C H A P T E R
1
History of Zebrafish Research
Judith S. Eisen
Institute of Neuroscience, University of Oregon, Eugene, OR, United States of America
Although PubMed (pubmed.gov) lists papers study-
ing zebrafish embryos as early as the 1950s (Fig. 1.1),
zebrafish research did not hit its stride until nearly a
decade after publication of the pioneering work by
George Streisinger in 1981 (Streisinger, Walker, Dower,
Knauber, & Singer, 1981). Why has this small tropical
fish become an important biomedical model and what
has made its popularity soar beginning from the mid
1990s?
Setting the Stage
Scientists have been fascinated with learning how
humans and other animals develop since well before
the common era. Hippocrates, the renown Greek physi-
cian who lived over 2500 years ago and is considered the
father of modern medicine, or at least his followers of
the Hippocratic school are so considered, provided an
overview of human embryology in several volumes
including the “Hippocratic Treatises ‘On Generation’
and ‘On the Nature of the Child,’” and noted the similar-
ity to chick embryology (Lonie, 1981; Needham, 1963).
Aristotle, the celebrated Greek scientist and philosopher
who lived about 200 years later also carried out exten-
sive observations of animal development based on dis-
sections at various developmental stages, which he
published in his books “On the Generation of Animals,”
“The History of Animals,” “On Respiration,” and “On
the Motion of Animals” (Gilbert, Barresi, 2016; Need-
ham, 1963). Fast forward 1700 years to when another his-
torical luminary, Leonardo da Vinci, one of the foremost
artists and inventors during the Italian Renaissance,
contributed significantly to our understanding of em-
bryonic development through drawings and quantita-
tive measurements of dissected avian and mammalian
embryos, including a human fetus (Needham, 1963).
Why the enduring fascination with embryology? Part
of it undoubtedly comes from the difficulty of
The Zebrafish in Biomedical Research
https://doi.org/10.1016/B978-0-12-812431-4.00001-4
understanding developmental processes that occur
deep inside a human fetus’s mother, and thus remain
mysterious. These invisible processes could be under-
stood if the development of other animals were discov-
ered to mirror that of humans. Finding that this is the
case would allow animals to serve as proxies, models
that we can investigate in ways in which we cannot
study ourselves, and would enable us to discover how
human development normally unfolds and how
abnormal development might lead to human birth de-
fects. The hypothesis that animal development could
serve as a model for human development was estab-
lished early, for example by comparisons between hu-
man and animal embryos made by the Hippocratic
school and by Aristotle (Lonie, 1981; Needham, 1963),
and reinforced more recently, for example in the late
1800s by Ernst Haeckel whose drawings of developing
animals revealed the similarities of their embryonic
body plans and how these morphed into related, but
distinct, species-specific, adult forms during develop-
ment (Fig. 1.2).
The idea that studying a variety of animals could
reveal the principles underlying development suggests
that it is reasonable to choose a model to study based
on its experimental tractability for addressing a partic-
ular research question. In this regard, teleost fish have
been particularly useful for many types of observations
(Oppenheimer, 1936; Wourms, 1997; Wourms, Whitt,
1981). For embryology, oviparous teleosts have been
particularly advantageous because fertilization, and
indeed their entire developmental process, unfolds in
the water column, and thus, they do not have to be
extracted from the mother for observation or experimen-
tation (Oppenheimer, 1936). In addition, some teleost
embryos show “striking lucidity,” providing an excep-
tional platform for microscopic observations of morpho-
genetic and cellular processes during the unfolding of
living embryos (Trinkaus, 1990).
3 © 2020 Elsevier Inc. All rights reserved.
4 1. History of Zebrafish Research
FIGURE 1.1 Since the 1990s there has been significant acceleration in publication of papers concerning zebrafish. Data from PubMed (https://
www.ncbi.nlm.nih.gov/pubmed/?term¼zebrafish).
Interestingly, Aristotle contributed not only to our un-
derstanding of embryology of terrestrial vertebrates, but
he also appears to have been the first contributor to our
recorded knowledge of teleost development, an area to
which there was little in the way of additional recorded
information until the 1700s (Oppenheimer, 1936;
Wourms, 1997). Although he was limited to observa-
tions that could be made without magnification, Aristo-
tle was still able to describe aspects of teleost egg
development and compare them with the development
of avian eggs (Oppenheimer, 1936). From the beginning
of the 18th century, a variety of teleost species have been
studied to uncover aspects of embryonic development
(Wourms, 1997); readers are referred to the excellent re-
views cited above to learn more about the breadth of
contributions made by these species to understanding
embryogenesis.
Notable among the fish that have contributed to un-
derstanding vertebrate embryonic development is the
killifish, Fundulus heteroclitus, commonly known as the
mummichog or simply Fundulus (Atz, 1986). This fish
played a prominent role in the early days of the now
world-renowned Embryology Course at the Marine Bio-
logical Laboratories in Wood Hole, Massachusetts (Atz,
1986). In the late 1800s and early 1900s, a number of sci-
entists developed exquisitely detailed descriptions of
Fundulus embryology. Their drawings provided a foun-
dation for experimental studies exploring the potential
of different parts of the developing embryo to respond
to a variety of perturbations, for example, extirpation
and grafting. A series of biology luminaries published
studies of Fundulus embryos during this time, including
Thomas Hunt Morgan, the father of genetics of the fruit
fly, Drosophila melanogaster, Jacques Loeb, who helped
I. Introduction
pioneer experimental biology (https://embryo.asu.
edu/pages/jacques-loeb-1859-1924) as well as compara-
tive brain physiology and psychology (http://www.
thecrimson.com/article/1965/2/11/jacques-loeb-
bridging-biology-and-metaphysics/), Philip Armstrong
and Julie Swope Child at the Woods Hole Marine Biolog-
ical Laboratories, Jane Oppenheimer, a pioneering pro-
fessor at Bryn Mawr, John Paul Trinkaus at Yale, and
William Ballard at Dartmouth (Loeb, 1916; Morgan,
1895; Oppenheimer, 1936, 1979; Trinkaus, 1990)
(https://embryo.asu.edu/featured/1549). These
studies provided important insights into the processes
occurring during vertebrate embryogenesis and laid
the groundwork for research carried out today.
During this same time frame, the rediscovery of Men-
del’s genetic experiments ushered in an era of concerted
effort to learn the genetic basis of inheritance (Gilbert,
1978). Initially, this work included understanding the
relationship between genes and development. However,
these disciplinesdembryology and geneticsdbecame
largely separate in the early 1900s and remained that
way for many decades (Gilbert, 1991, 1998). The study
of embryology and genetics began to reunite in the
1970s when a number of researchers started to use ge-
netic approaches in fruit flies to dissect the basis of
development. For example, as a step toward his dream
of building a bridge between developmental genetics
and molecular biology, Ernst Hadorn, developmental
genetics pioneer of the University of Zurich, organized
a conference on this topic in 1972 (Nothinger, 2002).
Another developmental genetics pioneer, Edward Lewis
at the California Institute of Technology, investigated
how genes controlled the development of specialized or-
gans from individual body segments, work for which he
 Establishing the Zebrafish Model
FIGURE 1.2 This cover of the August 1989 edition of Trends in
Genetics shows the similarity among vertebrate animals in a modified
drawing from Ernst Haeckel’s 1879 book “The Evolution of Man.” The
top row shows fish, salamander, tortoise, and chicken embryos at an
early stage of embryonic development when they are very similar. The
middle and bottom rows show the same animals at increasingly later
developmental stages; the last stage shows species-specific character-
istics. It is important to note that although the picture shown here has
been widely reproduced, it remains controversial because not all fea-
tures of embryos illustrated in the top row are conserved, as described
in more detail by Richardson et al. (1997). Cover image of Trends in
Genetics volume 5 issue 56 from August 1989 used with permission from
Elsevier.
was awarded a Nobel Prize in Physiology or Medicine in
1995 (https://www.nobelprize.org/nobel_prizes/
medicine/laureates/1995/press.html). Christiane
Nusslein-Volhard and Eric Wieschaus conducted a ge-
netic screen in fruit flies to learn the mechanisms under-
lying body patterning (Nusslein-Volhard & Wieschaus,
1980) and shared the 1995 Nobel Prize with Lewis. Sub-
sequent experiments in the late 1980s and early 1990s
uncovered the molecular nature of the mutations iso-
lated in the Nusslein-Volhard/Wieschaus screen
I. Introduction
5
(McGinnis & Krumlauf, 1992) and revealed that the ba-
sis for the similarities in embryogenesis among dispa-
rate animals is that development is orchestrated by
orthologs of the same genes in animals as diverse as fruit
flies, chickens, mice, and humans (Beddington & Smith,
1993; Carver & Stubbs, 1997; Davidson, 1994; Gellon &
McGinnis, 1998; Manak & Scott, 1994). This realization
cemented the idea that animals could be investigated
not only to unveil the secrets of their development but
also as models to reveal the secrets of human
development.
Establishing the Zebrafish Model
It is against this backdrop that George Streisinger, a
professor in the nascent Institute of Molecular Biology
at the University of Oregon, chose zebrafish, Danio rerio,
a small, tropical, fresh-water fish, as a model in which to
explore the genetic basis of vertebrate neural develop-
ment. Streisinger was a pioneering molecular biologist
who contributed significantly to the dawn of the modern
molecular era through his historic work on bacterio-
phage in the 1950s. Among his major contributions
were an understanding of the DNA triplet code,
including deciphering the first in vivo codon assign-
ments, as well as uncovering the structure of the bacte-
riophage genome (Grunwald & Eisen, 2002; Stahl,
1995). One of Streisinger’s close University of Oregon
colleagues, and an avid supporter of his zebrafish en-
deavors, was Franklin Stahl, another molecular biology
pioneer who discovered that DNA replication is semi-
conservative (Streisinger, 2004). By the mid-1960s,
many of the initial questions of molecular biology
were nearing resolution and attention was turning to-
ward using genetic approaches to understand the mech-
anisms underlying more complex systems, such as the
“almost mystical” questions of nervous system function
and the basis of behavior and cognition (Grunwald &
Eisen, 2002). In parallel with Streisinger’s choice to use
zebrafish to dissect nervous system function and devel-
opment, two other molecular biology pioneers estab-
lished other genetic models to investigate development
(Grunwald & Eisen, 2002). Sidney Brenner, codiscoverer
of messenger RNA, who also showed that the amino
acid sequence of a protein is determined by the RNA
nucleotide sequence from which it is translated, devel-
oped the roundworm, Caenorhabditis elegans as a new ge-
netic model, work for which he was awarded a Nobel
Prize in Physiology or Medicine in 2002 (http://www.
nobelprize.org/nobel_prizes/medicine/laureates/
2002/). Seymour Benzer, who used bacteriophage to
reveal the structure of genes and how they are aligned
within the genome, began studying nervous system
function and behavior using fruit flies.
6 1. History of Zebrafish Research
FIGURE 1.3 This cover of the July 1995 issue of Developmental
Dynamics shows a portion of the zebrafish staging series, a crucial
advance for standardizing conditions between different experiments
and laboratories. A fertilized egg is shown at the top, and development
progresses clockwise. All of the fish were imaged live (Kimmel,
Ballard, Kimmel, Ullmann, & Schilling, 1995). The fish in the middle is
3 days postfertilization (3 dpf) (an enlarged view of a fish of this stage
is also shown separately, below the cover image). By three dpf,
zebrafish have hatched and are referred to as larvae, whereas, at earlier
stages, before hatching, they are called embryos. The head of the three
dpf larva in the cover image is at the top; the head of the larva below
the cover image is to the left. The dark region of the head is the eye. The
heart appears red because it contains red blood cells. The large struc-
ture under the heart is the yolk, which will nourish the larva until it is
able to eat, starting about 1 day later than when this photograph was
taken. The embryo at the top of the cover image was just fertilized and
is shown within its chorion or eggshell. The chorions were removed
prior to taking the other photographs. The next embryo, going clock-
wise, is at the eight-cell stage. All 8 cells sit on top of the large yolk cell,
although only four of them are visible; four of them are hidden behind
the visible cells. The next embryo is at the 256-cell stage, followed by an
embryo at the 50% epiboly stage. During epiboly, the cells move from
sitting on top of the yolk to completely surrounding it. The next em-
bryo, at five o’clock in the cover image, is at the bud stage; “bud” refers
=
to the tailbud, the beginning of the tail, which is pointed toward the
bottom of the photograph. The embryo at six o’clock in this cover
image is at the four-somite stage; somites are the precursors of the
segmented body muscles that fish use for swimming. The embryo at
I. Introduction
Why did Streisinger choose zebrafish? Research on
Fundulus had already provided many insights into
vertebrate development, so Fundulus could have been
an obvious choice, despite lacking any previous history
of genetic research. There were, however, several other
fish species with considerable histories of genetic
research, including medaka (Shima & Mitani, 2004),
goldfish (Fu, 2016) and Xiphophorus (Kazianis, 2006).
Streisinger initially brought a number of tropical fish
species, including medaka, into his laboratory; however,
no written records describing the basis of his choice have
been uncovered (Grunwald & Eisen, 2002). Although it
is only speculation, perhaps the difficulty of removing
Fundulus embryos from the protective eggshell, or
chorion (Atz, 1986; Trinkaus, 1990), or the inclusion
within Fundulus and medaka embryos of oil droplets
that obscure visibility (Wourms, 1997), contributed to
Streisinger’s ultimate choice of zebrafish. During the
late 1950s and early 1960s, several other laboratories
also began investigating zebrafish embryology
(Hisaoka, 1958), including muscle and nervous system
development (van Raamsdonk, Mos, Smit-Onel, van
de Laarse, & Fehres, 1983; van Raamsdonk, Tekronnie,
Pool, & van de Laarse, 1980; Weis, 1968a, 1968b), as
well as using zebrafish in the aquaculture trade to study
toxicology (Laale, 1977). However, none of these studies
incorporated the type of genetic analysis that was cen-
tral to Streisinger’s approach to obtaining a mechanistic
understanding, and whether any of this work contrib-
uted to Streisinger’s choice of zebrafish is unknown.
Whatever the reasons for Streisinger’s choice of
zebrafish, it is now clear that zebrafish is an excellent
research model, based on several characteristics of its
development. First, in contrast to many fish, zebrafish
are not seasonal breeders; thus, embryos can be obtained
for study throughout the year. Second, like most fish,
fertilization, and all subsequent developmental pro-
cesses occur within the water column, not inside of the
mother; thus, zebrafish are amenable to both observa-
tion and manipulation throughout the developmental
period. Third, a single female zebrafish can produce
hundreds of eggs in a single spawning, making it
possible to have statistically significant numbers of
closely related embryos for even a single experiment.
Fourth, zebrafish embryos and early larvae are nearly
transparent, thus using appropriate microscopic
methods, it is possible to image essentially every cell
during development (Fig. 1.3). Fifth, zebrafish embryos
develop rapidly, hatching as predatory larvae in 3 days
seven o’clock is at the fifteen-somite stage, and the embryo at nine
o’clock is at the twenty five-somite stage; this embryo is nearly 22 h
postfertilization. The embryo at 11 o’clock is about 28 h postfertiliza-
tion. Cover image of Developmental Dynamics volume 203 number
three from July 1995 used with permission from Wiley.
 Synthesizing Genetics and Embryology
(Fig. 1.3), and thus development of individual cells or
structures deep inside a zebrafish embryo can be
observed in real time, or by time-lapse videography, as
they occur.
Streisinger’s focus on using genetics to uncover
developmental processes necessitated establishing tools
appropriate for this purpose. Vertebrate genetics is
cumbersome because, after mutagenesis, heterozygous
carriers need to be crossed to wild types to establish a
stock that will contain both wild types and heterozy-
gotes, and then individuals within that stock need to
be crossed to one another to identify those that carry
mutations of interest. This identification is accomplished
FIGURE 1.4 This issue of Nature from MayeJune 1981 published
the groundbreaking paper from George Streisinger showing that
zebrafish are amenable to genetic analysis (Grunwald & Eisen, 2002;
Streisinger et al., 1981). This cover illustrates zebrafish embryos,
enclosed within their chorions. Some of the embryos in this picture
have normal, black pigment cells in their skin and eyes, but some
lack black pigment cells in their skin and eyes, and thus, appear golden
in color. The difference in these embryos is the result of a mutation in a
single gene, which is called golden, after the appearance of the
mutant embryos. The discoverers of the molecular nature of the
zebrafish golden gene showed that the human ortholog is responsible
for 25%e40% of the difference in skin color between Europeans and
West Africans (Lamason et al., 2005). Cover image of Nature volume 291
issue 5813 from MayeJune 1981 used with permission from Springer Nature.
I. Introduction
7
by screening the offspring of the mated heterozygotes,
only one-quarter of which will show a phenotype
when a mutation is recessive. Streisinger reasoned that
he could facilitate the production of homozygous
offspring by circumventing the need to breed heterozy-
gous male and female partners (Grunwald & Eisen,
2002). He achieved this goal by adapting methods to
activate eggs without fertilization, as well as by methods
to produce gynogenotes, homozygous diploid embryos
whose entire genetic contribution derives from the
mother. These gynogenotes were useful not only for ge-
netic screens, but Cyrus Levinthal at Columbia Univer-
sity obtained some gynogenotes from Streisinger
(Charline Walker, personal communication) to investi-
gate axon trajectories of isogenic neurons (http://
www.nasonline.org/publications/biographical-
memoirs/memoir-pdfs/levinthal-cyrus.pdf). Streising-
er’s seminal work describing the cloning of zebrafish
was published in 1981 in Nature (Streisinger et al.,
1981) and heralded on the cover (Fig. 1.4), just 7 months
after publication in the same journal of the seminal work
on fruit flies (Nusslein-Volhard & Wieschaus, 1980) for
which Nusslein-Volhard and Wieschaus were awarded
the 1995 Nobel Prize in Physiology or Medicine that
they shared with Edward Lewis. Streisinger’s article
received national attention, including an editorial
cartoon published in the Chicago Tribune (Fig. 1.5).
Synthesizing Genetics and Embryology
Streisinger’s ability to develop zebrafish as a model
was fostered by the atmosphere of the University of Ore-
gon Institute of Molecular Biology. Founded in 1959, the
Institute nurtured a handful of the brightest young mo-
lecular biologists, in part by pooling resources for equip-
ment, facilities, and administrative support to help
FIGURE 1.5 This cartoon was published in the Chicago Tribune as
a response to George Streisinger’s seminal paper describing the clon-
ing of zebrafish (Streisinger et al., 1981). Wayne Stakyskal cartoon from
1981 used with permission from the Chicago Tribune.
8 1. History of Zebrafish Research
underwrite long-term projects, including Streisinger’s
zebrafish research program, which took over 10 years
to come to fruition (Grunwald & Eisen, 2002; Streisinger,
2004). Oregon also provided an extremely collegial at-
mosphere and even before zebrafish was ready for
prime time as a genetic model, Charles Kimmel, one of
Streisinger’s Oregon colleagues who made important
early contributions to understanding clonal selection in
the immune system (Tauber & Podolsky, 2000), realized
the potential of the zebrafish embryo for understanding
the cellular basis of neural development and initiated
this as a new direction in his research program. Kimmel
started his work in this area by describing the develop-
ment of an identified neuron, the Mauthner cell
(Kimmel, Sessions, & Kimmel, 1981), made famous by
work in other teleosts and in amphibians (Korn & Faber,
2005). Kimmel’s work revealed that morphologically
similar neurons were repeated in each hindbrain
segment (Metcalfe, Mendelson, & Kimmel, 1986), lead-
ing him to ask whether these neurons could be related
by cell lineage (Grunwald & Eisen, 2002). The optical
clarity and rapid development of the zebrafish embryo
make it ideally suited to carry out lineage studies. So
in the summer of 1982 Kimmel visited Michael Bennett’s
laboratory at the Marine Biological Laboratory (Kimmel,
personal communication) to learn how to inject individ-
ual cells, called blastomeres, in young Fundulus em-
bryos, using a fluorescent dye called Lucifer Yellow,
developed to the study passage of molecules between
cells via gap junctions (Kimmel, Spray, & Bennett,
1984; Stewart, 1981). Kimmel was later joined in his
zebrafish lineage tracing endeavors, by Oregon col-
leagues Monte Westerfield and Judith Eisen, both of
whom had backgrounds using Lucifer Yellow to label
and record from neurons, and who worked with
Kimmel to establish the use of another fluorescent dye,
fluorescein dextran, that did not pass through gap
junctions.
Kimmel’s lineage studies, many carried out with the
partnership of undergraduate students, were
completely captivating. No one knew the time course
of development, and with the instrumentation available
at the time, it was not possible to make the kind of
computer-controlled, time-lapse movies that are routine
today. Even if it were possible, no forum existed at that
time in which such movies could be published. So days
were divided up into manageable segments of four or
more hours, and teams of people sat during each
segment in a completely dark room, except for instru-
ment lights and what would be by today’s standards a
miniscule video monitor, and watched and took notes,
as cell movements and divisions were recorded onto a
videocassette recorder, and in many cases traced with
different colored markers onto sheets of acetate taped
to the video monitor screen. These studies provided
I. Introduction
unprecedented insights into vertebrate development
and enabled Kimmel to describe lineage contributions
to essentially all of the major cell types in the embryo
(Kimmel & Warga, 1988). During some of these sessions,
Eisen and Westerfield watched as the earliest-
developing spinal motoneurons extended axonsd
something never seen before in vertebrate embryosd
and were able to classify these neurons into distinct,
identifiable subtypes that could later be studied using
a genetic approach (Beattie, Raible, Henion, & Eisen,
1999; Eisen, Myers, & Westerfield, 1986).
This was a heady time for those involved. The clarity
of the zebrafish embryo was stunning and the applica-
tion of fluorescent dye technology, previously used
only for studies of gap junctional coupling in amphib-
ians and Fundulus (Kimmel et al., 1984; Spray, Harris,
& Bennett, 1979), lineage tracing and neuronal
morphology in the medicinal leech (Stewart, 1981; Weis-
blat, Zackson, Blair, & Young, 1980), and laser-ablation
in crustaceans (Marder & Eisen, 1984; Selverston &
Miller, 1980), meant that researchers learned something
new from every gorgeous experiment. And every exper-
iment revealed phenotypes that could be investigated by
combining these modern embryological and neurobio-
logical approaches with Streisinger’s newly developed
genetic tools. Casting a pall over the nascent field of
zebrafish developmental genetics, Streisinger died
completely unexpectedly in August 1984, and thus, he
never saw the blossoming of this new and exciting era.
Streisinger’s death could have been the end of the
new beginning for zebrafish research. However, his Ore-
gon colleagues, already deeply immersed in their
studies of zebrafish lineage and neural development,
took up the baton and began promoting zebrafish as
an outstanding new model in which to investigate the
genetic basis of developmental processes. A critical
aspect of support for zebrafish research came from the
National Institutes of Health, which allowed Kimmel
to become Principal Investigator of Streisinger’s grant
to study zebrafish genetics. David Grunwald, at that
time a postdoctoral fellow in Streisinger’s laboratory,
and Charline Walker, the research assistant who had
worked with Streisinger to develop his genetic tools,
were instrumental in helping keep up the momentum
of zebrafish research. The Oregon group’s studies had
already provided important new insights into the com-
monality of the developmental processes among verte-
brates, leading Kimmel to publish an influential article,
in the issue of Trends in Genetics shown in Fig. 1.2,
declaring “the fish is a frog . ..is a chicken . ..is a
mouse” that can be studied “for both detailed .
..embryogenesis and . ..genetic analysis” (Grun-
wald & Eisen, 2002; Kimmel, 1989).
By this time, a number of researchers prominent for
their studies of fruit flies, including Jose Campos-
 Synthesizing Genetics and Embryology 9

FIGURE 1.6 This issue of Development from December 1996 was devoted entirely to papers describing the first large scale genetic screens for
zebrafish mutations carried out in the Nusslein-Volhard laboratory at the Max-Planck-Institute fur Entwicklungsbiologie in Tubingen Germany
and the Driever and Fishman laboratories at Harvard in Boston Massachusetts. There were 37 papers describing the characterization of about 4000
mutations (Eisen, 1996; Grunwald & Eisen, 2002). Cover image of Development volume 123 from December 1996 used with permission from Devel-
opment and Robert Kelsh.

Ortega at the University of Cologne, as well as Nusslein-
Volhard, had taken note of the work being done in Ore-
gon and had begun to consider adding zebrafish studies
to their repertoire (Grunwald & Eisen, 2002). In 1990, the
Oregon group facilitated this possibility by hosting a
small meeting that brought together researchers from a
variety of fields and highlighted the utility of zebrafish
as a model. They also established an informal course
on zebrafish husbandry, genetics, and embryology, and
hosted numerous visiting scientists from around the
world who came to learn this new model system
(Grunwald & Eisen, 2002). Westerfield created the
“Zebrafish Book” (http://zfin.org/zf_info/zfbook/
zfbk.html), a primer on zebrafish methodology, and later
established the Zebrafish Information Network (zfin;
http://zfin.org/), which hosts the Zebrafish Model Or-
ganism Database. Westerfield also founded ZIRC, the
Zebrafish International Resource Center, the first zebra-
fish genetic repository. In 1994, Cold Spring Harbor
hosted the first open international meeting on zebrafish
development and genetics, and in 1998, a course on
zebrafish development and genetics was initiated at
the Marine Biological Laboratory in Woods Hole,
Massachusetts. In the ensuing years, there has been a
proliferation of zebrafish meetings, as well as courses
held in a variety of venues around the world.

I. Introduction
10 1. History of Zebrafish Research
Making a Big Splash
Although the Oregon group continued to use Strei-
singer’s methodology to conduct mutational screens,
uncovering genes involved in a variety of different
developmental processes, Nusslein-Volhard developed
a more ambitious plan, to essentially recapitulate in
zebrafish the screen she had carried out to discover
embryonic patterning mutants in fruit flies (Mullins,
Hammerschmidt, Haffter, & Nusslein-Volhard, 1994;
Mullins & Nusslein-Volhard, 1993). As she was devel-
oping new mutagenesis and fish-rearing procedures
for this endeavor in her laboratory at the Max Planck
Institute in Tubingen, Germany, her talented former
graduate student, Wolfgang Driever, who had spent a
year as a postdoctoral fellow with Westerfield in Ore-
gon, was recruited to Massachusetts General Hospital
by Marc Fishman, to establish a parallel screening effort.
In similar fashion to the earlier fruit fly mutational
screen, the “Big Screen” in zebrafish revealed mutations
that affected nearly every aspect of the embryonic body
plan and provided an exceptional window into the
genetic processes governing vertebrate development.
This was the largest forward mutational screen ever
undertaken in a vertebrate and a tour de force by the
Nusslein-Volhard and Driever laboratories. The screen
involved 65 people, including Nusslein-Volhard’s
colleague Friedrich Bonhoeffer and Driever’s colleague
Fishman, as well as many brilliant young students and
postdoctoral fellows who went on to establish their
own laboratories. Participants examined more than a
million and a half embryos over about a 2-year period,
resulting in the isolation of over 4000 mutations, about
half of which were characterized over the next year
(Driever et al., 1996; Eisen, 1996; Haffter et al., 1996).
Rather than publish these characterizations piece-
meal, as a series of papers describing mutations
affecting specific aspects of embryonic development,
the entire set of 37 screen papers was published as a
cohesive collection that constituted an entire, ectopic
volume of the journal Development (December 1996,
volume 123; Fig. 1.6), complete with a flipbook that
revealed the process of embryogenesis as viewed by
time-lapse microscopy (Karlstrom & Kane, 1996). This
was a historic decision because it facilitated the assess-
ment of the entire range of mutant phenotypes. The
brilliance of both the earlier fruit fly screen and also
the zebrafish “Big Screen” was categorizing mutants
into phenotypic groups. This organization allowed
gene functions to be ascribed to temporal and spatial
pathways even before the genes themselves had been
identified, and greatly facilitated elucidation of the
molecular pathways underlying developmental pro-
cesses. The “Big Screen” catapulted zebrafish to the
I. Introduction
forefront as an outstanding new model in which to
investigate the genetic underpinnings of essentially
every aspect of vertebrate development. Importantly,
these mutants were made available so that other labora-
tories could utilize zebrafish to begin or enhance inves-
tigations focused on particular developmental
processes.
Developing New Tools for Molecular Analyses
Understanding the mechanisms underlying mutant
phenotypes revealed by the “Big Screen” as well as other,
smaller screens (for example (Beattie et al., 1999)),
required learning which genes were affected and the mo-
lecular nature of the defects. Several years earlier, John
Postlethwait had anticipated the need for molecular land-
marks spread out across the genome and created the first
genetic linkage map for zebrafish (Johnson, Midson, Bal-
linger, & Postlethwait, 1994). Consistent with the Oregon
way of carrying out science, much of the mapping was
done by a team of exceptional undergraduate students.
The techniques used to establish the map (Postlethwait
& Talbot, 1997) made it relatively straightforward to
begin to clone the genes defined by mutant phenotypes,
and thus, to understand not only what had gone awry,
but also to learn how these mutated genes and resulting
phenotypes corresponded to the genotypes and pheno-
types of other organisms. In addition, these studies
revealed that the zebrafish lineage had experienced a
genome duplication event and that this event was shared
by all teleosts, an understanding that allowed accurate
connection of the zebrafish genome to the human
genome (Amores et al., 1998; Force et al., 1999). Perhaps
not surprisingly by this time, the cloning of zebrafish
mutations again reinforced the idea that similarities in
embryogenesis among different animals are governed
by orthologs of the same genes, opening the path to use
zebrafish to investigate genome architecture and evolu-
tion (Postlethwait et al., 1998), as well as to serve as a
model for human genetic diseases (Zon, 1999).
The momentum of zebrafish research was already
accelerating, and it was clear that additional resources
would enable many more laboratories to embrace this
model. The nascent zebrafish research community,
represented by John Postlethwait and Marc Fishman,
as well as Len Zon from Harvard Medical School and
Nancy Hopkins from the Massachusetts Institute of
Technology, worked with the then National Institutes
of Health Director, Harold Varmus, and in 1998, the
NIH established the “Trans-NIH Zebrafish Initiative”
in which many participating NIH institutes solicited
applications “to increase our support of the zebrafish
as an animal model for development, organ formation,
 Expanding Zebrafish Research Into New Areas
behavior, aging, and disease research” (https://grants.
nih.gov/grants/guide/pa-files/PA-01-095.html). It was
now evident that zebrafish had arrived as a model!
As more laboratories adopted the zebrafish model,
new resources were developed, and following in the
tradition of establishing an interactive research commu-
nity, these resources were readily shared. For example,
at one of the early Cold Spring Harbor Zebrafish Devel-
opment and Genetics Meetings, researchers from
Harvard brought and shared reagents, and later
Chi-Bin Chien from the University of Utah also shared
reagents at a Strategic Conference of Zebrafish Re-
searchers. In a similar fashion, Steve Ekker at the
Mayo Clinic developed and shared the antisense
methods for knocking out the function of nearly any
gene (Nasevicius & Ekker, 2000), and more recently a
number of laboratories have developed and shared
gene-editing strategies (Varshney, Sood, & Burgess,
2015), as well as new strategies for cloning existing mu-
tations (Hill et al., 2013; Miller, Obholzer, Shah, Mega-
son, & Moens, 2013).
Expanding Zebrafish Research Into New Areas
Zebrafish has continued to increase in popularity as a
model to investigate the cellular, molecular, and genetic
mechanisms underlying developmental processes (see
Chapter 45 by Pathak and Barresi). As of 2019, there
were over 1300 laboratories worldwide listed in ZFIN
(http://zfin.org/) that utilize zebrafish to investigate
many aspects of animal biology, including human
disease mechanisms. Below I describe some of the new
directions for zebrafish research that extend beyond
understanding the types of developmental mechanisms
illustrated above.
Learning how the nervous system orchestrates
behavior remains a significant challenge that is being
addressed by the international “Brain Initiative”
(https://www.braininitiative.nih.gov/). Zebrafish are
ideally suited for investigating the basic biological
mechanisms underlying neural function because of their
accessibility for behavioral measurements, in concert
with genetic and neural activity manipulation and imag-
ing studies (see Chapter 46 by Mcarthur and colleagues).
Zebrafish exhibit a variety of complex behaviors,
including, among many others, sleep and social interac-
tions (Orger & de Polavieja, 2017; Stednitz et al., 2018),
that should provide new insights into the underlying
mechanisms that can then be translated into under-
standing brain mechanisms and behavior in humans
under normal conditions and in disease states.
Understanding the molecular mechanisms of human
genetic disorders is a prerequisite for developing new
tools to diagnose and treat these diseases. The
I. Introduction
11
outstanding experimental attributes of zebrafish are
being increasingly leveraged to establish functional
models of human genetic diseases and to develop new
clinical tools for their diagnosis and treatment (Phillips
& Westerfield, 2014) (see Chapter 47 by Phillips and West-
erfield and Chapter 49 by Rissone and colleagues). The
list of human disease models to which zebrafish is mak-
ing important contributions includes cancer, tuberculosis,
neurodevelopmental, neurodegenerative and psychiatric
disorders, kidney diseases, cardiovascular diseases, skel-
etomuscular diseases, gastrointestinal tract dysfunctions;
this list continues to expand at a dizzying pace, as exem-
plified by many of the chapters within this volume.
Discovering new drugs is critical for developing ther-
apeutic approaches to human diseases. Zebrafish has
become an excellent model for drug discovery because
it is straightforward to use chemical screens, in which
zebrafish, typically embryos or larvae, are exposed to li-
braries containing many different molecules with phar-
maceutical potential, to identify novel therapeutic
agents (Peterson, Link, Dowling, & Schreiber, 2000)
(see Chapter 51 by Zhang and Peterson). In fact,
numerous chemical screens have been carried out and
are already providing important insights that have im-
plications for understanding human health (Rennekamp
& Peterson, 2015). This effort has been augmented by the
recent realization that human cancer cells can be
implanted into zebrafish, thus serving as “avatars”
that present highly sensitive platforms for developing
and testing pharmacological therapies matched to spe-
cific tumor behaviors (Fior et al., 2017; Leslie, 2017).
Another exciting new direction for zebrafish
research is as a model in which to study host-microbe
interactions (see Chapter 48 by Wiles and Guillemin).
In many contexts, microbes are thought of as infectious
agents that cause disease. The optical transparency and
genetic tractability of zebrafish have enabled it to
become an outstanding model for two infectious hu-
man diseases, tuberculosis (Ramakrishnan, 2013) and
leprosy (Madigan, Cameron, & Ramakrishnan, 2017),
that have historically been difficult to investigate in
mammalian models. These new studies are providing
surprising insights that are likely to lead to new thera-
peutic approaches.
A related aspect of understanding host-microbe
relationships is to learn about host interactions with
the microbial organisms associated with essentially
every plant and animal on the planet. A tenet of animal
development is that initial patterning of the body
depends on a maternal contribution of molecules pack-
aged into the egg, and then unfolds based on the regula-
tion of zygotic genes and their products (Chan et al.,
2009; Li, Lu, & Dean, 2013; Schier, 2007). It is increasingly
clear that after an embryo leaves the protective environ-
ment either inside its mother or inside its eggshell, it
12 1. History of Zebrafish Research
becomes associated with communities of microbes, often
referred to as microbiota that influence many aspects of
its subsequent development and physiology. However,
the extent to which host-associated microbiota modulate
the normal development and/or physiology of any ani-
mal species remains unknown. Zebrafish is ideally
suited as a model in this burgeoning new field, because
it is straightforward to rear zebrafish germ-free, in the
absence of any microbes, or gnotobiotically, with expo-
sure to only very specific microbes (Melancon et al.,
2017). At the same time, both host and microbial genetics
can be manipulated, and the exquisite optical properties
of zebrafish larvae enable real-time visualization of
microbe biogeography and reciprocal responses of
both the host and associated microbes, including normal
residents (Wiles et al., 2016), as well as invasive patho-
gens (Ramakrishnan, 2013).
In addition to the many features conserved with other
animals, zebrafish are able to accomplish something that
mammals, such as mice and humans, are unable to
achieve, which is to regenerate nearly any damaged
body part. Thus far, zebrafish have been shown to regen-
erate damaged limbs, hearts, retinas, brains, and spinal
cords (Gemberling, Bailey, Hyde, & Poss, 2013). This
feat is not unique to zebrafish, because other nonmam-
malian vertebrates, such as salamanders and axolotls,
can also regenerate limbs (Dall’Agnese & Puri, 2016).
However, the ease of studying zebrafish and manipu-
lating its genome has established it as an important
model that holds out the promise of discovering mecha-
nisms that might be exploited to aid regeneration in
humans (Mokalled & Poss, 2018).
As zebrafish research has expanded, there have been
a number of efforts to standardize zebrafish husbandry.
Beginning in the early days of zebrafish research,
several resources were developed, including “The
Zebrafish Book,” dedicated to George Streisinger. This
book was originally published by Monte Westerfield
in 1993 and printed by the University of Oregon Press.
This book is now in its fifth edition (Westerfield, 2007);
the fourth edition is available online from ZFIN (zfin.
org). Since that time, there have been additional books
detailing zebrafish husbandry methods for research
laboratories (Harper & Lawrence, 2011; Nusslein-
Volhard & Dahm, 2002). The importance of standard-
izing husbandry was made very clear recently when
the journal Zebrafish devoted an entire issue to health
and husbandry (http://online.liebertpub.com/toc/
zeb/13/S1). Zebrafish also regularly publishes a section
on this topic. The many chapters on health and hus-
bandry in this volume complement the chapters on
zebrafish research and provide state-of-the-art informa-
tion for those who rear zebrafish that will enhance the
utility of zebrafish as an outstanding research model.
I. Introduction
Conclusions
Streisinger’s groundbreaking paper describing ge-
netic tools for zebrafish marked a turning point for
developmental biology. Since that time, this small fish
has become established as a model for uncovering ge-
netic mechanisms underlying many aspects of develop-
ment, differentiation, physiology, regeneration, and
evolution. Zebrafish has also become a model in which
to investigate mechanisms by which environmental fac-
tors, such as animal-associated microbes, can influence
developmental and physiological processes by modu-
lating host genes. Research utilizing the zebrafish model
continues to accelerate, making the future bright for
even more important discoveries that will expand our
understanding of the genetic mechanisms that trans-
form a single cell, the fertilized egg, into a multicellular
animal with a backbone, as well as how vertebrate or-
gans function, how these mechanisms contribute to hu-
man health, and how they may result in human diseases
when they go awry.
References
Amores, A., Force, A., Yan, Y. L., Joly, L., Amemiya, C., Fritz, A., et al.
(1998). Zebrafish hox clusters and vertebrate genome evolution.
Science, 282(5394), 1711e1714.
Atz, J. W. (1986). Fundulus-heteroclitus in the laboratoryea history.
American Zoologist, 26(1), 111e120.
Beattie, C. E., Raible, D. W., Henion, P. D., & Eisen, J. S. (1999). Early
pressure screens. Methods in Cell Biology, 60, 71e86.
Beddington, R. S., & Smith, J. C. (1993). Control of vertebrate gastrula-
tion: Inducing signals and responding genes. Current Opinion in Ge-
netics and Development, 3(4), 655e661.
Carver, E. A., & Stubbs, L. (1997). Zooming in on the human-mouse
comparative map: Genome conservation re-examined on a high-
resolution scale. Genome Research, 7(12), 1123e1137.
Chan, T. M., Longabaugh, W., Bolouri, H., Chen, H. L., Tseng, W. F.,
Chao, C. H., et al. (2009). Developmental gene regulatory networks
in the zebrafish embryo. Biochimica et Biophysica Acta, 1789(4),
279e298. https://doi.org/10.1016/j.bbagrm.2008.09.005.
Dall’Agnese, A., & Puri, P. L. (2016). Could we also be regenerative su-
perheroes, like salamanders? BioEssays, 38(9), 917e926. https://
doi.org/10.1002/bies.201600015.
Davidson, E. H. (1994). Molecular biology of embryonic development:
How far have we come in the last ten years? BioEssays, 16(9),
603e615. https://doi.org/10.1002/bies.950160903.
Driever, W., Solnica-Krezel, L., Schier, A. F., Neuhauss, S. C., Malicki, J.,
Stemple, D. L., et al. (1996). A genetic screen for mutations affecting
embryogenesis in zebrafish. Development, 123, 37e46.
Eisen, J. S. (1996). Zebrafish make a big splash. Cell, 87(6), 969e977.
Eisen, J. S., Myers, P. Z., & Westerfield, M. (1986). Pathway selection by
growth cones of identified motoneurones in live zebra fish
embryos. Nature, 320(6059), 269e271. https://doi.org/10.1038/
320269a0.
Fior, R., Povoa, V., Mendes, R. V., Carvalho, T., Gomes, A.,
Figueiredo, N., et al. (2017). Single-cell functional and chemosensi-
tive profiling of combinatorial colorectal therapy in zebrafish
xenografts. Proceedings of the National Academy of Sciences of the
United States of America. https://doi.org/10.1073/pnas.1618389114.
 References
Force, A., Lynch, M., Pickett, F. B., Amores, A., Yan, Y. L., &
Postlethwait, J. (1999). Preservation of duplicate genes by comple-
mentary, degenerative mutations. Genetics, 151(4), 1531e1545.
Fu, L. (2016). Shisan C. Chen and his research on goldfish genetics.
Protein Cell, 7(2), 79e80. https://doi.org/10.1007/s13238-015-0236-3.
Gellon, G., & McGinnis, W. (1998). Shaping animal body plans in
development and evolution by modulation of Hox expression
patterns. BioEssays, 20(2), 116e125. https://doi.org/10.1002/
(SICI)1521-1878(199802)20:2<116::AID-BIES4>3.0.CO;2-R.
Gemberling, M., Bailey, T. J., Hyde, D. R., & Poss, K. D. (2013). The
zebrafish as a model for complex tissue regeneration. Trends in
Genetics, 29(11), 611e620. https://doi.org/10.1016/
j.tig.2013.07.003.
Gilbert, S. F. (1978). The embryological origins of the gene theory. Jour-
nal of the History of Biology, 11(2), 307e351.
Gilbert, S. F. (1991). Induction and the origins of developmental
genetics. Developmental Biology (N Y 1985), 7, 181e206.
Gilbert, S. F. (1998). Bearing crosses: A historiography of genetics and
embryology. American Journal of Medical Genetics, 76(2), 168e182.
Gilbert, S. F., & Barresi, M. J. F. (2016). Developmental biology (11th ed.).
Sunderland, MA: Sinauer.
Grunwald, D. J., & Eisen, J. S. (2002). Headwaters of the zebrafishd
emergence of a new model vertebrate. Nature Reviews Genetics,
3(9), 717e724. https://doi.org/10.1038/nrg892.
Haffter, P., Granato, M., Brand, M., Mullins, M. C.,
Hammerschmidt, M., Kane, D. A., et al. (1996). The identification
of genes with unique and essential functions in the development
of the zebrafish, Danio rerio. Development, 123, 1e36.
Harper, C., & Lawrence, C. (2011). The laboratory zebrafish (Vol. 15). Boca
Ratan, Florida: CRC Press.
Hill, J. T., Demarest, B. L., Bisgrove, B. W., Gorsi, B., Su, Y. C., &
Yost, H. J. (2013). MMAPPR: Mutation mapping analysis pipeline
for pooled RNA-seq. Genome Research, 23(4), 687e697. https://
doi.org/10.1101/gr.146936.112.
Hisaoka, K. K., & Battle, H. I. (1958). The normal developmental stages
of the zebrafish, Brachydanio rerio (Hamilton-buchanan). Journal of
Morphology, 102(2), 311e327.
Johnson, S. L., Midson, C. N., Ballinger, E. W., & Postlethwait, J. H.
(1994). Identification of RAPD primers that reveal extensive poly-
morphisms between laboratory strains of zebrafish. Genomics,
19(1), 152e156. https://doi.org/10.1006/geno.1994.1026.
Karlstrom, R. O., & Kane, D. A. (1996). A flipbook of zebrafish
embryogenesis. Development, 123, 461.
Kazianis, S. (2006). Historical, present, and future use of Xiphophorus
fishes for research. Zebrafish, 3(1), 9e10. https://doi.org/10.1089/
zeb.2006.3.9.
Kimmel, C. B. (1989). Genetics and early development of zebrafish.
Trends in Genetics, 5(8), 283e288.
Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., &
Schilling, T. F. (1995). Stages of embryonic development of the
zebrafish. Developmental Dynamics, 203(3), 253e310. https://
doi.org/10.1002/aja.1002030302.
Kimmel, C. B., Sessions, S. K., & Kimmel, R. J. (1981). Morphogenesis
and synaptogenesis of the zebrafish Mauthner neuron. The Journal
of Comparative Neurology, 198(1), 101e120. https://doi.org/
10.1002/cne.901980110.
Kimmel, C. B., Spray, D. C., & Bennett, M. V. (1984). Developmental
uncoupling between blastoderm and yolk cell in the embryo of
the teleost Fundulus. Developmental Biology, 102(2), 483e487.
Kimmel, C. B., & Warga, R. M. (1988). Cell lineage and developmental
potential of cells in the zebrafish embryo. Trends in Genetics, 4(3),
68e74.
Korn, H., & Faber, D. S. (2005). The Mauthner cell half a century later: A
neurobiological model for decision-making? Neuron, 47(1), 13e28.
https://doi.org/10.1016/j.neuron.2005.05.019.
I. Introduction
13
Laale, H. W. (1977). Biology and use of zebrafish, brachydanio-rerio in
fisheries researcheliterature-review. Journal of Fish Biology, 10(2),
121e173. https://doi.org/10.1111/j.1095-8649.1977.tb04049.x.
Lamason, R. L., Mohideen, M. A., Mest, J. R., Wong, A. C.,
Norton, H. L., Aros, M. C., et al. (2005). SLC24A5, a putative cation
exchanger, affects pigmentation in zebrafish and humans. Science,
310(5755), 1782e1786. https://doi.org/10.1126/science.1116238.
Leslie, M. (2017). Zebrafish larvae could help to personalize cancer
treatments. Science, 357(6353), 745. https://doi.org/10.1126/
science.357.6353.745.
Li, L., Lu, X., & Dean, J. (2013). The maternal to zygotic transition in
mammals. Molecular Aspects of Medicine, 34(5), 919e938. https://
doi.org/10.1016/j.mam.2013.01.003.
Loeb, J. (1916). The mechanism of diffusion of electrolytes through an-
imal membranes. Proceedings of the National Academy of Sciences of the
United States of America, 2(9), 511e516.
Lonie, I. M. (1981). The hippocratic treatises “on generation”, on the na-
ture of the Child. In “Diseases IV”: A commentary (Vol. 7)Berlin: Wal-
ter de Gruyter and Company.
Madigan, C. A., Cameron, J., & Ramakrishnan, L. (2017). A zebrafish
model of Mycobacterium leprae Granulomatous infection. The Journal
of Infectious Diseases, 216(6), 776e779. https://doi.org/10.1093/
infdis/jix329.
Manak, J. R., & Scott, M. P. (1994). A class act: Conservation of homeo-
domain protein functions. Development Supplement, 61e77.
Marder, E., & Eisen, J. S. (1984). Transmitter identification of pyloric
neurons: Electrically coupled neurons use different transmitters.
Journal of Neurophysiology, 51(6), 1345e1361.
McGinnis, W., & Krumlauf, R. (1992). Homeobox genes and axial
patterning. Cell, 68(2), 283e302.
Melancon, E., Gomez De La Torre Canny, S., Sichel, S., Kelly, M.,
Wiles, T. J., Rawls, J. F., et al. (2017). Best practices for germ-free
derivation and gnotobiotic zebrafish husbandry. Methods in Cell
Biology, 138, 61e100. https://doi.org/10.1016/bs.mcb.2016.11.005.
Metcalfe, W. K., Mendelson, B., & Kimmel, C. B. (1986). Segmental ho-
mologies among reticulospinal neurons in the hindbrain of the
zebrafish larva. The Journal of Comparative Neurology, 251(2),
147e159. https://doi.org/10.1002/cne.902510202.
Miller, A. C., Obholzer, N. D., Shah, A. N., Megason, S. G., &
Moens, C. B. (2013). RNA-seq-based mapping and candidate iden-
tification of mutations from forward genetic screens. Genome
Research, 23(4), 679e686. https://doi.org/10.1101/gr.147322.112.
Mokalled, M. H., & Poss, K. D. (2018). A regeneration toolkit. Develop-
mental Cell, 47(3), 267e280.
Morgan, T. H. (1895). The formation of the fish embryo. Journal of
Morphology, 10(2), 419e472.
Mullins, M. C., Hammerschmidt, M., Haffter, P., & Nusslein-
Volhard, C. (1994). Large-scale mutagenesis in the zebrafish: In
search of genes controlling development in a vertebrate. Current
Biology, 4(3), 189e202.
Mullins, M. C., & Nusslein-Volhard, C. (1993). Mutational approaches
to studying embryonic pattern formation in the zebrafish. Current
Opinion in Genetics and Development, 3(4), 648e654.
Nasevicius, A., & Ekker, S. C. (2000). Effective targeted gene ’knock-
down’ in zebrafish. Nature Genetics, 26(2), 216e220. https://
doi.org/10.1038/79951.
Needham, J. (1963). Chemical embryology (Vol. 1). New York: Hafner
Publishing Company.
Nothinger, R. (2002). Ernst Hadorn, a pioneer of developmental
genetics. International Journal of Developmental Biology, 46, 23e27.
Nusslein-Volhard, C., & Dahm, R. (2002). Zebrafish: Oxford: Oxford
University Press.
Nusslein-Volhard, C., & Wieschaus, E. (1980). Mutations affecting
segment number and polarity in Drosophila. Nature, 287(5785),
795e801.
14 1. History of Zebrafish Research
Oppenheimer, J. M. (1936). Historical introduction to the study of tele-
ostean development. Osiris, 2, 124e148.
Oppenheimer, J. M. (1979). 50 Years of Fundulus. Quarterly Review of
Biology, 54(4), 385e395. https://doi.org/10.1086/411443.
Orger, M. B., & de Polavieja, G. G. (2017). Zebrafish behavior: Oppor-
tunities and challenges. Annual Review of Neuroscience. https://
doi.org/10.1146/annurev-neuro-071714-033857.
Peterson, R. T., Link, B. A., Dowling, J. E., & Schreiber, S. L. (2000).
Small molecule developmental screens reveal the logic and timing
of vertebrate development. Proceedings of the National Academy of
Sciences of the United States of America, 97(24), 12965e12969.
https://doi.org/10.1073/pnas.97.24.12965.
Phillips, J. B., & Westerfield, M. (2014). Zebrafish models in transla-
tional research: Tipping the scales toward advancements in human
health. Disease Models and Mechanisms, 7(7), 739e743. https://
doi.org/10.1242/dmm.015545.
Postlethwait, J. H., & Talbot, W. S. (1997). Zebrafish genomics: From
mutants to genes. Trends in Genetics, 13(5), 183e190.
Postlethwait, J. H., Yan, Y. L., Gates, M. A., Horne, S., Amores, A.,
Brownlie, A., et al. (1998). Vertebrate genome evolution and the
zebrafish gene map. Nature Genetics, 18(4), 345e349. https://
doi.org/10.1038/ng0498-345.
van Raamsdonk, W., Mos, W., Smit-Onel, M. J., van de Laarse, W. J., &
Fehres, R. (1983). The development of the spinal motor column in
relation to the myotomal muscle fibers in the zebrafish (Brachydanio
rerio). I. Posthatching development. Anatomy and Embryology,
167(1), 125e139.
van Raamsdonk, W., Tekronnie, G., Pool, C. W., & van de Laarse, W.
(1980). An immune histochemical and enzymic characterization
of the muscle fibres in myotomal muscle of the teleost Brachydanio
rerio, Hamilton-Buchanan. Acta Histochemica, 67(2), 200e216.
Ramakrishnan, L. (2013). The zebrafish guide to tuberculosis immunity
and treatment. Cold Spring Harbor Symposia on Quantitative Biology,
78, 179e192. https://doi.org/10.1101/sqb.2013.78.023283.
Rennekamp, A. J., & Peterson, R. T. (2015). 15 years of zebrafish chem-
ical screening. Current Opinion in Chemical Biology, 24, 58e70.
https://doi.org/10.1016/j.cbpa.2014.10.025.
Richardson, M. K., Hanken, J., Gooneratne, M. L., Pieau, C.,
Raynaud, A., Selwood, L., et al. (1997). There is no highly conserved
embryonic stage in the vertebrates: Implications for current theories
of evolution and development. Anatomy and Embryology, 196(2),
91e106.
Schier, A. F. (2007). The maternal-zygotic transition: Death and birth of
RNAs. Science, 316(5823), 406e407. https://doi.org/10.1126/
science.1140693.
Selverston, A. I., & Miller, J. P. (1980). Mechanisms underlying pattern
generation in lobster stomatogastric ganglion as determined by se-
lective inactivation of identified neurons. I. Pyloric system. Journal
of Neurophysiology, 44(6), 1102e1121.
Shima, A., & Mitani, H. (2004). Medaka as a research organism: Past,
present and future. Mechanisms of Development, 121(7e8),
599e604. https://doi.org/10.1016/j.mod.2004.03.011.
I. Introduction
Spray, D. C., Harris, A. L., & Bennett, M. V. (1979). Voltage dependence
of junctional conductance in early amphibian embryos. Science,
204(4391), 432e434.
Stahl, F. (1995). George streisinger (Vol. 68). Washington DC: National
Academy Press.
Stednitz, S. J., McDermott, E. M., Ncube, D., Tallafuss, A., Eisen, J. S., &
Washbourne, P. (2018). Forebrain control of behaviorally driven so-
cial orienting in zebrafish. Current Biology, 28(15), 2445e2451.
https://doi.org/10.1016/j.cub.2018.06.016. e2443.
Stewart, W. W. (1981). Lucifer dyesdhighly fluorescent dyes for biolog-
ical tracing. Nature, 292(5818), 17e21.
Streisinger, L. (2004). From the sidelines. Eugene, Oregon: University of
Oregon Press.
Streisinger, G., Walker, C., Dower, N., Knauber, D., & Singer, F. (1981).
Production of clones of homozygous diploid zebra fish (Brachydanio
rerio). Nature, 291(5813), 293e296.
Tauber, A. I., & Podolsky, S. H. (2000). The generation of diversity clonal
selection theory and the rise of molecular immunology. Cambridge
MA: Harvard University Press.
Trinkaus, J. P. (1990). In H.-J. Marthy (Ed.), Some contributions of research
on early teleost embryogenesis to general problems of development. New
York: Plenum.
Varshney, G. K., Sood, R., & Burgess, S. M. (2015). Understanding and
editing the zebrafish genome. Advances in Genetics, 92, 1e52.
https://doi.org/10.1016/bs.adgen.2015.09.002.
Weis, J. S. (1968a). Analysis of the development of nervous system of
the zebrafish, Brachydanio rerio. I. The normal morphology
and development of the spinal cord and ganglia of the
zebrafish. Journal of Embryology and Experimental Morphology,
19(2), 109e119.
Weis, J. S. (1968b). Analysis of the development of the nervous system
of the zebrafish, Brachydanio rerio. II. The effect of nerve growth fac-
tor and its antiserum on the nervous system of the zebrafish. Journal
of Embryology and Experimental Morphology, 19(2), 121e135.
Weisblat, D. A., Zackson, S. L., Blair, S. S., & Young, J. D. (1980). Cell
lineage analysis by intracellular injection of fluorescent tracers.
Science, 209(4464), 1538e1541.
Westerfield, M. (2007). The zebrafish book (5th ed.). Eugene, Oregon: Uni-
versity of Oregon Press.
Wiles, T. J., Jemielita, M., Baker, R. P., Schlomann, B. H., Logan, S. L.,
Ganz, J., et al. (2016). Host gut motility promotes competitive exclu-
sion within a model intestinal microbiota. PLoS Biology, 14(7),
e1002517. https://doi.org/10.1371/journal.pbio.1002517.
Wourms, J. P. (1997). The rise of fish embryology in the nineteenth
century. American Zoologist, 37, 269e310.
Wourms, J. P., & Whitt, G. S. (1981). Future directions of research on the
developmental biology of fishes. American Zoologist, 21(2), 597e604.
Zon, L. I. (1999). Zebrafish: A new model for human disease. Genome
Research, 9(2), 99e100.