Preparation and Culture of Limb Blastema Stem Cells From Regenerating Larval and Adult Salamanders - Kumar and Godwin 2010 (1) - PDB - Prot5367 - Cold Spring Harbor Protocols

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Cite as: Cold Spring Harb. Protoc.; 2010; doi:10.1101/pdb.prot5367
Protocol
Preparation and Culture of Limb Blastema Stem Cells from Regenerating Larval
and Adult Salamanders
Anoop Kumar1,2,4 and James W. Godwin1,2,3,4
1 UCL Research Department of Structural and Molecular Biology, University College London,
London WC1E BT, UK
2 These authors contributed equally to this work.
3 Present address: Australian Regenerative Medicine Institute (ARMI), Monash University, Victoria 3800,
Australia.
4 Corresponding authors (anoop.kumar@ucl.ac.uk); (james.godwin@armi.monash.edu.au).
INTRODUCTION
Adult salamanders show extensive ability to regenerate their body parts, and this can be attributed at
least in part to their cellular plasticity. Cell reprogramming, dedifferentiation, proliferation and
redifferentiation occur naturally in adult salamanders in response to tissue damage or removal. Limb
regeneration proceeds by mesenchymal dedifferentiation and cell migration at the end of the stump. This
generates a cohort of mesenchymal stem cells called blastema cells, which accumulate under the wound
epithelium. The "limb blastema" is an autonomous structure that retains the positional identity of its
location of origin as well as morphogenetic cues for reconstructing a new limb. At the cellular level,
blastema stem cells maintain characteristics such as positional memory derived from their location, while
expressing new markers relevant to the process of limb regeneration. In this protocol, a nonenzymatic
method is described for the dissociation of blastema stem cells from newts (Notophthalmus viridescens)
and axolotls (Ambystoma mexicanum).
RELATED INFORMATION
The use of axolotls for regeneration studies has been described in Ambystoma mexicanum, the
Axolotl: A Versatile Amphibian Model for Regeneration, Development, and Evolution Studies
(Voss et al. 2009). For staging of newts, see Iten and Bryant (1973).
MATERIALS
Reagents
AMEM (1%)
Amphibian dissection medium
APBS
Ethanol (70%, in a spray bottle)
N. viridescens (adult newts) or A. mexicanum (axolotl larvae)
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Forelimb blastema of newts and axolotls and tail blastema of axolotls can be used for dissociation of
blastema cells. Axolotl larvae of 8-14 cm (snout-vent length) can be obtained from the Ambystoma
Genetic Stock Center (http://www.ambystoma.org/AGSC/) or local suppliers. Cells from six newt limb
blastema are required for one well in a 96-well plate and will yield 1000-1500 cells; variations have been
observed among batches of newts. Six limb blastema of 8-cm axolotls yield 1000-1500 cells. Six large
blastema from 14-cm axolotls can yield 3000-5000 cells. Some trials will be required initially to collect
enough cells. The number of animals can be increased to scale up the assay.
SF-AMEM
Sulfamerazine (0.5%, w/v in H2O; Sigma) (optional; see Step 3)
Tap H2O (dechlorinated and autoclaved)
Tricaine (0.1%, w/v in H2O; Sigma)
Prepare 500 mL of fresh solution with dechlorinated H2O available in the animal facility.
Virkon disinfectant (0.01%, w/v in autoclaved, dechlorinated tap H2O)
Equipment
Clean forceps and other dissection tools by soaking them in a 0.1% (w/v) Virkon solution for 30 min or in
an ultrasonic cleaner. Rinse the instruments in H2O and spray with 70% ethanol. Dry them with
laboratory wipes and wrap in aluminum foil for storage.
Aquarium for newts (temperature-controlled with circulating H2O, 24ºC) or temperature-controlled room
for axolotl larvae (temperature-controlled, 14ºC-18ºC) (see Step 5)
Beaker (500-mL)
Dish (glass-bottom, 50-mm; MatTek P50G-0-14-F)
Forceps (blunt-end)
Forceps (Dumont-Biological, #5, fine-tip, two pairs; Fine Science Tools)
Hood (sterile, for tissue culture)
Incubator (humidified, preset to 25°C, 2.5% CO2)
Laboratory wipes
Malleus nipper (angled down) (Fine Science Tools)
Micropipettes and tips (100-µL, 1-mL)
Microscope (inverted)
Pasteur pipette (fine-tip, sterile)
Petri dishes (150-mm, 92-mm, and 60-mm, bacterial, sterile)
Plastic wrap
Plate (96-well, clear bottom with lid, collagen-coated; Corning)
Coat the 96-well plate with collagen by pipetting 10 µL of collagen solution (Type I, Sigma C8919) into
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each well. Shake the plate to spread the solution evenly on the surface. Air-dry the plate for 2-3 h in an
oven at 45°C. Wash the plate twice with APBS before use. Collagen-coated dishes can be prepared in
advance and stored at 4ºC. Tissue culture dishes of other formats can also be used for collagen coating.
See Discussion.
Scissors (11.5-cm; Fine Science Tools)
Scissors (8-cm, Mini-Vannas; Fine Science Tools)
Spring scissors (12-cm; Fine Science Tools)
Stereomicroscope (Leica Z6)
Tubes (microcentrifuge, 1.5-mL, sterile)
Tweezers (#5)
METHOD
Preparation of Newts or Axolotls for Limb Blastema: Amputation of Limbs and Tails
The procedure for amputation of limbs and tails in newts and axolotls is similar. The routine for limb
amputation of 30 newts is described here.
1. Anesthetize the animals by placing them in 0.1% tricaine solution for 7-10 min. Preferably,
anesthetize the animals in two batches of 15 each.
2. Amputate the limbs:

i. Place an anesthetized newt on the stage of a stereomicroscope.
ii. Cut both forelimbs at the midhumerus level using 11.5-cm scissors.
iii. Push the muscle tissues attached to the bone backward with #5 tweezers, and trim the
protruding bone with the Malleus nipper.
iv. Trim the protruding muscle tissues with 12-cm spring scissors, and ensure that the
surface of the amputated limb is flat.
Any protruding bone will hamper the wound healing process and delay regeneration. This
can also cause rupture of the wound epithelium and fungal infection during limb
regeneration.
3. Transfer the animal to a solution of 0.5% sulfamerazine in H2O.
Axolotls can be kept in autoclaved, dechlorinated tap H2O instead of sulfamerazine solution.
4. Repeat Steps 2 and 3 for all of the remaining animals.
5. Change the H2O after 24 h, and keep the animals in normal maintenance conditions. Maintain
newts in an aquarium with circulating H2O at 24ºC. Maintain axolotl larvae in a
temperature-controlled room at 14°C-18°C.
Newts reach the blastema stage 12-14 d after amputation of the limb. Axolotl larvae regenerate
more synchronously and attain blastema stage in 7-12 d, depending on the size of the animals.
Dissociation of Blastema Cells
6. Anesthetize the animals by placing them in 0.1% tricaine solution for 7-10 min.
7. Spray 70% ethanol on the dissection area, including the stage of the stereomicroscope, and
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clean with a laboratory wipe. Spray ample quantities of 70% ethanol onto a few wipes and place
them in a 92-mm sterile Petri dish to keep the surgical instruments sterile.
8. Add 50 mL of autoclaved, dechlorinated tap H2O to a 150-mm sterile Petri dish and place near
the microscope.
9. Pipette 3 mL of amphibian dissection medium into a 60-mm sterile Petri dish and place near the
stereomicroscope.
The limb blastema will be collected and kept in this solution for dissection.
10. Disinfect the anesthetized animals:

i. Add 200 mL of 0.01% Virkon solution to a 500-mL beaker.
ii. Place the anesthetized animals into this solution, close the beaker with a sheet of plastic
wrap, and gently swirl the beaker for 30 sec. Decant the Virkon solution.
iii. Wash the animals three times in autoclaved, dechlorinated tap H2O to remove any traces
of the Virkon.
11. Place the animals in the dish of autoclaved, dechlorinated tap H2O from Step 8.
12. Use blunt-end forceps to pick one animal, and place it on the stage of the stereomicroscope.
Hold the right forelimb upward with #5 tweezers and remove the limb blastema using 8-cm
Vannas scissors.
Keep the scissors parallel to the edge of the limb that is vertically oriented under the microscope.
Cut only the limb blastema, which is clearly visible as a conical elevation from the stump tissue.
13. Transfer the blastema into the dish of amphibian dissection medium from Step 9.
14. Remove the left limb blastema as in Step 12 and place in the dish of amphibian dissection
medium.
15. Repeat Steps 12-14 for each animal.
16. Place the 50-mm glass-bottom dish on the stage of the stereomicroscope, and pipette 100 µL
of SF-AMEM onto the glass surface.
17. Pick one blastema from the amphibian dissection medium with forceps (#5, fine-tip), and place
it in the medium in the glass-bottom dish.
18. Use forceps (#5, fine-tip) to hold the blastema at the base (proximal), and insert the tip of the
8-cm Vannas scissors into the mesenchymal tissue and wound epithelium (see Fig. 1a ). Make a
midline cut along the proximal tissue to the tip of the blastema. This will open the blastema in two
halves (see Fig. 1b).
Figure 1. Schematic diagram of the microsurgical

procedure. (a) The green dotted line depicts the
proximo-distal axis of the limb blastema. (b) A
partially dissected blastema after surgical incision.
(c) The green dotted line indicates the proximal
boundary. The blastema cells can be released from
the mesenchymal cells distal to this region by
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gentle mechanical dissociation using a pair of
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fine-tip forceps.
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Take care not to completely separate the two halves of the blastema tissue.
19. Use fine-tip forceps to gently tap on the inner mesenchymal tissues of the distal blastema (see
Fig. 1c).
This procedure will release single cells from the mesenchymal tissues of the blastema into the
culture medium. Take care not to break the epithelial tissues or scoop the mesenchymal cells; this
action will result in cell clumps and epithelial tissues in culture. Do not try to tap the proximal
tissues. They are closely apposed and tend to release fewer cells, and disturbing them may also
increase the possibility of bacterial contamination.
20. Discard the blastema tissue. Repeat Steps 17-19 five times.
21. Transfer the culture medium containing the dissociated blastema cells into a microcentrifuge
tube. Repeat Steps 16-21 to collect cells from all the blastema tissues.
22. Gently mix the cell suspension several times using a 1-mL micropipette. Aliquot the cell
suspension equally into 10 wells of a collagen-coated, 96-well plate.
23. Incubate the 96-well plate overnight in a humidified CO2 incubator maintained at 25°C.
The cells will attach to the dish after 6-8 h of incubation. On average, 1000-1500 cells will attach in
each well.
Maintenance of Cell Culture
24. Remove the culture plate from the incubator, inspect the cells with an inverted microscope,
and place the plate in a sterile tissue culture hood.
25. Using a sterile, fine-tip Pasteur pipette, gently remove the medium from all culture wells.
26. Pipette 100 µL of APBS into each well. Use a fine-tippipette to gently draw the liquid up and
down a few times in each well to remove any debris attached to the cells. Discard the medium. Add
fresh 1% AMEM and return the culture to the incubator.
Serum is not critical for the maintenance of culture. For cell proliferation assays (e.g.,
bromodeoxyuridine [BrdU] incorporation), reduce serum concentration to 0.1%. See Discussion for
more details.
See Troubleshooting.
TROUBLESHOOTING
Problem: There are not enough cells in the 96-well plate after dissociation of the blastema.
[Step 26]
Solution: Some practice is required for successful dissociation of blastema cells. Observe an aliquot of the
cell suspension in Step 22 with an inverted microscope to monitor effectiveness of dissociation. To
increase the number of cells, the following remedies are possible:
1. Use midsize blastema.
2. If the use of early blastema is required, increase the number of blastema used per well.
Problem: The cell proliferation assay shows high background.
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[Step 26]
Solution: To reduce background in cell proliferation assays, try the following:
1. Reduce the serum concentration in the AMEM to 0.05%-0.1%.
2. Omit insulin from the AMEM.
3. Try a new batch of animals.
DISCUSSION
Blastema Cells in Culture
When newt blastema are isolated, ~1%-2% of the dissociated, resuspended cells adhere to the culture
substratum. In axolotls, more than 3% of blastema cells have been observed to attach to the culture
dishes. The cells can be maintained on collagen- or gelatin-coated culture dishes, as shown in Figures 2
and 3 . Blastema cells have also been found to attach to dishes commercially treated for tissue culture
(Nunc or Falcon brands), but the cells remain spindle-shaped, and long-term viability is compromised.
Figure 2. Photomicrograph of dissociated newt blastema cells in

culture attached to a 35-mm culture dish coated with collagen. (a)
Cells after 96 h in culture; (b) a culture maintained for 10 d. Scale
bar, 100 µm.
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Figure 3. Axolotl blastema cells in culture. Axolotl

blastema cells were isolated with a method similar to the
method described for newt blastema cells and cultured
on collagen-coated dishes. The method can also be
extended to dissociated cells from tail blastema of axolotl.
(a) Limb blastema cells after 7 d in culture; (b) the limb
blastema cells after 30 d in culture; (c) tail blastema cells
after 7 d in culture; (d) the tail blastema cells after 30 d
in culture. Scale bar, 100 µm.
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One striking feature of cultured blastema cells is their inability to survive in culture media containing
higher levels of serum. Extensive cell death occurs in cultures in which the serum concentration is 2%.
We procured serum certified for the culture of embryonic stem cells and compared it to the batch of
serum screened for the culture of newt cells, and under all conditions, the blastema cells undergo cell
death in response to increasing serum concentrations. We have maintained cell cultures in serum-free
media or media with 0.5%-1% serum for up to 30 d.
In cell proliferation assays using BrdU, 2%-10% of the blastema cells were observed to undergo cell cycle
re-entry in various batches of cultures. The percentage of mitotic cells in newt blastema cell culture at
day 10 was recorded as 0.24 (n = 2, assayed with anti-phospho-histone H3 antibody). The low level of cell
cycle re-entry is an attractive feature of these cells for assaying growth factor activity (Kumar et al.
2007). Although we have established the utility of our cultures for studying the properties of primary
blastema cells, it has not yet been possible to expand and passage them at a satisfactory yield.
Expression of Cellular Markers
Antigens 22/18, 22/31, and vimentin are expressed in cultured blastema cells (see Fig. 4 ). We detected
22/18 in 60%-70% of the cells, whereas nearly all the cells reacted with 22/31 and vimentin antibody, in
agreement with the previous observations (Ferretti and Brockes 1991).
Figure 4. Newt limb blastema cells express antigens associated with

regeneration and mesenchymal stem cell markers. (a) Expression of
cytokeratin 22/18 after 48 h in culture. More than 80% of cells stably
express 22/18. (b) The cells recognize an epitope (22/31) associated
with filamentous cytokeratin in blastema cells that is transiently
expressed in the blastema stage. (c) Blastema cells express the
mesenchymal marker vimentin. (a) Scale bar, 50 µm; (b,c) scale bars,
100 µm.
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version (44K):
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Gene Expression Profiling by Quantitative Polymerase Chain Reaction (qPCR)
We analyzed three genes that were characterized previously in the context of newt limb regeneration
(Tables 1 and 2). The homeobox gene Msx1 is expressed during limb regeneration in salamanders
(Carlson et al. 1998; Koshiba et al. 1998; Kumar et al. 2004). Msx1 transcript level is present in both
blastema tissues and cultured cells. The newt D-11 (NvHbox2) is a regeneration-specific homeobox gene
that is expressed in the limb blastema during regeneration (Brown and Brockes 1991). Newt D-11 is not
detectable in normal limb tissues. The transcript for D-11 is robustly expressed in both blastema tissues
and cultured blastema cells. It is notable that the D-11 transcript is absent in cultured B1H1 cells (Table
1). Prod 1 is a cell surface protein expressed in newt limbs in a proximo-distal gradient and implicated in
positional identity (Morais da Silva et al. 2002). The transcript level for Prod 1 is low both in an adult limb
and a regenerating limb. The blastema cells cultured for 24 h express Prod 1 at a relatively low level,
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whereas freshly dissociated blastema cells in suspension express Prod 1 at a 10-fold higher level.
Established newt cell cultures like B1H1 cells do not express Prod 1, which indicates that a cell surface
protein like Prod 1 is likely to be down-regulated during long-term culture.
ACKNOWLEDGMENTS
We thank Jeremy Brockes for encouragement and support, Phillip Gates and Robert Blassberg for
performing the PCR assay, and Kathrin Graβme for comments on the manuscript. The research is funded
by a Medical Research Council (MRC) Programme Grant to Jeremy P. Brockes.
REFERENCES
Brown R, Brockes JP. 1991. Identification and expression of a regeneration-specific homeobox gene in the
newt limb blastema. Development 111: 489–496.[Abstract]
Carlson MR, Bryant SV, Gardiner DM. 1998. Expression of Msx-2 during development, regeneration, and
wound healing in axolotl limbs. J Exp Zool 282: 715–723.[Medline]
Ferretti P, Brockes JP. 1991. Cell origin and identity in limb regeneration and development. Glia 4:
214–224.[Medline]
Iten LE, Bryant SV. 1973. Forelimb regeneration from different levels of amputation in the newt N.
viridescens. Length, rate and stages. Wilhem Roux Arch Dev Biol 173: 263–282.
Koshiba K, Kuroiwa A, Yamamoto H, Tamura K, Ide H. 1998. Expression of Msx genes in regenerating and
developing limbs of axolotl. J Exp Zool 282: 703–714.[Medline]
Kumar A, Velloso CP, Imokawa Y, Brockes JP. 2004. The regenerative plasticity of isolated urodele
myofibers and its dependence on Msx1. PLoS Biol 2: e218. doi: 10.1371/journal.pbio.0020218.[Medline]
Kumar A, Godwin JW, Gates PB, Garza-Garcia AA, Brockes JP. 2007. Molecular basis for the nerve
dependence of limb regeneration in an adult vertebrate. Science 318: 772–777.[Abstract/Free Full Text]
Morais da Silva S, Gates PB, Brockes JP. 2002. The newt ortholog of CD59 is implicated in proximodistal
identity during amphibian limb regeneration. Dev Cell 3: 547–555.[Medline]
Voss SR, Epperlein HH, Tanaka EM. 2009. Ambystoma mexicanum, the axolotl: A versatile amphibian
model for regeneration, development, and evolution studies. Cold Spring Harb Protoc doi:
10.1101/pdb.emo128.[Abstract/Free Full Text]
Caution
Sulfamerazine
Sulfamerazine is an irritant and may be harmful by inhalation, ingestion, or skin absorption. Wear
appropriate gloves and safety glasses.
Caution
Tricaine (Tricaine methanesulfonate)

Tricaine (tricaine methanesulfonate) is an irritant and may be harmful by inhalation, ingestion, or skin
absorption. Wear appropriate gloves and safety glasses.
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Caution
Virkon disinfectant
Virkon disinfectant is an irritant and may be harmful by inhalation, ingestion, or skin absorption. Wear
appropriate gloves and safety goggles. Do not breathe the dust.
Emerging Model Organisms
Ambystoma mexicanum, the Axolotl: A Versatile Amphibian Model for

Regeneration, Development, and Evolution Studies
S. Randal Voss1 , Hans H. Epperlein2 , and Elly M. Tanaka3,4,5
1 Department of Biology and Ambystoma Genetic Stock Center, University of Kentucky,

Lexington, KY 40506, USA
2 Institute of Anatomy, Medical Faculty, Dresden University of Technology, 01307 Dresden,
Germany
3 Center for Regenerative Therapies, Dresden University of Technology, 01307 Dresden,
Germany
4 Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany
5 Corresponding author (elly.tanaka@crt-dresden.de)
INTRODUCTION
Adult salamanders are best known for their capacity to regenerate an astounding range of body
structures including the whole limb and tail, the central nervous system, and tissues of the eye and
heart. The axolotl (Ambystoma mexicanum) represents the salamander species that is most easily bred in
the laboratory, and for which the most comprehensive genetic, genomic, and transgenesis tools have
been developed. As such, it serves as an important vertebrate model for studying regeneration and tissue
repair. Beyond regeneration, axolotls have a deep and rich history as primary amphibian models, especially
in research areas concerning embryonic development--most notably the inductive mode of germ cell
formation. The easily obtained oocytes, high quantities of embryos produced by each spawning, large size
of the embryo, and ability to graft tissues from individual to individual at any stage without rejection make
the axolotl an advantageous model system for the study of development, electrophysiology, and
regeneration.
RELATED INFORMATION
For further reading on axolotls, see the text by Armstrong and Malacinski (1989) and Developmental
Biology of Urodeles (1996).
BACKGROUND INFORMATION
The axolotl (Ambystoma mexicanum) is a tailed amphibian that is commonly called a salamander.
Salamanders represent one of the three major amphibian groups, the other two being caecilians and
anurans (frogs and toads). These groups have been evolving independently for >200 million years and
are highly divergent from one another. Thus, no single species within any of these groups provides a
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"typical" amphibian perspective. The axolotl is a member of the tiger salamander species complex within
the family Ambystomatidae. Tiger salamanders are a recently evolved group consisting of several species
and subspecies that are widely found in terrestrial and fresh-water aquatic habitat types of North America
(Petranka 1998). Like many salamanders, axolotls exhibit paedomorphosis, meaning that they fail to
undergo a metamorphosis and retain the gross morphology of larvae as reproductive adults. The
biological significance of salamander paedomorphosis, made famous in Gould’s (1977) opus Ontogeny and
Phylogeny, continues to serve as the model by which all other examples are judged, including those of
human neotenic traits.
Axolotls are thought to have originated from metamorphic tiger salamander ancestors that colonized large
lake systems once typifying the natural habitat of present day Mexico City (Brandon 1989). Historical
artifacts and documents clearly show that axolotls played a prominent role in Aztec culture and religion
(Smith 1989). Unfortunately, we are currently witnessing something akin to the fall of the Aztec
civilization in axolotls and, arguably, all amphibians. The axolotl population in Mexico City has been
decimated over the last few decades by human activities that are destroying natural habitats. Sadly,
there are more axolotls propagated in captivity than there are living in the few remaining canals of
Xochimilco. As a result, axolotls are recognized as endangered and are protected under the Convention on
International Trade in Endangered Species (CITES) agreement.
The axolotl is an example of a reemerging model organism, since it was originally introduced to European
researchers in the late 1800s and was used extensively by developmental biologists of that era. The
axolotl’s large eggs, which are twice as large as anuran eggs and five times larger than zebrafish eggs,
provided the raw material for many seminal experiments in experimental embryology during the early
20th century (Nieuwkoop 1996). In particular, large and accessible embryos allowed researchers to trace
the morphogenetic movements of cells with vital dye staining and tissue grafting. Later in the century,
when many developmental biologists turned to faster genetic models, axolotls maintained a loyal following
of researchers, especially in the area of tissue regeneration. In contrast to mammals, adult salamanders
regenerate many complex structures, including limbs, retina, spinal cord, heart, jaw, and lateral line
sensory cells, which are analogous to hair cells of the mammalian inner ear. Pioneering research efforts
into the basis of these various regeneration paradigms, coupled with recent genome resource
development, is opening a new chapter where axolotls will play a primary role in regenerative biology. As
described in more detail below, axolotls and other tiger salamanders also continue to be an important
laboratory model in multiple research areas, including olfaction, vision, cardiogenesis, embryogenesis, and
post-embryonic development (see, e.g., Zhang et al. 2006; Harlow and Barlow 2007; Gollisch and Meister
2008; Page et al. 2008).
SOURCES AND HUSBANDRY

Axolotls are available from the Ambystoma Genetic Stock Center (AGSC) at the University of Kentucky
(http://www.ambystoma.org/AGSC/). This is a historically significant living resource that is used actively
and widely in biological research. The collection traces its ancestry back to individuals that were collected
from Xochimilco in the 1800s. Additional individuals from nature and other domesticated strains have
been introduced into the colony. However, there has not been a significant introduction of genetic
material since Humphrey (1967) introduced the genome of a related tiger salamander species (A. t.
tigrinum) when introgressing the albino mutation into an axolotl strain. Since this time, many decades of
inbreeding have yielded a homogeneous genetic stock that thrives under laboratory conditions. As a
result of long-term domestication, axolotls are the only salamanders that are easily reared and cultured in
the laboratory. The AGSC collection is maintained as a large population of ~1000 breeding adults that are
segregating several color mutants (melanoid, white, albino, axanthic, and wild-type), two lethal mutations
affecting heart (cardiac) and limb development and renal function (short-toes), and a mutation affecting
eye development and fertility (eyeless). Transgenic axolotls that ubiquitously express green fluorescent
protein (GFP) in all cell types (Sobkow et al. 2006) are also available. The collection provides a standard
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source of research material (embryos, larvae, and adults) in support of a number of areas where axolotls
continue to be the best model organism. Although primarily a production center, the AGSC also serves as
a community hub, providing advice about axolotl care and available resources to better enable research
efforts. It is important to note that many developmental biologists maintain independent axolotl colonies
and there is a relatively large community of aquarists around the world that maintain axolotls as pets.
The AGSC website and edited volume by Armstrong and Malacinski (1989) provide instructions to rear
and breed axolotls. Static renewal procedures (manual water changes) are ideal for maintaining embryos
and larvae. In recent years, automated recirculating systems that were originally designed for Xenopus or
zebrafish have been successfully adapted for juvenile and adult axolotl care. These systems, which offer
biological, filtration, and UV sterilization, greatly decrease the labor required to maintain hundreds of
animals.
Axolotls are capable of breeding several times in a year after reaching maturity at 8-12 mo. A single
female can produce two to five clutches per year, generating hundreds of eggs per clutch; males can
mate every few weeks. Breeding is accomplished using natural or artificial methods (see Technical
Approaches). When male and female axolotls are paired together in the dark, progeny are generated in
~50% of mating attempts. The success rate varies, being lowest in August and highest in January.
During mating encounters, males deposit sperm packets on rocks or the bottom of the tank, and later
the female picks up the sperm packet with its cloaca. As eggs are released, they are fertilized internally.
The embryonic period is ~20 d at 20°C. The length of the embryonic period can be dramatically
manipulated, because embryos can tolerate temperatures from 5°C to 25°C. This allows precise
adjustment of developmental rate for experimental studies and also permits more efficient utilization of
embryonic material. Larvae are free-living and feed on brine shrimp until they attain a total body length of
~3 cm. After this point, larvae are fed larger prey items (e.g., blackworms, beef liver, or salmon pellets).
Mass-reared larvae typically bite off each other’s limbs and tails during the frenzy of feeding. These
injuries can be curtailed by reducing rearing densities or by rearing larvae separately; however, the latter
requires considerably more effort. Mass rearing is generally preferred because regeneration rapidly
restores bite-damaged tissues. After larvae attain 6 cm, they are reared separately. It is possible to
communally house female juvenile and adult axolotls, but because males are slightly more aggressive
toward one another, the AGSC houses males independently. Under standard conditions, axolotls are
known to live past 10 years. Occasionally, axolotls will undergo spontaneous metamorphosis, presumably
in response to stress and changes in diet. The frequency is <1% under standard laboratory conditions.
RELATED SPECIES
Axolotls are closely related to all other tiger salamander species in North America. In fact, it is possible to
artificially cross the axolotl to other tiger salamanders and even make crosses to distantly related
ambystomatid species (Voss and Shaffer 1996). The apparent lack of hybridization barriers has clearly
played an important role in the recent evolutionary history of tiger salamanders and is influencing genetic
structures of contemporary populations (Fitzpatrick and Shaffer 2007). Many ambystomatids are the
focus of ecological and evolutionary studies, most notably A. opacum, A. talpoidium, A. maculatum, A.
californiense, and a complex of unisexual ambystomatid lineages from the Great Lakes region. Further
emphasizing the importance of hybridization in ambystomatid biology, these unisexual lineages are
thought to have originated from an ancient, interspecific hybridization event (Robertson et al. 2006).
In terms of laboratory research, tiger salamanders (A. tigrinum) from natural populations continue to be
preferred over axolotls for electrophysiological studies of the retina. Much of the early experimental
embryological work was accomplished using A. maculatum (previously known as A. punctatum) embryos
collected from nature. Today, A. maculatum is primarily used in ecological and evolutionary studies,
though Kumar and Brockes (2007) recently developed a protocol using A. maculatum to prepare
dedifferentiated cells from muscle fibers. Of the various nonambystomatid salamanders that are used in
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laboratory research, the three most commonly used are newts: red-spotted newts (Notophthalmus
viridescens), Japanese newts (Cynops pyrrhogaster), and the European newt (Pleurodeles waltl). Newts
and axolotls share a common body plan but are quite different in an evolutionary sense, having diverged
from a common ancestor 30-50 million years ago. Thus, it should not be surprising to find that some
protocols and biological information will not be transferable between ambystomatids and newts. Moreover,
with amphibians showing global declines and laboratory axolotl resources available, it is becoming
increasingly difficult to justify ethically the collection of newts or any salamander from natural populations
for use in experimental laboratory studies.
USES OF THE AXOLOTL MODEL SYSTEM

As mentioned above, salamanders were a major model system for embryologists until the mid-20th
century due to the abundance and size (3-mm diameter) of embryos and the ease of grafting embryonic
tissues. After this time, Xenopus became a more broadly used embryological system because it was
possible to house animals at high density, and to easily perform in vitro fertilizations. Indeed, many
classical experiments were first performed in the salamander, such as the characterization of Spemann’s
organizer (Spemann and Mangold 1924). Whereas Xenopus and zebrafish have the advantage of
relatively rapid development, this entails evolutionary specializations that are not representative of the
great majority of species, and mammalian development in particular (Bolker 1995). Moreover, Xenopus
presents a range of morphological specializations not typical of other frogs. As a generalized amphibian
and tetrapod, axolotls provide better models for some aspects of vertebrate development that are not
available or difficult to study using Xenopus or zebrafish. Some of these examples are reviewed below.
Germ Cell Induction
It has long been recognized that control of germ cell formation varies among vertebrates. For example, in
Xenopus and zebrafish, a portion of the egg cytoplasm segregates to form a germ plasm that contains
special components required for forming germ cells. Subsequent subdivision of the egg distributes this
cytoplasm among cells that go on to form germ cells. In contrast, mammals like the mouse show no
evidence of germ plasm, and germ cells are induced by bone morphogenetic protein (BMP) signaling
during early embryogenesis (Lawson et al. 1999; Ying and Zhao 2001; Ying et al. 2001). Following on the
pioneering work by Nieuwkoop (1996) indicating that germ cells are induced during axolotl
embryogenesis, Johnson et al. (2003) have shown that the axolotl shares an inductive mode of germ cell
formation with the mouse. They propose that the inductive mode of germ formation is the ancestral
mechanism of germ cell formation in vertebrates, and that segregation of germ plasm was independently
evolved in other models such as Xenopus and fish.
Gastrulation
Comparative studies performed among vertebrates have found surprising variation in morphogenetic and
patterning mechanisms. For example, cellular movements during mesoderm formation differ markedly
among frogs, and it is generally acknowledged that Xenopus laevis differs from more generalized species
of frogs as well as salamanders (Nieuwkoop 1996). Similarly, mechanisms of gastrulation vary among
species of teleosts (Collazo et al. 1994). Shook et al. (2002) argued that cell movements occurring at the
organizer (or dorsal blastopore lip) in the axolotl share features with the primitive streak in the chicken.
Therefore, it appears that the axolotl is a less derived amphibian in several developmental aspects
compared with Xenopus, and thus is an important and valuable resource for comparative studies of
vertebrate development. In general, studies of axolotls provide an important test for assessing the
generality of developmental mechanisms elucidated through studies of other model organisms.
Neural Crest and Lateral Line Formation
Researchers continue to use axolotls to study particular aspects of embryonic development, mainly
because of the special properties of early embryos such as their large size, slow development, and ability
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to repair tissues. These studies include the neural crest (Löfberg et al. 1985) and the neural
crest-derived dorsal root ganglia (Detwiler 1937; DuShane 1938), lateral line (Northcutt et al. 1994;
Epperlein et al. 2007), and head mesenchyme (Cerny et al. 2004; Soukup et al. 2008). It is possible to
remove and cleanly graft cranial and/or trunk neural folds containing neural crest or trunk neural tubes
from one embryo into another. The use of grafts containing the neural crest has allowed its manipulation
in studying neural crest migration and differentiation during development (Epperlein et al. 2000, 2007).
Another reason for studying the neural crest in axolotl is the availability of the white mutant (d/d), where
the lateral migration of pigment cells (melanophores and xanthophores) is impaired (Armstrong and
Malacinski 1989). The genetic basis of the d/d locus is still being studied (Parichy et al. 1999). Meanwhile,
it was shown that BMP-4 and Noggin proteins interfere with the distribution of melanophores in the
axolotl trunk and that Noggin can stimulate melanophore migration in the white mutant embryo (Hess et
al. 2008). The neural crest is also the focus of those studying mesenchymal derivatives in the head,
either during pharynx (Cerny et al. 2004) or tooth development (Soukup et al. 2008).
Regeneration
The most enduring use of the axolotl model has been to study the ability of juveniles or adults to
regenerate entire limbs and tails. Important mechanistic insight into how regeneration occurs has been
gained through various experiments where tissue was transplanted from one animal to another (Stocum
1984). For example, the potency of tissues to produce a regenerating limb was first defined by blocking
regeneration by irradiating host limbs and then rescuing the limbs by transplanting in different types of
unirradiated tissues (Umanski 1938). In recent years, Bryant and coworkers (Endo et al. 2004) have
defined the "minimal requirements" for limb regeneration in their Accessory Limb Model, where deviation
of nerve to a lateral site combined with grafting of anterior and posterior limb skin together at that site is
sufficient to induce a limb to grow out of this lateral site. This capability highlights the remarkable feature
of this system in which grafted tissues are not rejected, even in sexually mature individuals. A modulation
of the immune system may be linked to this broad regenerative capacity (Mescher and Neff 2006). More
recently, transgenesis was combined with embryonic and tissue grafting to reliably track tissue cell fate
during regeneration. In these experiments, limb regeneration occurred without generating a pluripotent
stem cell intermediate (Kragl et al. 2009).
Techniques such as electroporation of ectopic genes and the knockdown of protein levels through
morpholinos, along with germline transgenesis, have significantly aided the study of the molecular basis of
limb regeneration. A recent highlight has been the study of proximal-distal positional identity during limb
regeneration, where the glycosyl phosphatidylinositol (GPI)-linked molecule Prod1 and the nuclear
homeodomain proteins Meis 1 and 2 were identified and tested to specify positional identity during limb
regeneration (da Silva et al. 2002; Echeverri and Tanaka 2005; Mercader et al. 2005). Viral infection
using vaccinia and adenovirus was effectively used to study the role of wnt and sonic hedgehog signaling
during regeneration (Roy et al. 2000; Kawakami et al. 2006).
Electrophysiology
The axolotl has long been used to study electrophysiology. Due to their large genome size, axolotl cells are
typically five to 10 times larger than their mammalian counterparts. This makes axolotl cells particularly
accessible for complex electrophysiology experiments. Recently, the axolotl oocyte was used as an
expression system to identify calcium-activated chloride channels (CaCCs) (Schroeder et al. 2008).
Although Xenopus oocytes are classically used for electrophysiology studies, the abundant expression of
the channel in Xenopus oocytes (important for the prevention of polyspermia in frog eggs) precluded
their use in this case, while oocytes from the polyspermic axolotl showed little endogenous expression of
the channel. Axolotl oocytes also have an advantage in the study of calcium channels or unselective cation
channels. These channels are difficult to measure in the Xenopus oocyte system using a two-electrode
voltage clamp, because endogenous channels are activated by calcium influx (Björn Schroeder, pers.
comm.). Again, this highlights the complementary nature of the two amphibian systems for biological
studies.
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Thyroid Hormone-Induced Metamorphosis
Amphibian metamorphosis has long served as a model for investigating aspects of post-embryonic
development that are dependent upon thyroid hormone. Axolotls can readily be induced to undergo
metamorphosis by adding thyroid hormone to the ambient water. In a few weeks, traits that allow for
aquatic existence, such as gills, pigmentation, and tail fins, are remodeled to allow for a more terrestrial
existence. In fact, a metamorphosed axolotl looks very much like an adult tiger salamander. Thyroid
hormone concentration alters the timing of skin metamorphosis initiation, but the subsequent gene
expression responses are remarkably reproducible (Page et al. 2008, 2009). Thus, developmental events
can be precisely induced in axolotls at arbitrary times during ontogeny. It is also possible to induce
metamorphosis in anurans; however, the interpretation of such experiments is confounded by the fact
that anurans are always developing toward a metamorphic outcome. Also, some of the molecular insights
provided by studies of Xenopus may be anuran-specific and not amphibian-specific (Page et al. 2008).
Continued studies of axolotls are likely to provide complementary and novel insights about amphibian
metamorphosis, and to develop this model further for ecological and ecotoxicological research (Page et al.
2007).
GENETICS, GENOMICS, AND ASSOCIATED RESOURCES

The axolotl has a deep history as a developmental genetic model. Many axolotl mutants were discovered
by crossing different domesticated strains of axolotl, or by crossing domesticated strains with individuals
collected directly from Xochimilco. However, most of these mutants are no longer propagated by the
AGSC (Malacinski 1989). Of all the mutants, albino is perhaps the best known. It was intricately
introgressed into the axolotl from the metamorphic Eastern tiger salamander (A. t. tigrinum), using
interspecific hybridization and nuclear transplantation (Humphrey 1967). This effort also provided
perspective about the paedomorphic condition of axolotls. Mendelian segregation was observed,
suggesting a single gene basis. Voss and Smith (2005) adopted this same interspecific crossing design
and subsequently mapped the genetic factor for the discrete expression of metamorphosis versus
paedomorphosis (met), and also showed that met contributes to quantitative variation in developmental
timing. Perhaps more importantly, these intraspecific crosses were used to build a linkage map of the
axolotl/tiger salamander genome and relate the structure of the axolotl genome to other fully sequenced
vertebrate genomes (Smith and Voss 2007). Over 1000 molecular markers have been mapped (Smith et
al. 2005a), the majority of which are expressed sequence tags (ESTs) that have been isolated from both
axolotls and tiger salamander (Habermann et al. 2004; Putta et al. 2004). Recently, 750,000 ESTs,
100,000 bacterial artificial chromosomes (BACs), and thousands of small RNAs were generated from
regenerating limbs of axolotls, and additional cDNA sequencing is ongoing (Monaghan et al. 2009; Smith
et al. 2009; SR Voss, unpubl.).
EST development has been prioritized over genome sequencing because the axolotl’s genome size is 10
times larger than the human genome. Information from ESTs is being used in various ways to improve
axolotl genome resources. An Affymetrix GeneChip has been developed and used to study a number of
different research paradigms, including limb and spinal cord regeneration, thyroid hormone-induced skin
metamorphosis, and the response of spleen cells to ranavirus infection (Monaghan et al. 2007, 2009;
Page et al. 2007, 2008; Cotter et al. 2008). A second-generation GeneChip will be produced in 2009 and
made available to the community. To date, ~9000 axolotl EST contigs have been assembled that
correspond to different human protein-coding loci. These EST data and information about molecular
markers and the Ambystoma linkage map are available via a community web portal (Sal-Site,
www.ambystoma.org; Smith et al. 2005b).
TECHNICAL APPROACHES
Transgenesis, Grafting, and Regeneration
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It is possible to generate transgenic animals with relative ease. Injection of plasmid and even BAC DNA
into the axolotl egg results in varying frequencies of integration (depending on the plasmid DNA) within
one of the first few cell divisions (Sobkow et al. 2006; S Khattak and EM Tanaka, unpubl.). The integration
efficiency is significantly increased if the plasmid contains SceI meganuclease sites and the DNA is
coinjected with SceI meganuclease, as in Generation of Transgenic Axolotls (Ambystoma
mexicanum) (Khattak et al. 2009). This method has been used to generate a germline transgenic
axolotl that expresses eGFP behind the ubiquitous CAGGs promoter, available through the Kentucky stock
center (Sobkow et al. 2006). Because the axolotl is so amenable to grafting in embryonic and
post-embryonic stages, it has proved useful for marking tissues during embryogenesis and regeneration
(Sobkow et al. 2006; Mchedlishvili et al. 2007; Soukup et al. 2008; Kragl et al. 2009). The Epperlein and
Tanaka labs have worked out many optimizations for the transplantation of tissues in embryonic axolotls,
described in Axolotl (Ambystoma mexicanum) Embryonic Transplantation Methods (Nacu et al.
2009). In addition, protocols for Grafting Axolotl (Ambystoma mexicanum) Limb Skin and
Cartilage from GFP+ Donors to Normal Hosts (Kragl and Tanaka 2009a) and Axolotl (Ambystoma
mexicanum) Limb and Tail Amputation (Kragl and Tanaka 2009b) are available.
Breeding
Breeding axolotls is normally performed by natural matings. Briefly, a male and female animal are paired
together in the dark. Approximately 50% of pairings produce progeny. First, sperm packets are laid by
the male on rocks or the bottom of the tank, and later the female picks up the sperm packet and
undergoes internal fertilization. The female will start to lay eggs 12-24 h after picking up the sperm
packet. A more complete description of natural breeding can be found at www.ambystoma.org and in the
book The Developmental Biology of the Axolotl (Armstrong et al. 1989). Alternatively, a protocol for
Axolotl (Ambystoma mexicanum) In Vitro Fertilization (Khattak and Tanaka 2009) is also available.
Axolotls rarely, if ever, metamorphose in the lab. However, if thyroid hormone is provided at an
appropriate concentration and developmental stage, it is possible to generate healthy and robust
terrestrial axolotls, as in Induction of Metamorphosis in Axolotls (Ambystoma mexicanum) (Page
and Voss 2009).
Electroporation
Techniques to transduce plasmids or morpholinos into somatic tissue are valuable for testing gene
function in animals beyond embryonic stages. Electroporation provides an appropriate method for this. It
has the advantage of being able to test gene function rapidly without making transgenic animals, and it
avoids the use of viruses. Potential disadvantages are the variability of electroporation efficiency and
damage to the samples. Used widely in chicken, electroporation was first applied to axolotl tissue by
Echeverri and Tanaka (2002). The voltages and exact conditions used are highly dependent on the
electroporation device and electrodes, but basic requirements and suggestions for electroporation of
axolotl tissue have been published by Echeverri and Tanaka (2003).
ACKNOWLEDGMENTS
We thank Björn Schroeder, Dunja Knapp, Eugen Nacu, Martin Kragl, and Robert Page for contributions to
the related protocols and for comments on the manuscript. The work described in this manuscript was
partially supported by grants from the Volkswagen Foundation (E.T.), the German Research Council grant
of the priority program 1109 and 1356 (E.T.), German Research Council grant SFB655 (E.T.), BMBF
Biofutures Award (E.T.), the Max-Planck Institute (E.T.), the Center for Regenerative Therapies Dresden
(E.T.), NIH-R24-RR016344 (S.R.V.), and NSF-DBI-0443496 (S.R.V.).
REFERENCES
Armstrong, JB and Malacinski, GM, eds. 1989. Developmental biology of the axolotl. Oxford University
15 of 21 7/1/10 11:18 AM
Press, NY.
Armstrong JB, Duhon ST, Malacinski GM. 1989. Raising the axolotl in captivity. In Developmental biology
of the axolotl (eds. JB Armstrong and GM Malacinski), pp. 220–227. Oxford University Press, New York.
Bolker JA. 1995. Model systems in developmental biology. Bioessays 17: 451–455.[Medline]
Brandon RA. 1989. Natural history of the axolotl and its relationship to other ambystomatid salamanders.
In Developmental biology of the axolotl (eds. JB Armstrong and GM Malacinski), pp. 13–21. Oxford
University Press, New York.
Cerny R, Lwigale P, Ericsson R, Meulemans D, Epperlein HH, Bronner-Fraser M. 2004. Developmental

origins and evolution of jaws: New interpretation of ‘maxillary’ and ‘mandibular.’ Dev Biol 276:
225–236.[Medline]
Collazo A, Fraser SE, Mabee PM. 1994. A dual embryonic origin for vertebrate mechanoreceptors. Science
264: 426–430.[Abstract/Free Full Text]
Cotter JD, Storfer A, Page RB, Beachy CK, Voss SR. 2008. Transcriptional response of Mexican axolotls to
Ambystoma tigrinum virus (ATV) infection. BMC Genomics 9: 493. doi:
10.1186/1471-2164-9-493.[Medline]
da Silva SM, Gates PB, Brockes JP. 2002. The newt ortholog of CD59 is implicated in proximodistal identity
during amphibian limb regeneration. Dev Cell 3: 547–555.[Medline]
Detwiler SR. 1937. Observations upon the migration of neural crest cells, and upon the development of
the spinal ganglia and vertebral arches in Ambystoma. Am J Anat 61: 63–94.
Developmental Biology of Urodeles. 1996, Int J Dev Biol 40: 613–916.[Medline]
DuShane GP. 1938. Neural fold derivatives in the amphibia: Pigment cells, spinal ganglia and Rohon-Beard
cells. J Exp Zool 78: 485–503.
Echeverri K, Tanaka EM. 2002. Ectoderm to mesoderm lineage switching during axolotl tail regeneration.
Science 298: 1993–1996.[Abstract/Free Full Text]
Echeverri K, Tanaka EM. 2003. Electroporation as a tool to study in vivo spinal cord regeneration. Dev Dyn
226: 418–425.[Medline]
Echeverri K, Tanaka EM. 2005. Proximodistal patterning during limb regeneration. Dev Biol 279:
391–401.[Medline]
Endo T, Bryant SV, Gardiner DM. 2004. A stepwise model system for limb regeneration. Dev Biol 270:
135–145.[Medline]
Epperlein HH, Meulemans D, Bronner-Fraser M, Steinbeisser H, Selleck MAJ. 2000. Analysis of cranial
neural crest migratory pathways in axolotl using cell markers and transplantation. Development 127:
2751–2761.[Abstract]
Epperlein HH, Selleck MA, Meulemans D, Mchedlishvili L, Cerny R, Sobkow L, Bronner-Fraser M. 2007.
Migratory patterns and developmental potential of trunk neural crest cells in the axolotl embryo. Dev Dyn
236: 389–403.[Medline]
Fitzpatrick BJ, Shaffer HB. 2007. Hybrid vigor between native and introduced salamanders raises new
challenges for conservation. Proc Natl Acad Sci 104: 15793–15798.[Abstract/Free Full Text]
Gollisch T, Meister M. 2008. Rapid neural coding in the retina with relative spike latencies. Science 319:
1108–1111.[Abstract/Free Full Text]
Gould SJ. 1977. Ontogeny and phylogeny. Harvard University Press, Cambridge, MA.
Habermann B, Bebin AG, Herklotz S, Volkmer M, Eckelt K, Pehlke K, Epperlein HH, Schackert HK, Wiebe G,
Tanaka EM. 2004. An Ambystoma mexicanum EST sequencing project: Analysis of 17,352 expressed
sequence tags from embryonic and regenerating blastema cDNA libraries. Genome Biol 5: R67. doi:
10.1186/gb-2004-5-9-r67.[Medline]
Harlow DE, Barlow LA. 2007. Embryonic origin of gustatory cranial sensory neurons. Dev Biol 310:
317–328.[Medline]
16 of 21 7/1/10 11:18 AM
Hess K, Steinbeisser H, Kurth T, Epperlein HH. 2008. Bone morphogenetic protein-4 and Noggin
signaling regulates pigment cell distribution in the axolotl trunk. Differentiation 76: 206–218.[Medline]
Humphrey RR. 1967. Albino axolotls from an albino tiger salamander through hybridization. J Hered 58:
95–101.[Free Full Text]
Johnson AD, Crother B, White ME, Patient R, Bachvarova RF, Drum M, Masi T. 2003. Regulative germ cell
specification in axolotl embryos: A primitive trait conserved in the mammalian lineage. Philos Trans R Soc
Lond B Biol Sci 358: 1371–1379.[Abstract/Free Full Text]
Kawakami Y, Rodriguez Esteban C, Raya M, Kawakami H, Martí M, Dubova I, Izpisúa Belmonte JC. 2006.
Wnt/β-catenin signaling regulates vertebrate limb regeneration. Genes & Dev 20:
Khattak S, Tanaka EM. 2009. Axolotl (Ambystoma mexicanum) in vitro fertilization. Cold Spring Harb
Protoc (this issue). doi: 10.1101/pdb.prot5263.[Abstract/Free Full Text]
Khattak S, Richter T, Tanaka EM. 2009. Generation of transgenic axolotls (Ambystoma mexicanum). Cold
Spring Harb Protoc (this issue). doi: 10.1101/pdb.prot5264.[Abstract/Free Full Text]
Kragl M, Tanaka EM. 2009a. Grafting axolotl (Ambystoma mexicanum) limb skin and cartilage from GFP+
donors to normal hosts. Cold Spring Harb Protoc (this issue). doi:
10.1101/pdb.prot5266.[Abstract/Free Full Text]
Kragl M, Tanaka EM. 2009b. Axolotl (Ambystoma mexicanum) limb and tail amputation. Cold Spring Harb
Protoc (this issue). doi: 10.1101/pdb.prot5267.[Abstract/Free Full Text]
Kragl M, Knapp D, Nacu E, Khattak S, Maden M, Epperlein HH, Tanaka EM. 2009. Cells keep a memory of
their tissue origin during axolotl limb regeneration. Nature 460: 60–65.[Medline]
Kumar A, Brockes JP. 2007. Preparation of cultured myofibers from larval salamander limbs for cellular
plasticity studies. Nat Protoc 2: 939–947.[Medline]
Lawson KA, Dunn NR, Roelen BA, Zeinstra LM, Davis AM, Wright CV, Korving JP, Hogan BL. 1999. Bmp4 is
required for the generation of primordial germ cells in the mouse embryo. Genes & Dev 13:
Löfberg JA, Nynäs-McCoy A, Olsson C, Jönsson L, Perris R. 1985. Stimulation of initial neural crest cell
migration in the axolotl embryo by tissue grafts and extracellular matrix transplanted on microcarriers.
Dev Biol 107: 442–459.[Medline]
Malacinski GM. 1989. Developmental genetics. In Developmental biology of the axolotl (eds. JB Armstrong
and GM Malacinski), pp. 102–109. Oxford University Press, New York.
Mchedlishvili L, Epperlein HH, Telzerow A, Tanaka EM. 2007. A clonal analysis of neural progenitors during
axolotl spinal cord regeneration reveals evidence for both spatially restricted and multipotent progenitors.
Development 134: 2083–2093.[Abstract/Free Full Text]
Mercader N, Tanaka EM, Torres M. 2005. Proximodistal identity during vertebrate limb regeneration is
regulated by Meis homeodomain proteins. Development 132: 4131–4142.[Abstract/Free Full Text]
Mescher AL, Neff AW. 2006. Limb regeneration in amphibians: Immunological considerations.
ScientificWorldJournal Suppl. 1 6: 1–11.
Monaghan JR, Walker JA, Page RB, Putta S, Beachy CK, Voss SR. 2007. Early gene expression during
natural spinal cord regeneration in the salamander Ambystoma mexicanum. J Neurochem 101:
27–40.[Medline]
Monaghan JR, Epp L, Putta S, Page RB, Walker JA, Zhu W, Pao GM, Verma IM, Hunter T, Bryant SV, et al.
2009. Microarray and DNA sequence analysis of transcription during nerve-dependent limb regeneration.
BMC Biology 7: 1. doi: 10.1186/1741-7007-7-1.[Medline]
Nacu E, Knapp D, Tanaka EM, Epperlein HH. 2009. Axolotl (Ambystoma mexicanum) embryonic
transplantation methods. Cold Spring Harb Protoc (this issue). doi:
10.1101/pdb.prot5265.[Abstract/Free Full Text]
Nieuwkoop PD. 1996. What are the key advantages and disadvantages of urodele species compared to
17 of 21 7/1/10 11:18 AM
anurans as a model system for experimental analysis of early development? Int J Dev Biol 40:
617–619.[Medline]
Northcutt RG, Catania KC, Criley BB. 1994. Development of lateral line organs in the axolotl. J Comp Neurol
340: 480–514.[Medline]
Page RB, Voss SR. 2009. Induction of metamorphosis in axolotls (Ambystoma mexicanum). Cold Spring
Harb Protoc (this issue). doi: 10.1101/pdb.prot5268.[Abstract/Free Full Text]
Page RB, Monaghan JR, Samuels AK, Smith JJ, Beachy CK, Voss SR. 2007. Microarray analysis identifies
keratin loci as sensitive biomarkers for thyroid hormone disruption in salamanders (Ambystoma). Comp
Biochem Physiol Part C 145: 15–27.
Page RB, Voss SR, Samuels AK, Smith JJ, Putta S, Beachy CK. 2008. Effect of thyroid hormone
concentration on the transcriptional response underlying induced metamorphosis in the Mexican axolotl
(Ambystoma). BMC Genomics 9: 78. doi: 10.1186/1471-2164-9-78.[Medline]
Page RB, Monaghan JR, Walker JA, Voss SR. 2009. An integrative model of transcriptional and
morphological changes during TH-induced metamorphosis of the Mexican axolotl. Gen Comp Endo 162:
219–232.
Parichy DM, Stigson M, Voss SR. 1999. Genetic analysis of steel and the PG-M/Versican-encoding gene
AxPG as candidates for the white (d) pigmentation mutant in the salamander Ambystoma mexicanum.
Dev Genes Evol 209: 349–356.[Medline]
Petranka JW. 1998. Salamanders of the United States and Canada. Smithsonian Institution Press,
Washington, DC.
Putta S, Smith JJ, Walker JA, Rondet M, Weisrock D, Monaghan JR, Samuels AK, Kump K, King DC,
Maness NJ, et al. 2004. From biomedicine to natural history research: Expressed sequence tag resources
for ambystomatid salamanders. BMC Genomics 5: 54. doi: 10.1186/1471-2164-5-54.[Medline]
Robertson AV, Ramsden C, Niedzwiecki J, Fu J, Bogart JP. 2006. An unexpected recent ancestor of
unisexual Ambystoma. Mol Ecol 15: 3339–3351.[Medline]
Roy S, Gardiner DM, Bryant SV. 2000. Vaccinia as a tool for functional analysis in regenerating limbs:
Ectopic expression of Shh. Dev Biol 218: 199–205.[Medline]
Schroeder BC, Cheng T, Jan YN, Jan LY. 2008. Expression cloning of TMEM16A as a calcium-activated
chloride channel subunit. Cell 134: 1019–1029.[Medline]
Shook DR, Majer C, Keller R. 2002. Urodeles remove mesoderm from the superficial layer by subduction
through a bilateral primitive streak. Dev Biol 248: 220–239.[Medline]
Smith HM. 1989. Discovery of the axolotl and its early history in biological research. In Developmental
biology of the axolotl (eds. JB Armstrong and GM Malacinski), pp. 3–12. Oxford University Press, New
York.
Smith JJ, Voss SR. 2007. Bird and mammal sex-chromosome orthologs map to the same autosomal
region in a salamander (ambystoma). Genetics 177: 607–613.[Abstract/Free Full Text]
Smith JJ, Kump K, Walker JA, Parichy DM, Voss SR. 2005a. A comprehensive expressed sequence tag
linkage map for tiger salamander and Mexican axolotl: Enabling gene mapping, comparative genomics,
and PCR marker development in Ambystoma. Genetics 171: 1161–1171.[Abstract/Free Full Text]
Smith JJ, Putta S, Walker JA, Kump DK, Samuels AK, Monaghan JR, Weisrock DW, Staben C, Voss SR.
2005b. Sal-Site: Integrating new and existing ambystomatid research and information resources. BMC
Genomics 6: 181. doi: 10.1186/1471-2164-6-181.[Medline]
Smith JJ, Putta S, Zhu W, Pao GM, Verma IM, Hunter T, Bryant SV, Gardiner DM, Harkins TT, Voss SR.
2009. Genic regions of a large salamander genome contain long introns and novel genes. BMC Genomics
10: 19. doi: 10.1186/1471-2164-10-19.[Medline]
Sobkow L, Epperlein HH, Herklotz S, Straube WL, Tanaka EM. 2006. A germline GFP transgenic axolotl and
its use to track cell fate: Dual origin of the fin mesenchyme during development and the fate of blood cells
during regeneration. Dev Biol 290: 386–397.[Medline]
Soukup V, Epperlein HH, Horácek I, Cerny R. 2008. Dual epithelial origin of vertebrate oral teeth. Nature
18 of 21 7/1/10 11:18 AM
455: 795–798.[Medline]
Spemann H, Mangold H. 1924. Über induktion von embryonalanlagen durch, implantation artfremder
organisatoren. Arch mikrosk Anat EntwMech 100: 599–638.
Stocum DL. 1984. The urodele limb regeneration blastema. Determination and organization of the
morphogenetic field. Differentiation 27: 13–28.[Medline]
Umanski EE. 1938. The regeneration potencies of axolotl skin studied by means of exclusion of the
regeneration capacity of tissues through exposure to X-rays. Bull Biol Med Exp USSR 6: 141–145.
Voss SR, Shaffer HB. 1996. What insights into the developmental traits of urodeles does the study of
interspecific hybrids provide? Int J Dev Biol 40: 885–893.[Medline]
Voss SR, Smith JJ. 2005. Evolution of salamander life cycles: A major-effect quantitative trait locus
contributes to discrete and continuous variation for metamorphic timing. Genetics 170:
Ying Y, Zhao GQ. 2001. Cooperation of endoderm-derived BMP2 and extraembryonic ectoderm-derived
BMP4 in primordial germ cell generation in the mouse. Dev Biol 232: 484–492.[Medline]
Ying Y, Qi X, Zhao GQ. 2001. Induction of primordial germ cells from murine epiblasts by synergistic
action of BMP4 and BMP8B signaling pathways. Proc Natl Acad Sci 98:
Zhang C, Pietras KM, Sferrazza GF, Jia P, Athauda G, Rueda-de-Leon E, Maier JA, Dube DK, Lemanski SL,
Lemanski LF. 2006. Molecular and immunohistochemical analyses of cardiac troponin T during cardiac
development in the Mexican axolotl, Ambystoma mexicanum. J Cell Biochem 100: 1–15.
Recipe
AMEM (1%)
24 mL H2O (sterile; Merck)
1 mL antibiotic-antimycotic solution (AA solution; Sigma)
1 mL glutamine (Invitrogen)
1 mL insulin solution for amphibians
100 µL gentamicin (50 mg/mL; Sigma)
1 mL fetal calf serum, embryonic stem (ES) cell-qualified, heat-inactivated (Invitrogen) (see Note below)
72 mL MEM (1X; Invitrogen)
Prepare the medium in a sterile reagent bottle (e.g., a 75-cm2 tissue culture flask). Combine the reagents
in the order listed. The resulting culture medium is MEM-adjusted to amphibian osmolarity plus serum
for growth.
To heat-inactivate the complement system in the serum, defrost the stock bottle and incubate for 30 min
at 56ºC. Cool the bottle to room temperature and store as 10-mL aliquots at –20ºC.
Recipe
APBS
19 of 21 7/1/10 11:18 AM
100 mL PBS (phosphate-buffered saline; Invitrogen)
In this recipe, PBS is adjusted to amphibian osmolarity (225 ± 5 mOsm/L) by the addition of H2O.
Recipe
Amphibian dissection medium
Reagent Amount to add Final concentration
APBS 5 mL
50 mg/mL gentamicin (Sigma) 5 µL 50 µg/mL
Recipe
SF-AMEM
1 mL antibiotic-antimycotic solution (AA solution; Sigma)
1 mL glutamine (Invitrogen)
1 mL insulin solution for amphibians
100 µL gentamicin (50 mg/mL; Sigma)
73 mL 1X MEM (Invitrogen)
Prepare the medium in a sterile reagent bottle (e.g., a 75-cm2 tissue culture flask). Combine the reagents
in the order listed. The resulting culture medium is serum-free MEM adjusted to amphibian osmolarity.
Table
Table 1. Gene expression profiling in newt blastema cells by qPCR
Sample Msx1 D-11 Prod 1
Blastema tissue 14.2 49.0 1
Normal limb 1 1 1
20 of 21 7/1/10 11:18 AM
Blastema cells (from culture) 18.7 85.3 0.007
Blastema cells (from suspension) -- -- 0.071
B1H1 cells 3.1 0 0
The mRNA from cultured blastema cells was prepared using cell lysis buffer from Ambion. Trizol
reagent (Sigma) was used for the preparation of mRNA from normal newt limb and limb blastema.
The values are normalized to newt EF-1 in all samples and expressed as relative units.
Table
Table 2. List of gene-specific primers for Msx 1, D-11, Prod1, and EF-1
Newt msx1 fwd 5'-AAGTTCTCTCCACCCTCAGC-3'
Newt msx1 rev 5'-GCTTCCGGTTGGTCTTGT-3'
Newt msx1 FAM/BHQ1 5'-TGCATGCACCCTTCGGAAGC-3'
Newt D-11 rev 5'-GCTTCCGGTTGGTCTTGT-3'
Newt D-11 FAM/BHQ1 5'-TGCATGCACCCTTCGGAAGC-3'
Newt D-11 fwd 5'-GTGGGCTCTCTGGACAAGAC-3'
Newt D-11 rev 5'-GGCAGCTAGTTCACGTGTTG-3'
Newt D-11 FAM/BHQ1 5'-TGCGCCCATCTTCTTGTCCG-3'
Prod1 fwd 5'-TTCCCTAGAATTTGGGAACG-3'
Prod1 rev 5'-GGCAGCTAGTTCACGTGTTG-3'
Prod1 FAM/BHQ1 5'-TGACTGGTGTCCTCACACAACCACC-3'
EF-1 fwd 5'-TAGAGTGCAGGTGACGATCC-3'
EF-1 rev 5'-AGTCACCAAGTCTGCCATCA-3'
EF-1 FAM/BHQ1 5'-AGTAGCGCCAGCCACACCCA-3'
Copyright © 2010 by Cold Spring Harbor Laboratory Press. Online ISSN: 1559-6095 Terms of Service All rights reserved. Anyone
using the procedures outlined in these protocols does so at their own risk. Cold Spring Harbor Laboratory makes no representations
or warranties with respect to the material set forth in these protocols and has no liability in connection with their use. All materials
used in these protocols, but not limited to those highlighted with the Warning icon, may be considered hazardous and should be
used with caution. For a full listing of cautions, click here.
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Preparation and Culture of Limb Blastema Stem Cells From Regenerating Larval and Adult Salamanders - Kumar and Godwin 2010 (1) - PDB - Prot5367 - Cold Spring Harbor Protocols

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Preparation and Culture of Limb Blastema Stem Cells From Regenerating Larval and Adult Salamanders - Kumar and Godwin 2010 (1) - PDB - Prot5367 - Cold Spring Harbor Protocols

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Preparation and Culture of Limb Blastema Stem Cells from Regen... http://cshprotocols.cshlp.org/cgi/content/full/2010/1/pdb.prot5367...

Cite as: Cold Spring Harb. Protoc.; 2010; doi:10.1101/pdb.prot5367

4 Corresponding authors (anoop.kumar@ucl.ac.uk); (james.godwin@armi.monash.edu.au).

Amphibian dissection medium

Ethanol (70%, in a spray bottle)

N. viridescens (adult newts) or A. mexicanum (axolotl larvae)

Sulfamerazine (0.5%, w/v in H2O; Sigma) (optional; see Step 3)

Tap H2O (dechlorinated and autoclaved)

Tricaine (0.1%, w/v in H2O; Sigma)

Virkon disinfectant (0.01%, w/v in autoclaved, dechlorinated tap H2O)

Dish (glass-bottom, 50-mm; MatTek P50G-0-14-F)

Forceps (Dumont-Biological, #5, fine-tip, two pairs; Fine Science Tools)

Hood (sterile, for tissue culture)

Incubator (humidified, preset to 25°C, 2.5% CO2)

Malleus nipper (angled down) (Fine Science Tools)

Micropipettes and tips (100-µL, 1-mL)

Pasteur pipette (fine-tip, sterile)

Petri dishes (150-mm, 92-mm, and 60-mm, bacterial, sterile)

Plate (96-well, clear bottom with lid, collagen-coated; Corning)

Scissors (11.5-cm; Fine Science Tools)

Scissors (8-cm, Mini-Vannas; Fine Science Tools)

Spring scissors (12-cm; Fine Science Tools)

Stereomicroscope (Leica Z6)

Tubes (microcentrifuge, 1.5-mL, sterile)

2. Amputate the limbs:

4. Repeat Steps 2 and 3 for all of the remaining animals.

Dissociation of Blastema Cells

10. Disinfect the anesthetized animals:

15. Repeat Steps 12-14 for each animal.

Figure 1. Schematic diagram of the microsurgical

Maintenance of Cell Culture

1. Use midsize blastema.

Problem: The cell proliferation assay shows high background.

Solution: To reduce background in cell proliferation assays, try the following:

1. Reduce the serum concentration in the AMEM to 0.05%-0.1%.

2. Omit insulin from the AMEM.

3. Try a new batch of animals.

Figure 2. Photomicrograph of dissociated newt blastema cells in

View larger version

Figure 3. Axolotl blastema cells in culture. Axolotl

View larger version (137K):

Expression of Cellular Markers

Figure 4. Newt limb blastema cells express antigens associated with

Gene Expression Profiling by Quantitative Polymerase Chain Reaction (qPCR)

Tricaine (Tricaine methanesulfonate)

Emerging Model Organisms

Ambystoma mexicanum, the Axolotl: A Versatile Amphibian Model for

1 Department of Biology and Ambystoma Genetic Stock Center, University of Kentucky,

5 Corresponding author (elly.tanaka@crt-dresden.de)

SOURCES AND HUSBANDRY

USES OF THE AXOLOTL MODEL SYSTEM

Germ Cell Induction

Neural Crest and Lateral Line Formation

Thyroid Hormone-Induced Metamorphosis

GENETICS, GENOMICS, AND ASSOCIATED RESOURCES

Cerny R, Lwigale P, Ericsson R, Meulemans D, Epperlein HH, Bronner-Fraser M. 2004. Developmental

Developmental Biology of Urodeles. 1996, Int J Dev Biol 40: 613–916.[Medline]

1 mL antibiotic-antimycotic solution (AA solution; Sigma)

1 mL insulin solution for amphibians

100 µL gentamicin (50 mg/mL; Sigma)

72 mL MEM (1X; Invitrogen)

25 mL H2O (sterile; Merck)