Axolotl Development

Embryology - 2 Dec 2023        Expand to Translate
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Introduction

Adult Axolotl

Axolotls (Ambystoma mexicanum) are the larval form of the Mexican Salamander amphibian and are an animal model used in limb regeneration studies. Axolotls take about 12 months to reach sexual maturity, males release spermatophore into the water and the female may take them up, eventually laying around 200-600 eggs on plants. Egg development takes two weeks, the tadpole-like young remain attached to the plants for a further two weeks. Loss or amputation of the axolotl limb leads to the regeneration of the lost limb from trunk tissue, thereby repeating a developmental sequence as a repair process.

The sequence of axolotl embryonic developmental stages was characterised in the late 1980's.

Links: Category:Axolotl | Category:Salamander | Animal Development

Some Recent Findings

Neural control of growth and size in the axolotl limb regenerate "Upon the completion of the developmental stages of regeneration, when the regenerative organ known as the blastema completes patterning and differentiation, the limb regenerate is proportionally small in size. It then undergoes a phase of regeneration that we have called the 'tiny-limb' stage, which is defined by rapid growth until the regenerate reaches the proportionally appropriate size. In the current study we have characterized this growth and have found that signaling from the limb nerves is required for its maintenance. Using the regenerative assay known as the accessory limb model (ALM), we have found that growth and size of the limb positively correlates with nerve abundance. We have additionally developed a new regenerative assay called the neural modified-ALM (NM-ALM), which decouples the source of the nerves from the regenerating host environment. Using the NM-ALM we discovered that non-neural extrinsic factors from differently sized host animals do not play a prominent role in determining the size of the regenerating limb. We have also discovered that the regulation of limb size is not autonomously regulated by the limb nerves. Together, these observations show that the limb nerves provide essential cues to regulate ontogenetic allometric growth and the final size of the regenerating limb." BMP signaling is essential for sustaining proximo-distal progression in regenerating axolotl limbs "Amputation of a salamander limb triggers a regeneration process that is perfect. A limited number of genes have been studied in this context and even fewer have been analyzed functionally. In this work, we use the BMP signaling inhibitor LDN193189 on Ambystoma mexicanum to explore the role of BMPs in regeneration. We find that BMP signaling is required for proper expression of various patterning genes and that its inhibition causes major defects in the regenerated limbs. Fgf8 is down-regulated when BMP signaling is blocked, but ectopic injection of either human or axolotl protein did not rescue the defects. By administering LDN193189 treatments at different time points during regeneration, we show clearly that limb regeneration progresses in a proximal to distal fashion. This demonstrates that BMPs play a major role in patterning of regenerated limbs and that regeneration is a progressive process like development." Rediscovering the Axolotl as a Model for thyroid Hormone Dependent Development "The Mexican axolotl (Ambystoma mexicanum) is an important model organism in biomedical research. Much current attention is focused on the axolotl's amazing ability to regenerate tissues and whole organs after injury. However, not forgotten is the axolotl's equally amazing ability to thwart aspects of tissue maturation and retain juvenile morphology into the adult phase of life. Unlike close tiger salamander relatives that undergo a thyroid hormone regulated metamorphosis, the axolotl does not typically undergo a metamorphosis. Instead, the axolotl exhibits a paedomorphic mode of development that enables a completely aquatic life cycle. The evolution of paedomorphosis allowed axolotls to exploit relatively permanent habitats in Mexico, and preadapted axolotls for domestication and laboratory study. In this perspective, we first introduce the axolotl and the various meanings of paedomorphosis, and then stress the need to move beyond endocrinology-guided approaches to understand the axolotl's hypothyroid state. With the recent completion of the axolotl genome assembly and established methods to manipulate gene functions, the axolotl is poised to provide new insights about paedomorphosis and the role of thyroid hormone in development and evolution."

More recent papers

This table allows an automated computer search of the external PubMed database using the listed "Search term" text link. This search now requires a manual link as the original PubMed extension has been disabled. The displayed list of references do not reflect any editorial selection of material based on content or relevance. References also appear on this list based upon the date of the actual page viewing. References listed on the rest of the content page and the associated discussion page (listed under the publication year sub-headings) do include some editorial selection based upon both relevance and availability. More? References | Discussion Page | Journal Searches | 2019 References | 2020 References Search term: Axolotl Development | Axolotl Embryology | Axolotl Limb Development

Older papers

These papers originally appeared in the Some Recent Findings table, but as that list grew in length have now been shuffled down to this collapsible table. See also the Discussion Page for other references listed by year and References on this current page. Acquisition and reconstruction of 4D surfaces of axolotl embryos with the flipping stage robotic microscope "We have designed and constructed a Flipping Stage for a light microscope that can view the whole exterior surface of a 2 mm diameter developing axolotl salamander embryo. It works by rapidly inverting the bottom-heavy embryo, imaging it as it rights itself. The images are then montaged to reconstruct the whole 3D surface versus time, for a full 4D record of the surface. Imaging early stage axolotl development will help discover how cell differentiation and movement takes place in the early embryo. For example, the switch from ectodermal to neural plate cells takes place on the top, animal surface portion the egg/embryo and can be observed using the flipping stage microscope. Detailed pictures of the whole surface need to be obtained so that cell tracking and event histories, such as cell divisions and participation in differentiation waves, of individual cells can be recorded. Imaging the whole exterior of the eggs/embryos will allow for the analysis of cell behavior and the forces the cells experience in their natural setting in the intact or manipulated embryo." The axolotl genome and the evolution of key tissue formation regulators "Salamanders serve as important tetrapod models for developmental, regeneration and evolutionary studies. An extensive molecular toolkit makes the Mexican axolotl (Ambystoma mexicanum) a key representative salamander for molecular investigations. Here we report the sequencing and assembly of the 32-gigabase-pair axolotl genome using an approach that combined long-read sequencing, optical mapping and development of a new genome assembler (MARVEL). We observed a size expansion of introns and intergenic regions, largely attributable to multiplication of long terminal repeat retroelements. We provide evidence that intron size in developmental genes is under constraint and that species-restricted genes may contribute to limb regeneration. The axolotl genome assembly does not contain the essential developmental gene Pax3. However, mutation of the axolotl Pax3 paralogue Pax7 resulted in an axolotl phenotype that was similar to those seen in Pax3-/- and Pax7-/- mutant mice." Pax Morphological and transcriptomic analyses reveal three discrete primary stages of postembryonic development in the common fire salamander, Salamandra salamandra "The postembryonic development of amphibians has been characterized as divided into three predominant periods, hereafter named primary developmental stages: premetamorphosis (PreM), prometamorphosis (ProM), metamorphic climax (Meta), and completion of metamorphosis (PostM), largely based on examination of anuran development. ...Our results support that primary stages of postembryonic development in caudates are homologous to those of anurans, and offer a baseline for the study of the evolution of developmental modes." Dual embryonic origin and patterning of the pharyngeal skeleton in the axolotl "The impressive morphological diversification of vertebrates was achieved in part by innovation and modification of the pharyngeal skeleton. Extensive fate mapping in amniote models has revealed a primarily cranial neural crest derivation of the pharyngeal skeleton....Our fate map confirms a dual embryonic origin of the pharyngeal skeleton in urodeles, including derivation of basibranchial 2 from mesoderm closely associated with the second heart field. Additionally, heterotopic transplantation experiments reveal lineage restriction of mesodermal cells that contribute to pharyngeal cartilage. The mesoderm-derived component of the pharyngeal skeleton appears to be particularly sensitive to retinoic acid (RA): administration of exogenous RA leads to loss of the second basibranchial, but not the first. Neural crest was undoubtedly critical in the evolution of the vertebrate pharyngeal skeleton, but mesoderm may have played a central role in forming ventromedial elements, in particular. When and how many times during vertebrate phylogeny a mesodermal contribution to the pharyngeal skeleton evolved remain to be resolved." Stochastic specification of primordial germ cells from mesoderm precursors in axolotl embryos "A common feature of development in most vertebrate models is the early segregation of the germ line from the soma. For example, in Xenopus and zebrafish embryos primordial germ cells (PGCs) are specified by germ plasm that is inherited from the egg; in mice, Blimp1 expression in the epiblast mediates the commitment of cells to the germ line. How these disparate mechanisms of PGC specification evolved is unknown. Here, in order to identify the ancestral mechanism of PGC specification in vertebrates, we studied PGC specification in embryos from the axolotl (Mexican salamander), a model for the tetrapod ancestor. In the axolotl, PGCs develop within mesoderm, and classic studies have reported their induction from primitive ectoderm (animal cap). We used an axolotl animal cap system to demonstrate that signalling through FGF and BMP4 induces PGCs. The role of FGF was then confirmed in vivo. We also showed PGC induction by Brachyury, in the presence of BMP4. These conditions induced pluripotent mesodermal precursors that give rise to a variety of somatic cell types, in addition to PGCs. Irreversible restriction of the germ line did not occur until the mid-tailbud stage, days after the somatic germ layers are established. Before this, germline potential was maintained by MAP kinase signalling. We propose that this stochastic mechanism of PGC specification, from mesodermal precursors, is conserved in vertebrates." Regeneration of Limb Joints in the Axolotl "In spite of numerous investigations of regenerating salamander limbs, little attention has been paid to the details of how joints are reformed. An understanding of the process and mechanisms of joint regeneration in this model system for tetrapod limb regeneration would provide insights into developing novel therapies for inducing joint regeneration in humans. To this end, we have used the axolotl (Mexican Salamander) model of limb regeneration to describe the morphology and the expression patterns of marker genes during joint regeneration in response to limb amputation. These data are consistent with the hypothesis that the mechanisms of joint formation whether it be development or regeneration are conserved. We also have determined that defects in the epiphyseal region of both forelimbs and hind limbs in the axolotl are regenerated only when the defect is small. As is the case with defects in the diaphysis, there is a critical size above which the endogenous regenerative response is not sufficient to regenerate the joint. This non-regenerative response in an animal that has the ability to regenerate perfectly provides the opportunity to screen for the signaling pathways to induce regeneration of articular cartilage and joints." Multiple sequences and factors are involved in stability/degradation of Awnt-1, Awnt-5A and Awnt-5B mRNAs during axolotl development. "Following fertilization in amphibian, early cleavage stages are maternally controlled at a post-transcriptional level before initiation of zygotic transcriptions at the mid blastula transition (MBT). ...Altogether, these results show that oocyte maturation and late cleavages following MBT are two important periods when axolotl Wnt RNAs are highly regulated." The axolotl limb: a model for bone development, regeneration and fracture healing. "Among vertebrates, urodele amphibians (e.g., axolotls) have the unique ability to perfectly regenerate complex body parts after amputation." Expression of heat-shock protein 70 during limb development and regeneration in the axolotl. "Using molecular biology and biochemical techniques, we have characterized both the spatiotemporal and quantitative expression patterns of Hsp-70 in axolotl development and regeneration. Our results show that Hsp-70 is expressed and regulated during axolotl development as in other vertebrates. Our data also demonstrate an up-regulation of the RNA transcript for Hsp-70 during limb regeneration as early as 24 hr after amputation that is maintained up to early differentiation. We also demonstrate a similar pattern of expression for the protein during regeneration. Finally, we show that axolotl Hsp-70 is induced threefold after heat-shock as observed in other vertebrates."

Developmental Stages

The following stage information is based upon published staging data.

Axolotl Timeline

Stage	Time (hours)	Description
1	0	Freshly laid egg in jellycoat.
2		First appearance of the first cleavage furrow and the animal pole.
3	2.5	Four cells.
4	4	Eight cells.
5-6	5.5	16 cells.
6	6.5 - 7	32 cells
7	8 - 9	64 cells
8	16	Early blastula (fall of mitotic index in animal blastomeres).
9	21	Late blastula, surface is smooth.
10	26	Early gastrula I, first sign of dorsal blastopore lip.
11	38	Middle gastrula II, Blastopore covers three quadrants. Lateral lips are formed; ventral lip is marked only by pigment accumulation. Yolk plug reaches its maximum diameter.
12	47	Late gastrula II, Blastopore has an oval or circular shape.
14	58	Early neurula II: Neural plate is broad. Neural folds are outlined and begin to rise above the surface in the head region. Embryo is slightly elongated.
15-16	59 - 63	Early neurula III and middle neurula: Neural plate is shield-shaped and becomes sunken; neural folds are raised and bound all regions of the neural plate.
17	64	Late Neurula I: Neural folds are higher; especially in the head region. Further narrowing and deepening of the neural plate occur both in the head and in the spinal regions. Hyomandibular furrow limiting the mandibular arch is slightly outlined. The segmentation of the mesodermal material begins. There are two pairs of somites.
18	66	Late neurula II: Neural plate is deeply sunken. Neural folds are closing and are especially high in the head region where three slight bulges corresponding to fore-, mid- and hindbrain vesicles are outlined. The neural folds of the spinal region are almost in contact. Hyomandibular furrow is more marked. There are two pairs of somites.
19	69	Late neurula III: Neural folds are in contact throughout, but are not yet fused. Brain curvature is quite distinct in profile; fore-, mid- and hindbrain vesicles are also distinct. The swelling of optic vesicles is outlined (barely visible in these pictures because of unfortunate perspective). Hymandibular furrows are deeper. There are three pairs of somites.
20	70	Late Neurula IV: Späte Neurulation IV: Neural folds are fused in spinal region (or are starting this process in these pictures); in brain region, they are only in contact. Optical vesicles are destinct and becoming larger. Grooves in ectoderm appear at the level of the hindbrain. A very slight swelling marks the future gill region. Mandibular arch becomes prominent and four pairs of somites are present.
22-23	73-74	Neural folds are completely fused. The gill region and the pronephros are distinct; the tailbud is slightly outlined. Five to six pairs of somites are present. Primordium of the ear is outlined as a shallow depression in the ectoderm in the region above the future hyoid arch. The hyobranchial furrow appears, outlining the boundary between the hyoid arch and the first branchial arch.
24	80	Ear pit is outlined and becomes more distinct. The hyobranchial furrow lengthens ventrally. The prophenic swelling is clearly outlined, and both the pronephros itself and the beginning of the pronephric duct are clearly visible Eight or nine pairs of somites are visible.
29		Since first cleavage, up to 97 hours have passed (sorry, I missed some hours here... ;-) ). Ear pit becomes quite distinct. The gill region is clearly outlined. The pronephric duct is clearly visible along six somites at least. The primordium of the olfactory organ appears as a tubercle on the anterior part of the head; the tailbud is gradually enlarging in all stages. Up to 16 pairs of somites are present.
30-31	110	The body of the embryo continues to straighten; the tailbud enlarges. Dorsal finfold begins at somite 14-12. A groove appears in the region of the lens primordium (visual system - i.e. eyes) The third branchial furrow becomes apparent in the dorsal part of the gill region.
32-34	115	The dorsal fin develops until it begins at somite 10.
35	122	From this stage on, the body axis from the hindbrain to the tail base are quite straight. Three external gills show ad nodules on the surface of the gill swelling. The lateral line reaches to the sixth somite. The dorsal fin begins at the fifth somite. The first chromatophores appear; heart pulsation begins.
36-37	177	Gills elongate and push venventroposteriorly. No limb buds are yet visible.
38		Filament sprouts appear as two nodules on each gill. The primordium of the operculum is visible as a fold upon the hyoid arch. Neither of the two rudiments of the perculum reaches the midline. The limb buds are slightly outlined.
39	220	The first gills have two pairs of filament sprouts; the second and third have three pairs each. The gills cover the limb buds. Both rudiments of the operculum approach the midline. The angle of the mouth begins to show.
40	240	The gills are longer and the number of filaments increases (four pairs in the first gills, six or seven pairs on the second or third gills). The rudiments of the operculum join at the middline. The angles of the mouth are marked more distinctly and limb buds protrude slightly.
41	265	The gills contunie to elongate, the number of filaments increases and they become longer. The mouth is distinctly outlined. The second lateral line runs along the flank toward the limb bud and bypasses it on the ventral side. Hatching begins.
42	296	The gills extend far beyond the forelimb buds. The mouth is completely outlined, but is not broken through.
43		With galvanic and convulsive movements the larva breaks free of the jelly coat. The mouth either is already opened or will open within the next 24 - 72 hours.
Data

Thyroid Hormone Effects

The effect of thyroxine on the early larval development of the axolotl. The same control and 30 nM T4-treated (TH) sibling animals were photographed at the days postfertilization noted. T4 was added from day 14. (Bar = 1 cm.)

Limb Development

Axolotl developing limb Bmp2 and Sox9 expression and cartilage staining.

Teeth Development

A study has used the axolotl as a model for tooth development. Using transgenic axolotls and fate-mapping approaches they found "evidence of oral teeth derived from both the ectoderm and endoderm and, moreover, demonstrate teeth with a mixed ecto/endodermal origin. Despite the enamel epithelia having a different embryonic source, oral teeth in the axolotl display striking developmental uniformities and are otherwise identical. This suggests a dominant role for the neural crest mesenchyme over epithelia in tooth initiation and, from an evolutionary point of view, that an essential factor in teeth evolution was the odontogenic capacity of neural crest cells, regardless of possible 'outside-in' or 'inside-out' influx of the epithelium."

Additional Images

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  Axolotl brain ventricular zone proliferative activity

Historic Images

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  Prophases of the Heterotype Division in the Male Axolotl

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  The maturation divisions in the female (Axolotl)

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  Development of the optic cup and lens in Siredon pisciformis

References

Reviews

Desnitskiy AG. (2018). Cell cycles during early steps of amphibian embryogenesis: A review. BioSystems , 173, 100-103. PMID: 30240720 DOI.

Payzin-Dogru D & Whited JL. (2018). An integrative framework for salamander and mouse limb regeneration. Int. J. Dev. Biol. , 62, 393-402. PMID: 29943379 DOI.

Nowoshilow S, Schloissnig S, Fei JF, Dahl A, Pang AWC, Pippel M, Winkler S, Hastie AR, Young G, Roscito JG, Falcon F, Knapp D, Powell S, Cruz A, Cao H, Habermann B, Hiller M, Tanaka EM & Myers EW. (2018). The axolotl genome and the evolution of key tissue formation regulators. Nature , 554, 50-55. PMID: 29364872 DOI.

Maden M. (2008). Axolotl/newt. Methods Mol. Biol. , 461, 467-80. PMID: 19030817 DOI.

Roy S & Gatien S. (2008). Regeneration in axolotls: a model to aim for!. Exp. Gerontol. , 43, 968-73. PMID: 18814845 DOI.

Frost-Mason SK & Mason KA. (1996). What insights into vertebrate pigmentation has the axolotl model system provided?. Int. J. Dev. Biol. , 40, 685-93. PMID: 8877441

Epperlein HH, Löfberg J & Olsson L. (1996). Neural crest cell migration and pigment pattern formation in urodele amphibians. Int. J. Dev. Biol. , 40, 229-38. PMID: 8735933

Epperlein HH & Löfberg J. (1993). The development of the neural crest in amphibians. Ann. Anat. , 175, 483-99. PMID: 8297037 DOI.

Articles

Brooks GC, Gorman TA, Jiao Y & Haas CA. (2020). Reconciling larval and adult sampling methods to model growth across life-stages. PLoS ONE , 15, e0237737. PMID: 32822355 DOI.

Fei JF, Lou WP, Knapp D, Murawala P, Gerber T, Taniguchi Y, Nowoshilow S, Khattak S & Tanaka EM. (2018). Application and optimization of CRISPR-Cas9-mediated genome engineering in axolotl (Ambystoma mexicanum). Nat Protoc , 13, 2908-2943. PMID: 30429597 DOI.

Nowoshilow S, Schloissnig S, Fei JF, Dahl A, Pang AWC, Pippel M, Winkler S, Hastie AR, Young G, Roscito JG, Falcon F, Knapp D, Powell S, Cruz A, Cao H, Habermann B, Hiller M, Tanaka EM & Myers EW. (2018). The axolotl genome and the evolution of key tissue formation regulators. Nature , 554, 50-55. PMID: 29364872 DOI.

Simon HG. (2012). Salamanders and fish can regenerate lost structures--why can't we?. BMC Biol. , 10, 15. PMID: 22369645 DOI.

Caulet S, Pelczar H & Andéol Y. (2010). Multiple sequences and factors are involved in stability/degradation of Awnt-1, Awnt-5A and Awnt-5B mRNAs during axolotl development. Dev. Growth Differ. , 52, 209-22. PMID: 20151991 DOI.

Hutchison C, Pilote M & Roy S. (2007). The axolotl limb: a model for bone development, regeneration and fracture healing. Bone , 40, 45-56. PMID: 16920050 DOI.

Lévesque M, Guimond JC, Pilote M, Leclerc S, Moldovan F & Roy S. (2005). Expression of heat-shock protein 70 during limb development and regeneration in the axolotl. Dev. Dyn. , 233, 1525-34. PMID: 15965983 DOI.

Nye HL, Cameron JA, Chernoff EA & Stocum DL. (2003). Extending the table of stages of normal development of the axolotl: limb development. Dev. Dyn. , 226, 555-60. PMID: 12619140 DOI.

Meuler DC & Malacinski GM. (1985). An analysis of protein synthesis patterns during early embryogenesis of the urodele--Ambystoma mexicanum. J Embryol Exp Morphol , 89, 71-92. PMID: 4093754

Signoret J. (1980). Evidence of the first genetic activity required in axolotl development. Results Probl Cell Differ , 11, 71-4. PMID: 7444204

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External Links

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• Axolotl - Developmental stages
• Axolotl Org - Developmental stages
• Oxford University Press Developmental Biology of the Axolotl, John B. Armstrong and George M. Malacinski
• [www.axolotl-omics.org Axolotl-omics] website that allows rapid ortholog searches, and access to genome and transcriptomic resources.

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