), because there are a number of
transgenic lines available highlighting endodermal progenitors, liver, pancreas, or
intestine (Table I). FACS allows the direct quantification of cells in a single
embryo and can also be used to isolate cell populations for further genomic
analyses to identify novel regulators of endoderm and gut formation, either in
conjunction with the fluorescent protein Kaede (Brown et al., 2008), or in gut:
GFP (Tg(XlEef1a1:GFP)) embryos (Stuckenholz et al., 2009). FACS instruments
with analysis-only capacity are typically user-operated, while machines capable of
cell sorting often require a dedicated, experienced operator. Recent protocols also
10. Endoderm Specification, Liver Development, and Regeneration 213

Table I
Available transgenic reporter lines to highlight endodermal organs

Transgenic line Target organ

Tg(-5.0sox17:EGFP)zf99 Endodermal progenitors


Tg(XlEef1a1:GFP)s854 (gut:GFP) Endoderm, intestine, liver, pancreas
Tg(-2.8fabp10:EGFP)as3 Liver (hepatocytes)
Tg(fabp10:dsRed)gz4 Liver (hepatocytes)
Tg(fabp10:RFP,ela3l:EGFP)gz12 Dual labeling of liver in red and exocrine
Pancreas in green
Tg(gata6:GFP)ae5 Gut, liver, pancreas
Tg(P0-pax6b:DsRed)ulg302 Pancreas
Tg(ptf1a:eGFP)jh1 Pancreatic duct, acinar cells
Tg(wt1b:EGFP)li1 Exocrine pancreas
Tg(-6.5pdx1:GFP)zf6 Pancreas
Tg(ela3l:GFP)gz2 Exocrine pancreas
Tg(-4.0ins:GFP)zf5 Endocrine pancreas
Tg(ins:dsRed)m1018 Endocrine pancreas
Tg(nkx2.2a:mEGFP)vu16 Endocrine pancreas
Tg(fabp2:DsRed)zf129 Intestine
Tg(h2afv:GFP)kca66 Gut epithelium

enable the FACS-based analysis of cell cycle status, apoptosis (Annexin V), and
proliferation (EdU incorporation).
Protocol – FACS Quantification of Fluorescent Cells:
1. Separate nonfluorescent and fluorescent embryos under the microscope; nonflu-
orescent embryos will serve as gating controls.
2. Pool equal number of control and fluorescent embryos into separate 1.5 mL
microfuge tubes. Typically, 5–10 embryos are sufficient, depending on age, but
single-embryo analysis is possible.
3. Remove all excess fish water.
4. Add 50 ml 0.9 Dulbecco’s buffer.
5. Homogenize embryos using a tight-fitting pestle with twirl and lift motion. Grind
embryos against sides of walls until they appear macroscopically well homogenized.
(Note: Overly aggressive homogenization may result in cell shearing and death.)
6. Wash remaining cells off pestle into microfuge tube using 150 ml of 0.9
Dulbecco’s buffer.
7. Strain homogenate through Falcon FACS tubes with 40 mm filter top and perform
FACS analysis.

III. Liver Injury and Regeneration Protocols


Liver regeneration is a universal phenomenon in many vertebrate organisms,
including zebrafish. In contrast to regeneration of other organs that is not conserved
214 Trista E. North and Wolfram Goessling

between zebrafish and mammals, such as the fin or the heart, the parallels between
zebrafish and mammalian liver regeneration can be utilized to identify common
pathways involved in repair to better understand and enhance the regenerative
process for clinical purposes. There are several models of hepatic injury and regen-
eration due to chemical, physical, and genetic insults that enable the identification of
novel mechanisms of liver injury and repair.

A. Acetaminophen Injury
Acetaminophen (N-acetyl-p-aminophenol; APAP) is one of the most commonly
used medications to alleviate pain and fever. It is safe at normal therapeutic doses,
but accidental or suicidal overdose can cause severe liver damage. APAP is the most
common cause for liver transplantation for toxin-induced fulminant hepatic failure
and results in more than 300 deaths annually in the USA (Lai et al., 2006). The
toxicity of APAP results from a hepatotoxic metabolite, N-acetyl-p-benzoquinone
imine (NAPQI) that is produced by cytochrome P450 enzymes. NAPQI is efficiently
inactivated in the liver by glutathione conjugation at therapeutic doses
(Mitchell et al., 1973). At higher doses, increased production of NAPQI causes
oxidative stress and mitochondrial damage. We have recently developed a zebrafish
model of APAP toxicity (North et al., 2010). Exposure of adult zebrafish to APAP in
fish water leads to dose-dependent toxicity that can be assessed by increasing serum
liver enzymes, histological changes with necrosis and hemorrhage, and mortality.
Exposure of fish for 24 h to 10 mM APAP diminishes liver-specific fluorescence in
transgenic reporter fish and causes death in 50% of fish (LD50). In addition,
zebrafish respond like mammals do to the FDA-approved clinical antidote N-acet-
ylcysteine, documenting significant physiological parallels between zebrafish and
humans. The effects of APAP toxicity are also present in larval zebrafish, as soon as
hepatocytes, which are responsible for the formation of the toxic APAP metabolites,
develop. In larvae exposed to APAP from 96 to 120 hpf, expression of liver-specific
genes is depressed as assessed by in situ hybridization and in liver reporter fish,
leading to substantial mortality. The conservation of the toxic phenotype between
adult fish and larvae enables a focused chemical screen to detect novel modifiers of
APAP toxicity. This revealed that prostaglandin E2 improves survival in both
embryos and adults (North et al., 2010). Other models of common hepatotoxins
have recently emerged; for example, acute exposure of 4-day old fish to 2% ethanol
resulted in an enlarged liver and fat accumulation with concomitant upregulation of
genes involved in lipid metabolism (Passeri et al., 2009). Zebrafish have also been
developed to examine the effects of less frequently used hepatic toxins such as
mercury (Ung et al., 2010) and thioacetamide (Amali et al., 2006) on the liver.
Protocol – Acetaminophen Exposure in Adult Fish:
1. Prepare fresh 2.5 M acetaminophen solution in DMSO or dH2O (3.78 g per
10 mL).
10. Endoderm Specification, Liver Development, and Regeneration 215

2. Transfer up to 10 wild-type adult zebrafish to small plastic containers (beakers or


inert plastic cups) with 200 mL of fish water. This volume will be sufficient for
this number of fish, while minimizing the amount of compound required. (Note:
Do not feed the fish while they are maintained in this small volume of water.)
3. Add 800 mL of acetaminophen stock solution to each container (10 mM final
concentration).
4. Expose fish for 24 h to hepatotoxin. Check regularly for death and remove dead
fish if necessary.
5. After 24 h, pour off the APAP solution and exchange water with fresh zebrafish
water and move to a larger container.
6. Record survival every 4–6 h post exposure.
7. Fish may also be analyzed by histology. Sacrifice fish with tricaine overdose, fix
with 4% of fresh paraformaldehyde solution. (Note: Modifications of this pro-
tocol allow for the treatment of fish with additional compounds of interest either
concurrent with, prior to, or after APAP exposure.)

B. Mechanical Injury
Surgical liver resection in mice and rats has been a principal model to study
mammalian liver regeneration for decades, enabling quantitative, functional, and
genomic assessments of organ recovery. Several groups have recently introduced
surgical resection of the adult zebrafish liver to demonstrate the importance of
specific signaling pathways in liver regeneration. The adult zebrafish liver is trilo-
bar, with a ventral lobe located directly beneath the ventral abdominal wall and a
lateral lobe on each side. Our group used resection of the ventral lobe to examine the
role of wnt signaling in liver regrowth, assessing both quantitative length measure-
ments and qualitative markers of cell proliferation (Goessling et al., 2008).
Following resection, the fish are sacrificed at specified time points throughout the
course of complete organ recovery of 7 days, which corresponds to the length of liver
regeneration in mice and men. The endodermal organs are dissected en bloc, and the
length of the regenerating lower lobe is measured. The ratio of the complete lower
lobe length to the length of the remnant stump, typically identified by a remaining
blood clot, is used to calculate the regenerative index as a measure of regenerative
capacity independent of organ size. This assessment can most easily be performed in
a freshly dissected specimen, but also in sagittal histological sections. In wild-type
fish, the regenerative index is 2.0 at day 3 postresection. If WNT signaling is
inhibited by heat shock induction of dominant-negative tcf3 in Tg(hsp70l:tcf3-GFP)
w26 embryos, the regenerative index is close to 1.0, indicating the absence of any
regenerative activity. In contrast, genetic activation of WNT by induction of wnt8a
(Tg(hsp70l:wnt8a-GFP)w34) or in apchu745/+ zebrafish enhances regenerative
capacity. Direct evolutionary conservation of the role of WNT signaling was dem-
onstrated in APCMin mice following partial hepatectomy. We used the same approach
to document the requirement of topoisomerase II alpha for liver regeneration
(Dovey et al., 2009). To assess the importance of other developmentally relevant
216 Trista E. North and Wolfram Goessling

genes, Sadler et al. (2007) documented the requirement of the ubiquitin-like, con-
taining PHD and RING finger domains 1 (uhrf1) gene during organ recovery. More
recently, Kan et al. (2009) also used ventral lobe resection to evaluate the role of
BMP and FGF signaling during liver regeneration, devising a method to measure
liver:body weight ratios, which revealed larger ratios for female fish. The rapidity
with which these resections can be performed, allowing 30–60 procedures per
hour, its safety with a survival rate of >95%, which is similar to murine resections,
and the high conservation with mammalian models allows the rapid assessment of
chemical and genetic modulation in organ injury and repair.
Protocol – Liver Resection
1. Anesthetize adult zebrafish with tricaine.
2. Transfer anesthetized fish to lid of 10 cm culture dish, located under dissection
stereoscope.
3. Using McPHERSON-VANNAS microdissecting spring scissors (Biomedical
Research Instruments 11–1050) and #55 forceps, make an 3 mm incision on
the ventral abdomen, just caudal to opercula.
4. Remove the inferior liver lobe, using gentle tugging motion. The zebrafish liver
has no capsule and can easily tear. Use caution not to injure the intestine as this
will result in higher mortality.
5. No wound closure is required after the completion of the liver resection because
the scales will cover over the resection site.
6. Return fish to fresh zebrafish water. (Note: Care should be taken to complete the
procedure in a timely manner; if the fish does not exhibit gill movement after
prolonged anesthesia, then manual perfusion of the gills with a transfer pipette
may accelerate recovery.)
7. Sacrifice zebrafish at defined time interval after resection with approved meth-
ods, such as tricaine overdose. Either process entire fish for histological evalu-
ation or dissect out endodermal organs for length measurements of inferior lobe
and remnant stump (see schematic in Fig. 1).

C. Genetic Ablation
Another method for targeted and timed hepatocyte injury was recently introduced
by Curado et al. (2007). This approach has also been demonstrated in other cell
types, including pancreatic beta cells, cardiomyocytes, retina, and testis. Here,
transgenic fish are created that express the Escherichia coli enzyme nitroreductase
under the control of organ-specific promoters, in this case fabp10a (Tg(fabp10:
CFP-NTR)s891). Although nitroreductase expression has no effect on the target
organ by itself, its enzymatic activity reduces metronidazole (1-(2-hydroxyethyl)-
2-methyl-5-nitroimidazole), a commonly used and typically nontoxic antibiotic, to
form a potent DNA interstrand cross-linking agent, which causes cell death. This
technology can be applied to fish of all ages: embryos, larvae, juveniles, and adults.
10. Endoderm Specification, Liver Development, and Regeneration 217
[(Fig._1)TD$IG]

Fig. 1 Schematic depiction of zebrafish liver resection. The inferior liver lobe is resected.
Regenerative capacity can be analyzed after en bloc dissection of the endodermal organs and measure-
ment of the total inferior lobe length (indicated in black) and the length of the remnant liver stump
(indicated in grey, often identified by blood clots). The ratio of total:remnant lengths indicates the
regenerative index.

In this model, toxicity and cell death are exclusively limited to the nitroreductase-
expressing cells, potentially allowing highly targeted cellular injury. However, this
also makes this system extremely dependent on a suitable promoter, efficient trans-
genic expression, and availability of metronidazole to the cells to achieve significant
damage of the entire organ (Curado et al., 2008) and may not allow complete
ablation of hepatic tissue (Curado et al., 2010).

D. Transient Genetically Induced Apoptosis


Another model of hepatic recovery was also introduced by Curado and colleagues
(2010). This approach is based on the observation of a novel genetic mutant, oliver,
with a mutation of the mitochondrial protein translocase of the outer mitochondrial
membrane 22 homolog (tomm22) gene. These mutants exhibit a smaller liver con-
sisting of mainly biliary tissue caused by hepatocyte apoptosis. Transient knock-
down of tomm22 by morpholino injection results initially in a corresponding phe-
notype; however, with waning efficacy of the morpholino, the liver recovers. The
morphant provides a model for organ recovery after apoptotic cell death in larval
stages where the role and impact of other signaling pathways can be tested, which has
been done for WNT and FGF. This model may be amenable to chemical modifier
screens on a larger scale.

IV. Assessment of Liver Function


In recent years, several approaches have been developed to assess clinical and
functional parameters of hepatic physiology and damage in zebrafish. These
218 Trista E. North and Wolfram Goessling

methods are identical to or reflect the assessment of liver damage and function
obtained in patients suspected of having liver disease. These measures enable the
correlation of zebrafish phenotypes with clinical liver disease.

A. Liver Enzymes
Liver enzyme tests are the cornerstone of the clinical assessment of hepatobiliary
damage. The assessment of serum parameters in the adult zebrafish has been limited
due to the small volume of blood that can be obtained (Murtha et al., 2003). By direct
cardiac puncture using micropipettes, however, blood samples can be pooled to
obtain minimum quantities (50 ml) of serum for analysis (100 ml whole blood).
This can be processed in a clinical laboratory to obtain ‘‘individual’’ cohort test
values. We were able to demonstrate APAP-induced liver damage in adult fish by an
increase in circulating alanine aminotransferase levels (ALT) (North et al., 2010).
Other relevant parameters are the assessment of aspartate aminotransferase for
hepatocyte damage, alkaline phosphatase for biliary injury, and total bilirubin for
the metabolic capacity of the liver. In humans, albumin is routinely measured to
assess protein synthesis in the liver; however, recent genomic (Noel et al., 2010) and
proteomic analyses (North et al., 2010) demonstrate the absence of albumin in the
zebrafish. Overall, this method is resource-intensive, because 10–25 fish are
required to obtain one pooled sample sufficient for analysis. Despite this, it can
serve as a resource when demonstrating the clinical importance of zebrafish studies,
using therapeutically relevant parameters.

B. Lipid Metabolism
In addition to the evaluation of early liver morphology and its regenerative
capacity, recent studies have demonstrated the feasibility of evaluating functional
aspects of the intestine, pancreas, and liver in vivo. One technique is intravital
staining with Nile red which, like oil-red-O in histological sections, stains neutral
lipids, which could be used in small molecule screens to discover novel antiobesity
drugs (Jones et al., 2008). Although Nile red stains all lipid-rich tissues, it cannot
distinguish between cholesterol and fatty acids. The development of BODIPY
fluorophores has enabled the dynamic assessment of lipid metabolism in vivo.
One compound, N-([6-(2,4-dinitro-phenyl)amino]hexanoyl)-1-palmitoyl-2-
BODIPY-FL-pentanoyl-sn-glycerol-3-phosphoethanolamine (PED6), has recently
been used to demonstrate lipase activity in vivo (Farber et al., 2001). Zebrafish
larvae exposed to PED6 exhibit green fluorescence in the intestine, gall bladder,
and liver. This approach has enabled genetic screens to identify modulators of lipid
metabolism, such as in the mutant fat-free (c11orf2, also called ffr) (Farber et al.,
2001) and others in medaka (Watanabe et al., 2004). More recently, PED6 has been
combined with a quenched protease reporter, EnzChek (Invitrogen), which can
assess exocrine pancreas function, and with microspheres, which determine
10. Endoderm Specification, Liver Development, and Regeneration 219

swallowing and intestinal capacity, giving a comprehensive functional in vivo assess-


ment of gastrointestinal physiology (Hama et al., 2009).

V. Summary
Over the past decade, many new aspects of early endoderm formation, liver
specification, differentiation, and growth have been discovered in the zebrafish. In
zebrafish the liver does not function as a hematopoietic organ; therefore, genetic
and chemical effects that lead to impaired liver formation can be studied without
the interference of death from anemia. This has revealed the interaction of central
signaling pathways in liver formation, and a direct assessment of their function in
vivo. In addition, the study of larval and adult zebrafish has enabled the devel-
opment of models of liver injury and regeneration as well as in vivo assessment of
organ function, with a high conservation between fish and humans of toxic and
therapeutic drug effects. These findings indicate the potential of the zebrafish for
discovery of disease-relevant aspects of endoderm and liver structure and
function.

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CHAPTER 11

Morphogenesis of the Zebrafish Jaw:


Development Beyond the Embryo
Kevin J. Parsons,* Viktoria Andreeva,y W. James Cooper,*
Pamela C. Yelicky and R. Craig Albertson*
*
Department of Biology, Syracuse University, Syracuse NY, Tufts University, Boston, Massachusetts
y
Department of Oral and Maxillofacial Pathology, Tufts University, Boston, Massachusetts

Abstract
I. Postembryonic Development – Framing the Questions and Understanding the Challenges
II. Obtaining Phenotypes
A. Postembryonic Mutagenesis Screens in Zebrafish
B. Heterozygous Craniofacial Phenotypes in Previously Identified Homozygous
Recessive Early Lethal Zebrafish Mutants
C. Rescue of Homozygous Recessive Lethal Mutations to Examine Postembryonic
Defects
III. Quantitative Methods for Studying Adult Phenotypes
A. Geometric Morphometrics
B. Data Collection
C. General Data Analysis
D. Analysis of Shape
E. Analysis of Shape Variation
F. Rates of Shape Change
G. Trajectories of Shape in Zebrafish Lines
H. Morphological Integration
I. Synthesis of Quantitative Data
IV. A Complementary Approach: The Use of Natural Variation to Complement that
Generated in the Lab for Understanding Jaw Morphogenesis
V. Implications and Conclusions
References

Abstract
The zebrafish has emerged as an important model for vertebrate development as it
relates to human health and disease. Work in this system has provided significant
METHODS IN CELL BIOLOGY, VOL 101 0091-679X/10 $35.00
Copyright 2011. Elsevier Inc. All rights reserved. 225 DOI 10.1016/B978-0-12-387036-0.00011-6
226 Kevin J. Parsons et al.

insights into the variety of genetic signals that direct the cellular activities and tissue
interactions necessary for proper assembly of the pharyngeal skeleton.
Unfortunately our understanding of craniofacial development beyond embryonic
stages is far less complete. Stated another way, we know a great deal about the early
patterning of the skull, but we know comparatively little about how mature cranio-
facial shape is determined and maintained over time. Here we propose ways to
expand the current molecular genetic paradigm beyond the embryo to gain an
understanding of the processes and mechanisms that guide growth and remodeling
of mineralized craniofacial, skeletal, and dental tissues. First, we discuss sources of
adult mutant phenotypes that can be used to study of postembryonic development.
Next, we review salient quantitative methods that are necessary to define complex
adult phenotypes. We also discuss how other organismal systems can be used to
inform and complement studies in zebrafish. We conclude by discussing the impli-
cations for such studies within the context of furthering an understanding of the
etiology and pathophysiology of human craniofacial malformations, as well as
informing an understanding of adaptive craniofacial variation among natural
populations.

I. Postembryonic Development – Framing the Questions


and Understanding the Challenges

In stark contrast to the extensive body of literature focused on understanding the


basis of early craniofacial patterning, relatively little is known about the mechanisms
that underlie skeletal development beyond embryonic stages. Craniofacial develop-
ment can be broadly divided into chronological stages based on the appearance of
tissues, the formation of structures, and the initiation of various morphogenic pro-
cesses. The cranial neural crest (CNC) cells are the first to arise during embryonic
development that directly contribute to the craniofacial skeleton. A PubMed liter-
ature search (May 12, 2010) for CNC identified nearly 1400 peer-reviewed research
articles. By early larval stages of development the CNC cells condense and differ-
entiate to form craniofacial cartilages, which soon thereafter develop into pharyn-
geal bones and other components of the skull (Hall, 1999). A search for craniofacial
‘‘cartilage development’’ returned over 600 articles, whereas craniofacial ‘‘bone
development’’ identified 200 references. Bone growth and remodeling are initiated
during larval development, and continue throughout juvenile and adult stages. A
PubMed search for craniofacial ‘‘bone growth’’ and ‘‘bone remodeling’’ each iden-
tified fewer than 100 articles.
This brief survey of the literature reveals a dearth of peer-reviewed articles that
explore craniofacial development beyond embryonic stages. It also reveals an
inverse relationship between developmental chronology and our understanding of
the mechanisms that regulate this process. The main purpose of this chapter is to
describe ways in which the experimentally tractable zebrafish can be used to pro-
mote a more robust understanding of craniofacial development beyond the embryo.
11. Morphogenesis of the Zebrafish Jaw: Development Beyond the Embryo 227

The zebrafish is an excellent vertebrate model with which we study skeletogenesis


at all stages of development. They are easily bred and maintained, have short
generation times, and large numbers of progeny can be obtained from a single mating
(200–500/week). The zebrafish genome has been sequenced and zebrafish devel-
opmental genetics are easily manipulated through functional analyses, transplanta-
tion, and transgenics. Zebrafish are also touted for having transparent embryos,
allowing various developmental processes to be observed in the living embryo with
minimal processing or manipulation. This attribute also holds true for postembryo-
nic zebrafish, particularly for the skeletal system. Most craniofacial elements lie just
underneath a relatively thin layer of tissue and are easily observed in live or fixed
animals with minimal processing (Fig. 1).
Another relevant attribute of the zebrafish is that, like most teleosts, they exhibit
dynamic patterns of allometric growth, whereby anatomical changes accompany
their increase in size (Fuiman, 1983; Hernandez, 2000; Osse, 1990; Weatherly,
1990). The life cycles of bony fishes are often characterized by dramatic shifts in
ecology and behavior, accompanied by concomitant changes in the body shape (Loy
et al., 2001; Zelditch and Fink, 1995). Allometric growth is even considered to be a
defining characteristic of fish early life histories (Hernandez, 2000). The use of a
model that exhibits pronounced size-related shape change will likely facilitate our
efforts to understand the mechanisms that underlie skeletal growth and remodeling.
Zebrafish craniofacial mutants have traditionally been characterized using qual-
itative methods (i.e., the gross presence or absence of structures) up to, and rarely
past, 5 days postfertilization (dpf). We have argued previously (Albertson and Yelick,
2004, 2007; Cooper and Albertson, 2008) that a shift from qualitative to quantitative
descriptions of phenotype will facilitate a more comprehensive understanding of
craniofacial development, particularly as it pertains to development beyond larval
stages. The impetus for this assertion stems, in part, from the fact that the
adult skeleton is significantly more complex than that of the larvae. Fig. 2A depicts
a 5-day larval skeleton, while Fig. 2B shows the adult complex. The most obvious

[(Fig._1)TD$IG]

Fig. 1 Skeletal preparations in two adult zebrafish. (A) A living fish stained with the fluorescent
compound, Calcein (Sigma), which binds to free calcium (note the cranial sutures). (B) An adult AB
zebrafish that was fixed, enzymatically cleared, and stained with Alizarin red that also binds to calcium
(Sigma, St. Louis, MO). In both specimens detailed skeletal anatomy is apparent.
228 Kevin J. Parsons et al.

[(Fig._2)TD$IG]

Fig. 2 Ontogenetic changes in craniofacial bone in AB zebrafish. (A) depicts a 5-day larval skeleton,
while (B) shows the adult complex. The most obvious difference between the two structures is that the
larval skeleton is only barely ossified as indicated by the predominance of alcian blue staining.

difference between the two structures is that the larval skeleton is minimally ossified.
For the most part, only cartilages are present, and thus only a fraction of the elements
that comprise the adult complex is evident. In fact, entire functional units are absent
from the larval form, including the upper jaws and portions of the cranium
(Harris et al., 2008). So while significant progress has been made toward an under-
standing of the molecular/genetic factors that regulate development of larval pha-
ryngeal cartilage morphology, we know virtually nothing about major functional
complexes of the skeleton that appear later in development (i.e., mineralized jaws).
We contend that a major challenge facing developmental biologists is to extend
the current developmental genetic paradigm beyond the embryo. This challenge can
be framed by asking two basic questions: (1) What are the molecular determinants of
craniofacial development beyond embryonic stages? and (2) What is the molecular
basis for quantitative shifts in craniofacial shape? The answer to these and related
questions will inform a broad range of scientific disciplines, as we seek a better
understanding of both normal and abnormal variation in craniofacial form.

II. Obtaining Phenotypes

A. Postembryonic Mutagenesis Screens in Zebrafish


Over the years, the zebrafish has proven to be an excellent subject for forward-
genetic mutagenesis screens. By screening for early-lethal phenotypes, a large
number of mutants relevant to human development and disease have been discov-
ered, including a variety of craniofacial mutants (Neuhauss et al., 1996; Piotrowski
et al., 1996; Schilling et al., 1996). These studies have shed significant light on our
understanding of molecular events regulating early craniofacial and tooth develop-
ment. Our current focus is to move beyond the embryonic stages in order to identify
and characterize genes and signaling pathways that regulate the growth and
11. Morphogenesis of the Zebrafish Jaw: Development Beyond the Embryo 229

remodeling of adult mineralized tissues. Studies of viable, adult mineralized cra-


niofacial tissue phenotypes are particularly relevant to determining the genetic basis
of human craniofacial pathophysiology and disease. Several recent publications
have demonstrated the strengths of the zebrafish model for adult craniofacial and
skeletal morphogenesis. For example, Fisher et al. (2003) utilized a radiographic
analysis of adult ENU-mutagenized F1 fish to identify the dominant skeletal dys-
plasia chihuahua (chi) mutant. The chi mutant zebrafish harbors a dominant muta-
tion in the type I collagen gene (col1a1) which leads to uneven mineralization,
fragility and defects in bone growth (Fisher et al., 2003), features that are associated
with human skeletal dysplasia osteogenesis imperfecta (OI). OI is a dominant
heritable disorder of connective tissues in humans, caused in approximately 90%
of individuals by mutations in type I collagen genes (col1a1 and col1a2), for which
there currently is no effective clinical treatment (reviewed in Basel and Steiner,
2009). Thus, the zebrafish chi mutant is a valuable tool and model with which we can
extend our knowledge of OI, and hopefully identify effective clinical treatments.
Another large-scale mutagenesis screen for defects in adult structures led to the
discovery of two similar mutants finless (fls) and Nackt (Nkt), both of which exhibited
defects in the skeletal elements of the skull, fins, scales, and teeth (Harris et al., 2008).
It has been shown that Nkt harbors a mutation in the ectodysplasin (eda) gene,and that
mutations of the ectodysplasin receptor (edar) gene were found in fls mutants
(Harris et al., 2008). Remarkably, mutations of eda and edar genes are responsible
for the majority of hypohydrotic ectodermal dysplasia (HED) cases in humans
(Mikkola, 2009). HED is a hereditary disorder characterized by hair and teeth defects,
as well as defects in a number of other ectodermal organs (Mikkola, 2009).
In the Yelick laboratory, we have identified several mutants that exhibit craniofa-
cial and bone defects in adult fish, by applying high-throughput Alizarin red (AR)

[(Fig._3)TD$IG]

Fig. 3 Analysis of bka and knz mutants. (A) AR staining of bka mutant and wild-type control. (B) GM
analysis of bka mutant and wild-type control. The arrows in (A) and (B) point to reduced upper jaw in bka
mutant. (C) AR staining of knz mutant and wild-type control. (D) GM analysis of knz mutant and wild-
type control. The arrows in (C) and (D) point to changes in upper jaw morphology of knz mutant.
230 Kevin J. Parsons et al.

staining of ENU-mutagenized F3 generations at 6–8 weeks postfertilization


(Stewart-Swift et al., 2010; 9th International Zebrafish Development and Genetics
Conference, Madison, WI, June16–20, 2010). One of them, homozygous recessive
mutant belkal (38N), exhibits a midface hypoplasia phenotype, and reduced and
fused maxilla (Fig. 3A, B). In addition, approximately 50% of sqr mutants develop
scoliosis. A second mutant, which seems to be heterozygous semidominant, knjaz’
(78N), displays craniofacial shape defects including the shape of preorbital region
possibly due to a caudal displacement of the upper jaw in addition to scoliosis in the
tail region, as detected by AR staining, and as confirmed by geometric morphometric
(GM) analyses, the methodology for this will be explained in the following (Fig. 3C,
D). Efforts to map each of these mutations, and to identify the genetic mutations
responsible for these phenotypes, are currently underway.

B. Heterozygous Craniofacial Phenotypes in Previously Identified Homozygous Recessive


Early Lethal Zebrafish Mutants
Another approach to identify adult craniofacial and bone mutants is to use AR
staining and GM analyses to look for heterozygous phenotypes previously described
early-lethal homozygous recessive zebrafish mutants – particularly those harboring
gene mutations in craniofacial and tooth-expressed genes. One example is the acer-
ebellar (ace) mutant, caused by a mutation in the fibroblast growth factor (fgf8) gene.
The ace/fgf8 homozyougous mutants exhibit an asymmetric craniofacial pharyngeal
skeleton, among other defects (Albertson and Yelick, 2005), and die at approximately
7 dpf (Brand et al., 1996). Our study of heterozygous ace/fgf8 zebrafish allowed
investigations of craniofacial development to be extended beyond the early embryonic
stages, and identified roles for fgf8 in adult craniofacial form and function (Albertson
and Yelick, 2007). Our geometric shape GM analyses revealed that fgf8 haploinsuffi-
cency leads to craniofacial asymmetries and defects in cranial sutures, and staining for
alkaline phosphatase and tartrate resistant acid phosphatase activities showed aberrant
osteoblast and osteoclast activities, respectively, which contributed to abnormal bone
formation and remodeling in the lower jaw (Albertson and Yelick, 2007). Thus, these
studies demonstrated that subtle but highly informative phenotypes can be revealed by
investigations of viable adult heterozygous mutant zebrafish. Analyses of the viable
adult heterozygous zebrafish mutants have high clinical relevance, since the majority
of human hereditary disorders are induced by mutations in a single copy of the gene.
For instance, single allele mutations in fibroblast growth factor receptors (fgfr)types
1–3, have been found in several human syndromes that are characterized by cranio-
facial defects including craniosynostosis, or premature fusion of cranial sutures, as
well as defects in bone formation and growth (Morriss-Kay and Wilkie, 2005).

C. Rescue of Homozygous Recessive Lethal Mutations to Examine Postembryonic Defects


An additional way to extend our knowledge of craniofacial morphogenesis beyond
embryonic stages is by rescuing homozygous recessive mutants from early lethality
11. Morphogenesis of the Zebrafish Jaw: Development Beyond the Embryo 231

using wild-type mRNA injections. This approach was successfully used by


LeClair et al. (2009) to discover the effects of glypican 4 (gpc4) on craniofacial
organization in adult zebrafish (LeClair et al. (2009). gpc4 belongs to the family of
extracellular glycoproteins that can modulate Wnt, bmp, and fgf signaling (Filmus
and Selleck, 2001; Fransson, 2003). The gpc4/knypek homozygous mutant embryos
normally die around 5–7 dpf, and exhibit defects in convergence and extension
movements in the ectoderm and mesoderm (Topczewski et al., 2001). However,
when investigators rescued gpc4/knypek homozygous mutant embryos by single cell
injections of gpc4 mRNA, and then allowed the rescued mutants to develop for up to
1 year, they discovered the loss or rearrangement of several adult craniofacial bones,
defects in craniofacial cartilages, and changes in the shape of neurocranium
(LeClair et al., 2009). These elegant studies have shown that adult craniofacial
phenotypes can be identified and examined in previously identified homozygous
recessive lethal mutants, if they are allowed to survive beyond critical early devel-
opmental stages.

III. Quantitative Methods for Studying Adult Phenotypes


A. Geometric Morphometrics
Shape is a fundamentally important feature of organisms but has historically been
very challenging for biologists to quantify. Until recently morphometric analyses have
largely involved measuring sets of linear distances on a form, applying a chosen
method of size correction to these distances, and finally applying a multivariate
statistical test to these data to discern patterns. While these methods are still used,
they are fraught with disagreements over what size corrections to apply, and difficul-
ties in determining the biological meaning of statistical results (Parsons et al., 2003).
The field of morphometrics is undergoing a radical transformation through the
introduction and expansion of GM (Adams et al., 2004; Rohlf and Marcus, 1993).
These powerful techniques have now become the standard method to quantify the
shape and offer researchers a greater ability to discern and interpret trends in shape
variation.
Simply put, we contend that morphometrics is a quantitative approach to compare
shape differences that zebrafish biologists have been describing qualitatively for
years. Moreover, these newer methods are powerful enough that they can extract
meaningful information from situations where only subtle or continuous variation
occurs, even when these patterns are not apparent to the naked eye.
Although the use of GM in zebrafish research is still in its infancy, there is
enormous potential for biologists to adopt these methods to study development
beyond the embryo (Albertson et al., 2005; Albertson and Yelick, 2007; Cooper
and Albertson, 2008; LeClair et al., 2009). We recognize that GM can seem math-
ematically complex, abstract, or even obscure, and that this perception could easily
lead to discouragement. Accordingly, we have written the following sections as a
primer for zebrafish biologists, with largely heuristic explanations of the mathematics
232 Kevin J. Parsons et al.

and statistics being applied. We will also illustrate how data from GM can be
collected and analyzed in a developmental context using samples from the commonly
available AB and TL lines of zebrafish. Specifically, we will describe techniques that
are useful for assessing developmental robustness (i.e., canalization), the trajectory of
morphological development (i.e., allometric repatterning), the rate of shape change
over development (i.e., heterochrony), and morphological integration.

B. Data Collection
The beginning of any GM analysis involves the selection and photographic imag-
ing of samples, and the collection of x,y Cartesian landmark coordinates. Samples
can be chosen based on their membership to a particular group of interest (i.e., wild-
type vs. mutant zebrafish), and at multiple stages of ontogeny. Imaging is a relatively
straightforward but extremely important process whereby samples are photographed
in a reproducibly standard position in the presence of a scale bar. These images are
then used as a source from which x, y landmark data are collected. Currently the most
popular software for x, y data collection is TPSdig2 (Rohlf, 2010), which is used in
coordination with TPSutil (Rohlf, 2010). TPSutil creates a file that allows images to
be read sequentially into TPSdig2, using the ‘‘build tps files from images’’ function
so that the coordinate locations of homologous landmarks can be recorded on images
(available at http://life.bio.sunysb.edu/morph/). It is extremely important that x, y
coordinates are collected in the same sequence at homologous points on each
individual so that they are recognized correctly. Ultimately, these data will be used
to statistically test for shape differences between groups or ontogenetic stages.
The quality of any statistical test depends upon the quality and amount of data
collected. We therefore recommend the use of multiple individuals per group, and

[(Fig._4)TD$IG]

Fig. 4 Three stages of ontogeny in AB and TL lines of zebrafish analyzed for morphometric variation.
Note that the seven landmarks used in this study are in homologous positions throughout ontogeny.
11. Morphogenesis of the Zebrafish Jaw: Development Beyond the Embryo 233

the collection of as many landmarks as are reasonable for any GM study. Here we use
a total of 146 specimens and seven landmarks across three stages of ontogeny (10 dpf
n = 24 AB, 20 TL; 30 dpf n = 30 AB, 26 TL; and adult n = 25 AB, 21TL) (Fig. 4).

C. General Data Analysis


A first step toward analyzing x, y coordinate data is to eliminate variance that is
due to differences in the size of specimens, their orientation, and position. Several
superimposition algorithms have been developed but the most common approach is a
generalized procrustes analysis (GPA), which is available seperately or automatically
implemented in a number of morphometric programs (Zelditch et al., 2004). This
method superimposes landmark configurations through a process of translation,
rotation, and scaling for size (see Fig. 5), in order to minimize the sum of squared
distances between corresponding landmarks across specimens (Adams et al., 2004).

[(Fig._5)TD$IG]

Fig. 5 Processes carried out during a generalized Procrustes analysis.


234 Kevin J. Parsons et al.

Once performed, this analysis opens the gateway for a variety of further statistical
methods. However, it is important that researchers comparing multiple groups use a
single GPA across all specimens, as the comparison of data from separate GPAs can
create misleading results.

D. Analysis of Shape
A landmark-based approach to shape analysis is far more powerful than traditional
methods used to discern and describe patterns of shape variation (Parsons et al.,
2003). In the simplest terms a geometric approach can double the number of traits
examined as compared to traditional linear methods. For example, consider three
distances on the head of a zebrafish: the depth of the eye, the length of the lower jaw,
and the overall length of the skull. These measurements represent three traits, but if
one were simply to place landmarks at the ends of each linear distances, the number
of traits would double to six. Moreover, in a landmark-based analysis variation at
each landmark is evaluated relative to every other landmark, leading to a more
comprehensive picture of how the geometry of a given shape varies between exper-
imental manipulations or over time.
After GPA, GM shape analysis typically involves performing a thin-plate spline
(TPS) analysis. The analogy that is typically used in the field is that TPS analysis
models a starting form (e.g., that of a wild-type zebrafish) as an infinitely thin metal
plate that is constrained at some combination of points (i.e., landmarks), but is
otherwise free to adopt a target form (e.g., that of a mutant zebrafish) in a way that
minimizes bending energy (Bookstein, 1991). Bending energy can be thought of as
the degree of deformation that is required to bend the Cartesian coordinate (x, y)
system such that two landmark configurations can be superimposed on top of one
another (Bookstein, 1991). The total deformation of the TPS can be decomposed into
geometrically orthogonal components based on scale/bending energy such that less
energy is required to bend a metal plate while holding it at distantly positioned
points, whereas much more energy is required to bend the plate to the same vertical
extend while holding two points in close proximity (Rohlf and Marcus, 1993). These
components (partial warps) can be localized to describe precisely what aspects of
shape are different. Because the number of partial warps can be large, as they scale
with the number of landmarks, a data reduction analysis is typically performed,
principal component analysis (PCA) being the most common. Performing a PCA on
partial warp scores is formally referred to as relative warp analysis (Zelditch et al.,
2004), and can be an effective method to identify the major axes of shape variation.
By applying these methods, we are able to show that adult AB and TL strains of
zebrafish significantly differ in craniofacial geometry along PC1 (one-way ANOVA:
F-ratio = 35.32, p < 0.001). Differences are largely restricted to the anterior head,
and show that TL zebrafish on average have longer upper (yellow region) and lower
(purple) jaws than AB wild-type fish, whereas the retroarticular process is longer in
ABs than it is in TLs (Fig. 6). TL fish harbor a mutation that results in the expression
of their characteristic long tails. It is interesting that, while a craniofacial phenotype
11. Morphogenesis of the Zebrafish Jaw: Development Beyond the Embryo 235
[(Fig._6)TD$IG]

Fig. 6 Results of a relative warps analysis on craniofacial shape in adult AB and TL lines.
Morphological variation between lines along RW1 (the horizontal axis) and RW 2 (the vertical axis).
The scatter plot suggests that RW1, the major axis of variation, distinguishes AB and TL lines.
Deformation grids along this axis show that AB fish have a relatively smaller maxillae (yellow) and
shorter mandible (purple). However, the length of the retroarticular process is larger in ABs. Note that this
analysis utilizes 15 landmarks in contrast to the seven used in our ontogenetic analysis because more
homologous regions were apparent at adult stages. However, despite this reduction in the amount of shape
variation collected AB and TL lines are still distinguishable with seven landmarks. (See Plate no. 12 in the
Color Plate Section.)

has never been noted for this stain, TL fish (at least in our lab) also possess elongated
dermal bones in the jaw. These results demonstrate the power of GM to discriminate
subtle but discrete differences in craniofacial morphology.

E. Analysis of Shape Variation


Zebrafish biologists may also be interested in the effects elicited by an experi-
mental treatment or mutation with respect to shape variation. Magnitudes of shape
236 Kevin J. Parsons et al.

variation may also change over the course of ontogeny (Zelditch et al., 2004). With
GM it is possible to quantify the degree to which a sample varies and statistically test
for differences in the magnitude of variation among groups. While many biologists
are familiar with how variance is calculated for univariate traits, shape is a more
complex multivariate trait that is not compatible with these more traditional meth-
ods. Fortunately, a metric for calculating variance in multivariate traits exists; instead
of ‘‘variance’’ the term ‘‘disparity’’ is used. Disparity is calculated using the
approach of Foote (1993) as follows:
X 2
D¼ ðdi Þ=ðN1Þ
Here di represents the Procrustes distance of the centroid of individual i from the
centroid of all N individuals. Procrustes distance is normally calculated by compar-
ing an individual specimen to the average shape of a group (which is called the
consensus) to provide a standard metric of shape difference. Here we test for
differences in measures of disparity across ontogenetic stages between our AB
and TL lines.
To test for differences in disparity, 1000 permutations of the difference in the
disparities between stages within AB and TL lines, as well as between these lines at
the same stage were performed. For the permutation tests, residuals of the Procrustes
fit were randomly assigned within each treatment group, and then recombined with
the mean form of that group to calculate the disparity of the permuted group. The
observed results were then compared with the range of values obtained via permu-
tation to determine if the observed values were significant. These tests were per-
formed using the program DisparityBox6 (available at www3.canisius.edu/sheets/
morphsoft.html).
Our analyses show that overall disparity decreases over time in both lines of
zebrafish. However, in ABs this decrease was statistically significant between 10
and 30 dpf, whereas in TLs there was a significant reduction in disparity that
occurred between the 30 dpf and adult stages (Table I). This suggests that although
reductions in shape disparity take place in both lines, these changes are initiated at
different times between lines. Comparisons between lines over ontogeny show that
initially the AB line is approximately twice as disparate than the TL line after 10 dpf
(p < 0.001, Table I), but no significant difference in disparity was observed at 30 dpf
or in adults (all p > 0.16). In fact, levels of disparity are nearly equal at adult stages

Table I
Changes in craniofacial shape disparity over ontogeny in AB and TL lines

Disparity Ontogenetic comparisons p-values from 1000 permutations

Line 10 dpf 30 dpf Adult 10 to 30 dpf 30 dpf to adult


AB 0.011 0.005 0.004 < 0.0001 0.108
TL 0.005 0.008 0.004 0.388 0.047
11. Morphogenesis of the Zebrafish Jaw: Development Beyond the Embryo 237

Table II
The rate of morphological development as measured by Partial Procrustes distancesa

Partial Procrustes distances, F, and p-value from comparisons between ontogenetic stages

Line 10 to 30 dpf 30 dpf to adult 10 dpf to adult


AB 0.125, F = 28.24, p = 0.001 0.129, F = 53.21, p = 0.001 0.218, F = 82.44, p = 0.001
TL 0.138, F = 31.08, p = 0.001 0.140, F = 37.10, p = 0.001 0.239, F = 132.11, p = 0.001

a
The F and p-values from a test performed between developmental stages within lines is presented. Note that partial Procrustes
distances are similar between AB and TL lines and did not differ significantly.

suggesting that an equally canalized phenotype has been achieved in both lines by
adulthood (Table I, Fig. 7).

F. Rates of Shape Change


Heterochrony has most often been studied in the context of evolutionary biology
and is usually defined as a change in the developmental rate or timing of the
appearance of features that create parallels between ontogeny and phylogeny
(Gould, 1977). More general definitions also exist that do not consider this paral-
lelism, and only require that a developmental event occurs at a different time or rate
in a descendant relative to an ancestor (McKinney and McNamara, 1991;

[(Fig._7)TD$IG]

Fig. 7 Plotted GPA superimposed landmarks for each measured stage (10 dpf, 30 dpf, and adult stages)
of craniofacial development in AB and TL lines of zebrafish. Note that the scatter of landmarks tends to
decrease over ontogeny, happening earlier in the AB line during the period between 10 and 30 dpf, while
for TLs a reduction in scatter does not occur until the period between 30 dpf and adult stages.
238 Kevin J. Parsons et al.

McNamara, 1995, 1997). In other words, heterochrony causes a decoupling of shape


from developmental time and may also be a theory of interest to developmental
biologists comparing wild-type (ancestor) to mutant (descendant) phenotypes. Since
any developmental process is temporal in nature, thereby making it susceptible to
heterochronic modifications, it is likely that heterochrony is a widespread phenom-
enon for zebrafish mutants relative to their respective wild-type ancestors.
The rate of morphological development over ontogeny can be determined empir-
ically in the absence of trajectories, but we recommend that researchers use the
approaches for quantifying trajectories discussed below in a complementary fashion
to gain a comprehensive idea of morphological development. Determination of the
ontogenetic rate of morphological development is based on the morphometric dis-
tance between shapes at a given ontogenetic stage in relation to the average specimen
(of a treatment group at one stage of the experiment), and this is calculated as the
partial Procrustes distance (Bookstein, 1996; Dryden and Mardia, 1998; Zelditch
et al., 2004). We tested whether rates of morphological development differed over
ontogeny among AB and TL zebrafish lines (Table II). Ontogenetic tests for differ-
ences in the amount of shape change in morphometric distance between 10 and
30 dpf, 30 dpf and adult, and 10 dpf and adult specimens were performed between
lines using 900 bootstrap replicates while stage specific tests between lines were
conducted by performing Goodall’s F-test in the program TwoGroup6h (available at
http://www3.canisius.edu/sheets/morphsoft.html). Goodall’s F-test compares the
difference in the mean shape between two samples relative to the shape variation
found within the samples.
Our analysis showed that rates of morphological development did not differ
between our zebrafish lines at any of the ontogenetic intervals tested. However,
stage specific tests did reveal that significant differences in the position of AB and
TL lines in shape space (distance) were present at all three stages of the experiment,
indicating that lines did differ in shape at each stage. Taken together these data
suggest that while rates of shape change do not differ between lines at any stage, both
lines are characterized by marked shifts in rates of morphological development over
time, which may be a common feature of zebrafish craniofacial development.

G. Trajectories of Shape in Zebrafish Lines


Mutations may also modify the direction of ontogenetic shape change in zebra-
fish. This aspect of morphological development is distinct from changes in shape
due to alterations in the rate or timing of development (i.e., heterochrony). Whereas
heterochrony refers to changes in the timing of developmental events, allometric
repatterning refers to changes in the trajectory of development. Allometric repat-
terning can gradually develop over long intervals of ontogeny whereas heterochrony
may be sudden. Since both heterochrony and allometric repatterning can affect
shape, it is useful to consider both phenomena in relation to each other.
We can quantitatively test whether allometric repatterning has occurred between
AB and TL zebrafish lines by measuring their shape trajectories in relation to a
11. Morphogenesis of the Zebrafish Jaw: Development Beyond the Embryo 239

Table III
The angles between ontogenetic trajectories of craniofacial shape in AB and TL linesa

Angle between ontogenetic stages

Line 10 to 30 dpf 30 dpf to adult 10 dpf to adult


AB 84.1 * 127.0 * 101.5 *
TL 140.5 * 76.5 101.5

a
Trajectories are measured between all stages including 10 dpf, 30 dpf, and adults.

continuous variable (ln centroid size in this case). We accomplish this here by
measuring ontogenetic growth angles, which are the arc cosines of vector correla-
tions (Zelditch et al., 2000), between AB and TL zebrafish at 10 dpf, 30 dpf, and
adult stages. Similar to the analysis of disparity described above the calculation of
angle is simply a metric and there is no true statistical model. We therefore compared
the observed angle between ontogenetic vectors to bootstrapped estimates of these
angles. The null hypothesis was an angle of zero, which is biologically equivalent to
the hypothesis of a conserved ontogenetic trajectory of shape between AB and TL
lines. To test this null hypothesis, 95% confidence intervals for the angles were
created through a procedure involving 900 bootstrapped replicates. The angle
between the vectors would be considered statistically significant if they exceeded
those drawn from a bootstrapped distribution within a group. In other words the
differences between groups of interest would have to exceed those generated from
bootstrapping within both groups. This explains how smaller overall differences in
trajectories may sometimes be significant, whereas larger differences are not.
Growth angles and bootstrap estimates of 95% confidence intervals were calculated
in VecCompar6c (available at http://www3.canisius.edu/sheets/morphsoft.html).