Nerve-induced ectopic limb blastemas in the Axolotl are equivalent to amputation-induced blastemas.

Adult urodeles (salamanders) are unique in their ability to regenerate complex organs perfectly. The recently developed Accessory Limb Model (ALM) in the axolotl provides an opportunity to identify and characterize the essential signaling events that control the early steps in limb regeneration. The ALM demonstrates that limb regeneration progresses in a stepwise fashion that is dependent on signals from the wound epidermis, nerves and dermal fibroblasts from opposite sides of the limb. When all the signals are present, a limb is formed de novo. The ALM thus provides an opportunity to identify and characterize the signaling pathways that control blastema morphogenesis and limb regeneration. Our previous study provided data on cell contribution, cell migration and nerve dependency indicating that an ectopic blastema is equivalent to an amputation-induced blastema. In the present study, we have determined that formation of both ectopic blastemas and amputation-induced blastemas is regulated by the same molecular mechanisms, and that both types of blastema cells exhibit the same functions in controlling growth and pattern formation. We have identified and validated five marker genes for the early stages of wound healing, dedifferentiation and blastema formation, and have discovered that the expression of each of these markers is the same for both ectopic and amputation-induced blastemas. In addition, ectopic blastema cells interact coordinately with amputation-induced blastema cells to form a regenerated limb. Therefore, the ALM is appropriate for identifying the signaling pathways regulating the early events of tetrapod limb regeneration.

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.

The development of transgenesis in axolotls is crucial for studying development and regeneration as it would allow for long-term cell fate tracing as well as gene expression analysis. We demonstrate here that plasmid injection into the one-cell stage axolotl embryo generates mosaic transgenic animals that display germline transmission of the transgene. The inclusion of SceI meganuclease in the injections (Thermes, V., Grabher, C., Ristoratore, F., Bourrat, F., Choulika, A., Wittbrodt, J., Joly, J.S., 2002. I-SceI meganuclease mediates highly efficient transgenesis in fish. Mech. Dev. 118, 91-98) resulted in a higher percentage of F0 animals displaying strong expression throughout the body. This represents the first demonstration in the axolotl of germline transmission of a transgene. Using this technique we have generated a germline transgenic animal expressing GFP ubiquitously in all tissues examined. We have used this animal to study cell fate in the dorsal fin during development. We have uncovered a contribution of somite cells to dorsal fin mesenchyme in the axolotl, which was previously assumed to derive solely from neural crest. We have also studied the role of blood during tail regeneration by transplanting the ventral blood-forming region from GFP+ embryos into unlabeled hosts. During tail regeneration, we do not observe GFP+ cells contributing to muscle or nerve, suggesting that during tail regeneration blood stem cells do not undergo significant plasticity.

Gene expression in the axolotl germ line: Axdazl, Axvh, Axoct-4, and Axkit.

Primordial germ cells (PGCs) in embryos of mammals and urodele amphibians are formed by induction in the absence of germ plasm. We describe expression of four germ cell-related genes through the germ cell cycle of the axolotl. The orthologs of vasa and daz-like are up-regulated in PGCs of tail bud embryos before the gonad forms and are expressed throughout the female germ cell cycle. Mammalian Oct-4 is a marker of pluripotency in embryonic cells. Axolotl Oct-4 has higher homology to Oct-4 than that found in other vertebrates. It is expressed in the equivalent of the mouse epiblast, in the posterior mesoderm of late gastrulae that gives rise to PGCs, and in diplotene growing oocytes, but not in presumptive PGCs after gastrulation. Finally, a c-kit homolog is expressed in gonadal oogonia and growing oocytes as in mice but is also not found in PGCs. The expression pattern in urodele gonadal germ cells is similar to that of other vertebrates, although the pattern in pregonadal PGCs is distinctly different from that of mice. We conclude that PGCs are restricted to the germ line later in urodeles than in mice or lack migration and proliferation programs.

Expression of Axwnt-8 and Axszl in the urodele, axolotl: comparison with Xenopus.

In both the urodele axolotl and the anuran Xenopus, Wnt-8 is expressed in posterior lateral plate mesoderm (LPM) in neurula and tailbud stages. In contrast to Xenopus, expression in axolotl is more prominent in gastrula endoderm, is not initiated in mesoderm until late gastrulation, and is present in the tailbud and in the brain at tailbud stages. Sizzled is expressed in axolotl in the ventral region, similar to its pattern in Xenopus. In axolotl, the Wnt-8-expressing LPM remains relatively dorsal through tailbud stages, while ventral blood island (VBI) markers appear in a wide ventral arc.

Lateral line placodes are induced during neurulation in the axolotl.

In order to determine the time window for induction of lateral line placodes in the axolotl, we performed two series of heterotopic and isochronic transplantations from pigmented to albino embryos at different stages of embryogenesis and assessed the distribution of pigmented neuromasts in the hosts at later stages. First, ectoderm from the prospective placodal region was transplanted to the belly between early neurula and mid tailbud stages (stages 13-27). Whereas grafts from early neurulae typically differentiated only into epidermis, grafts from late neural fold stages on reliably resulted in differentiation of ectopic pigmented neuromasts. Second, belly ectoderm was transplanted to the prospective placodal region between early neurula and tailbud stages (stages 13-35). Normal lateral lines containing pigmented neuromasts formed in most embryos when grafts were performed prior to early tailbud stages (stage 24) but not when they were performed later. Our findings indicate that lateral line placodes, from which neuromasts originate, are already determined at late neural fold stages (first series of grafts) but are inducible until early tailbud stages (second series of grafts). A further series of heterochronic transplantations demonstrated that the decline of inducibility at mid tailbud stages is mainly due to the loss of ectodermal competence.

Immunohistochemical demonstration of hyaluronan and its possible involvement in axolotl neural crest cell migration.

Hyaluronan (HA), an extracellular matrix component, is involved mainly in the control of cell proliferation, neural crest and tumor cell migration, and wound repair. We investigated the effect of hyaluronan on neural crest (NC) cell migration and its ultrastructural localization in dark (wild-type) and white mutant embryos of the Mexican axolotl (Ambystoma mexicanum, Amphibia). The axolotl system is an accepted model for studying mechanisms of NC cell migration. Using a biotinylated hyaluronan binding protein (HABP), major extracellular matrix (ECM) spaces, including those of NC cell migration, reacted equally positive on cryosections through dark and white embryos. Since neural crest-derived pigment cells migrate only in subepidermal spaces of dark embryos, HA does not seem to influence crest cell migration in vivo. However, when tested on different alternating substrates in vitro, migrating NC cells in dark and white embryos prefer HA to fibronectin. In vivo, such an HA migration stimulating effect might exist as well, but be counteracted to differing degrees in dark and white embryos. The ultrastructural localization of HA was studied by means of transmission electron microscopic immunohistochemistry using HABP and different protocols of standard chemical fixation, cryofixation, embedding, and immunolabeling. The binding reaction of HA to HABP was strong and showed an equal distribution throughout ECM spaces after both standard chemical fixation/freeze substitution and cryofixation. A preference for the somite or subepidermal side was not observed. Following standard fixation/freeze substitution HABP-labeled "honeycomb"-like networks reminiscent of fixation artifacts were more prominent than labeled fibrillar or irregular net-like structures. The latter predominated in adequately frozen specimens following high-pressure freezing/freeze substitution. For this reason fibrillar or irregular net-like structures very likely represent hyaluronan in the complex subepidermal matrix of the axolotl embryo in its native arrangement.

Elongation of axolotl tailbud embryos requires GPI-linked proteins and organizer-induced, active, ventral trunk endoderm cell rearrangements.

Application of phosphatidylinositol-specific phospholipase C to early tailbud stage axolotl embryos reveals that a specific subset of morphogenetic movements requires glycosylphosphatidylinositol (GPI)-linked cell-surface proteins. These include pronephric duct extension, "gill bulge" formation, and embryonic elongation along the anteroposterior axis. The work of Kitchin (1949, J. Exp. Zool. 112, 393-416) led to the conclusion that extension of the notochord provided the motive force driving anteroposterior stretching in axolotl embryos, elongation of other tissues being a passive response. We therefore conjectured that axial mesoderm cells might display the GPI-linked proteins required for elongation of the embryo. However, we show here that removal of most of the neural plate and axial and paraxial mesoderm prior to neural tube closure does not prevent elongation of ventrolateral tissues. Tissue-extirpation and tissue-marking experiments indicate that elongation of the ventral trunk occurs via active, directed tissue rearrangements within the endoderm, directed by signals emanating from the blastopore region. Extension of both dorsal and ventral tissues requires GPI-linked proteins. We conclude that elongation of axolotl embryos requires active cell rearrangements within ventral as well as axial tissues. The fact that both types of elongation are prevented by removal of GPI-linked proteins implies that they share a common molecular mechanism.

Cloning of a complementary DNA encoding an Ambystoma mexicanum metallothionein, AmMT, and expression of the gene during early development.

We have used a polymerase chain reaction strategy to isolate a metallothionein (MT) cDNA from the amphibian Ambystoma mexicanum (axolotl). This cDNA is 875-bp long and encodes a 60 amino acid protein, AmMT, typical for family 1 MTs. It contains 20 cysteine (Cys) residues that can be aligned with those of other vertebrate MTs. The overall structure of the protein is unique among vertebrates in having only two amino acid residues before the first Cys at the amino-terminal end. Northern analyses showed that AmMT is expressed throughout embryogenesis, giving rise to three mRNA species of 650, 750, and 1,600 nucleotides (nt). The 750 and 1,600 nt transcripts appear to result from differential use of polyadenylation signals, whereas the 650 nt RNA could arise from deadenylation of the 750-nt transcript. Both the 750- and 1,600-nt RNAs were presented in embryos before the mid-blastula transition (MBT). After the MBT, the 750-nt RNA was replaced by the 650-nt RNA which was gradually degraded to undetectable levels in post-neurulation embryos. Levels of the 1,600-nt transcript increased at gastrulation and reach a maximum in Stage 30 embryos. In adult animals, levels of the 750-nt RNA were high in liver and testes, and very low in lung, gut, skin, and oviducts, whereas levels of the 1,600-nt transcript were similar and moderately elevated in all tissues examined. In contrast, in Xenopus laevis, Northern analysis did not detect XIMT-A mRNA in embryos before late neurulation (Stage 24). XIMT-A mRNA levels then increased sharply in Stage 36 hatched embryos at levels similar to those found in adult livers. These results show that AmMT presents a unique expression pattern among metazoans being transcribed as two transcripts differing in the length of their 3' untranslated regions, the levels of which vary during embryogenesis and in adult tissues.

Improved preservation of the subepidermal extracellular matrix in axolotl embryos using electron microscopical techniques based on cryoimmobilization.

The purpose of this metholdological survey was to find optimal methods for the fixation and demonstration of glycosaminoglycans, mainly hyaluronan, and proteoglycans, in subepidermal extracellular matrix (ECM) regions of axolotl embryos. We compared living ECM in the laser-scanning microscope (LSM) with chemically fixed or cryoimmobilized extracellular matrix in the transmission (TEM) and scanning electron microscope (SEM). The gel-like structure of living extracellular matrix in the LSM undoubtedly provides the most natural state, whereas shrinkage of the extracellular matrix occurs during conventional fixation and dehydration for TEM or SEM. Among the methods used for fixation and processing of subepidermal extracellular matrices for SEM, plunge-freezing/freeze-drying is to be preferred. Still more satisfying, however, are results obtained with high-pressure frozen/freeze-substituted ECM material in the TEM, for which 10% polyvinyl pyrrolidon +7% methanol was used as a cryoprotectant before high-pressure freezing. In these specimens, no freeze-damage could be observed and they could be regarded as adequately frozen. Conversely, the yield in adequately frozen specimens without cryoprotection was insufficient. In these specimens, the ECM contained honeycomb-like structures which, in the current literature, are regarded as hyaluronan.

Ambient UV-B radiation causes deformities in amphibian embryos.

There has been a great deal of recent attention on the suspected increase in amphibian deformities. However, most reports of amphibian deformities have been anecdotal, and no experiments in the field under natural conditions have been performed to investigate this phenomenon. Under laboratory conditions, a variety of agents can induce deformities in amphibians. We investigated one of these agents, UV-B radiation, in field experiments, as a cause for amphibian deformities. We monitored hatching success and development in long-toed salamanders under UV-B shields and in regimes that allowed UV-B radiation. Embryos under UV-B shields had a significantly higher hatching rate and fewer deformities, and developed more quickly than those exposed to UV-B. Deformities may contribute directly to embryo mortality, and they may affect an individual's subsequent survival after hatching.

Differential expression of C-protein isoforms in the developing heart of normal and cardiac lethal mutant axolotls (Ambystoma mexicanum).

Regulated assembly of contractile proteins into sarcomeric structures, such as A- and I-bands, is still currently being defined. The presence of distinct isoforms of several muscle proteins suggests a possible mechanism by which myocytes regulate assembly during myofibrillogenesis. Of several muscle isoforms located within the A-band, myosin binding proteins (MyBP) are reported to be involved in the regulation and stabilization of thick filaments during sarcomere assembly. The present confocal study characterizes the expression of one of these myosin binding proteins, C-protein (MyBP-C) in wild-type and cardiac lethal mutant embryos of the axolotl, Ambystoma mexicanum. C-protein isoforms are also detected in distinct temporal patterns in whole-mounted heart tubes and thoracic skeletal muscles. Confocal analysis of axolotl embryos shows both cardiac and skeletal muscles to regulate the expression of C-protein isoforms over a specific developmental window. Although the CPROAxslow isoform is present during the initial heartbeat stage, its expression is not retained in the adult heart. C-protein isoforms are simultaneously expressed in both cardiac and skeletal muscle during embryogenesis.

Effects of extracellular matrix molecules on subepidermal neural crest cell migration in wild type and white mutant (dd) axolotl embryos.

Migration of neural crest (NC) derived pigment cells is restricted in the white mutant (dd) axolotl embryo (Ambystoma mexicanum). Transplantations between mutant and wild type embryos show that the extracellular matrix (ECM) of the white mutant is unable to support the migration of prospective pigment cells in wild type embryos (Löfberg et al., 1989, Dev. Biol. 131:168-181). In the present study, we test the effects of various purified ECM molecules on NC cell migration in the subepidermal migratory pathway of wild type (D/-) and white mutant (dd) axolotl embryos. We adsorbed the ECM molecules onto membrane microcarriers, which were then implanted under the epidermis. Fibronectin (FN), tenascin (TN), collagens I and VI, and a chick aggrecan stimulated migration in both types of embryos. Laminin-nidogen, rat chondrosarcoma aggrecan, and shark aggrecan stimulated migration in dd embryos but did not affect migration in D/- embryos. Collagen III, fibromodulin and bovine aggrecan had no effect on migration in either type of embryo. NC cells did not migrate on control microcarriers, which lacked ECM molecules. Some cells observed contacting, and presumably migrating on, coated microcarriers could be identified as pigment cells by their ultrastructure. Enzymatic digestion in vivo with chondroitinase ABC had no effect on NC cell migration. The neutral or stimulatory effect of the aggrecans is surprising; when tested in vitro they inhibited NC cell migration. The effect of three-dimensionality and other molecules present either in the embryonic ECM or in solution may overcome the inhibitory effect of aggrecans.

Effects of the antiepileptic drug valproic acid on the development of the axolotl (Ambystoma mexicanum): histological investigations.

In the amphibian Ambystoma mexicanum, valproic acid (VPA) causes retarded development and malformations including neural tube defects. Some of the observed abnormalities resemble exencephaly. We present the light microscopic characteristics of VPA-induced effects on the developing central nervous system (CNS) as well as on other developing tissues of this species. To induce malformations, various concentrations of VPA were applied to embryos from blastula stage on, either as 24-h pulse or as continuous exposure. In treated embryos the abnormal development of the CNS was indicated by retarded neurulation and disturbed closure of the neural folds. Furthermore, the neural epithelium was disorganized and its cells were less elongated than normal. Two ventricle lumina of various shapes arose in the neural tube and the neural epithelium extended laterally, whereas in the control animals the neural tube was small and overlaid the notochord only. In treated embryos intercellular spaces in neural epithelium as well as in connective tissue were enlarged and cell adhesion seemed disturbed in both tissues. The notochord underlying the neural plate appeared to be normally organized but oversized somites appeared, which periodically filled the space between notochord and notoplate. VPA also affected neural crest cells. Additional effects occurring were edema and liver damage. A high concentration of VPA even stopped development in early gastrulation. Characteristics of induced effects indicate an interaction between the drug and components of the extracellular matrix and the cytoskeleton.

Regulation of HoxA expression in developing and regenerating axolotl limbs.

Homeobox genes are important in the regulation of outgrowth and pattern formation during limb development. It is likely that homeobox genes play an equally important role during limb regeneration. We have isolated and identified 17 different homeobox-containing genes expressed by cells of regenerating axolotl limbs. Of these, nearly half of the clones represent genes belonging to the HoxA complex, which are thought to be involved in pattern formation along the proximal-distal limb axis. In this paper we report on the expression patterns of two 5' members of this complex, HoxA13 and HoxA9. These genes are expressed in cells of developing limb buds and regenerating blastemas. The pattern of expression in developing axolotl limb buds is comparable to that in mouse and chick limb buds; the expression domain of HoxA13 is more distally restricted than that of HoxA9. As in developing mouse and chick limbs, HoxA13 likely functions in the specification of distal limb structures, and HoxA9 in the specification of more proximal structures. In contrast, during regeneration, HoxA13 and HoxA9 do not follow the rule of spatial colinearity observed in developing limbs. Instead, both genes are initially expressed in the same population of stump cells, giving them a distal Hox code regardless of the level of amputation. In addition, both are reexpressed within 24 hours after amputation, suggesting that reexpression may be synchronous rather than temporally colinear. Treatment with retinoic acid alters this Hox code to that of a more proximal region by the rapid and differential downregulation of HoxA13, at the same time that expression of HoxA9 is unaffected. HoxA reexpression occurs prior to blastema formation, 24-48 hours after amputation, and is an early molecular marker for dedifferentiation.

Electroreceptors and mechanosensory lateral line organs arise from single placodes in axolotls.

The lateral line system in salamanders consists of mechanoreceptive neuromasts and pit organs, distributed in lines on the head and trunk, and electroreceptive ampullary organs located adjacent to the cephalic lines of mechanoreceptors. Although numerous studies have documented that neuromast and pit organs and the cranial nerves that innervate these receptors arise from a dorsolateral series of placodes, there is no agreement concerning the number of these placodes, the specific groups of receptors that arise from them, or the embryonic origin of ampullary organs. A developmental model was recently proposed (Northcutt et al., 1994) in which all these placodes, except for the most posterior one, elongate to form sensory ridges whose central zones initially form neuromast and pit organ primordia and whose lateral zones subsequently form ampullary primordia. To test this model, individual placodes were unilaterally extirpated, or placodes from pigmented wild-type axolotl embryos were homotopically or heterotopically transplanted into albino hosts. Extirpation resulted in the loss of all three receptor classes, and both homotopic and heterotopic transplants produced pigmented receptors of all three classes in albino hosts. The receptors in the heterotopic transplants still formed lines which occasionally retained their normal orientation despite differentiating in an ectopic environment. These experiments demonstrated that, as previously postulated, specific lines of neuromasts and pit organs do arise from each placode, and ampullary organs also arise from many of the same placodes. The distribution of receptors that develop following incomplete extirpation or heterotopic transplantation also indicates that each placode is patterned regarding receptor classes and orientation prior to sensory ridge formation.

Reinvestigation of DNA ligase I in axolotl and Pleurodeles development.

We have recently shown that the exclusion process causing the replacement of DNA ligases II by DNA ligase I in amphibian eggs after fertilization does not occur in the case of Xenopus laevis [Hardy, S., Aoufouchi, S., Thiebaud, P., and Prigent, C., (1991) Nucleic Acids Res. 19, 701-705]. Since this result is in contradiction with the situation reported in axolotl and Pleurodeles we decided to reinvestigate such results in both species. Three different approaches have been used: (1) the substrate specificity of DNA ligase I; (2) the DNA ligase-AMP adduct reaction and (3) the immunological detection using antibodies raised against the X.laevis DNA ligase I. Our results clearly demonstrate that DNA ligase I activity is associated with a single polypeptide which is present in oocyte, unfertilized egg and embryo of both amphibians. Therefore, the hypothesis of a change in DNA ligase forms, resulting from an expression of the DNA ligase I gene in axolotl and Pleurodeles early development must be rejected. We also show that, in contradiction with published data, the unfertilized sea urchin egg contains a DNA ligase activity able to join blunt ended DNA molecules.

Calcium in the developing Ambystoma neural axis shown by 3H and fluorescent chlortetracycline and atomic absorption spectrometry.

The calcium ion has been implicated in the mediation of the morphogenetic movements that occur during neural tube formation. The present study identifies high levels of calcium in the neuroepithelium of the neural plate, folds, and tube. These levels are substantially higher than those discerned elsewhere in the embryo. The calcium is localized in morphogenetically active regions by using the antibiotic chlortetracycline (CTC) which chelates calcium and is demonstrated in this investigation by both autoradiography and calcium-linked fluorescence. The specificity of CTC reaction for calcium in the developing neural axis is confirmed by EGTA competition. A comparison of the actual calcium levels in the developing neural axis (dorsal) with equivalently weighted ventral tissues was obtained by atomic absorption spectrometry (AAS). This method provides a total count of the calcium without any loss during tissue processing. For AAS, living tissues were precisely excised and immediately dessicated. Each tissue sample (dry weight 1.5 mg) was then solubilized for analysis. The spectrometric data reveal that the embryonic dorsal aspect forming the neural tube contains 57% more calcium than an equivalent weight of the ventral aspect.

Regenerated hair cells can originate from supporting cell progeny: evidence from phototoxicity and laser ablation experiments in the lateral line system.

The mechanisms that lead to the production of sensory hair cells during regeneration have been investigated by using 2 different procedures to ablate preexisting hair cells in individual neuromast sensory epithelia of the lateral line in the tails of salamanders, then monitoring the responses of surviving cells. In one series of experiments, fluorescent excitation was used to cause the phototoxic death of hair cells that selectively take up the pyridinium dye DASPEI. In the other experiments, the ultraviolet output of a pulsed neodymium-YAG laser was focused to a microbeam through a quartz objective lens in epi-illumination mode and used to selectively kill individual unlabeled hair cells while the cells were simultaneously imaged by transmitted light DIC microscopy. Through observation of the treated neuromasts in vivo, these experiments demonstrated that mature sensory epithelia that have been completely depleted of hair cells can still generate new hair cells. Preexisting hair cells are not necessary for regeneration. Immediately after the ablations the only resident cells in the sensory epithelia were supporting cells. These cells were observed to divide at rates that were increased over control values, and eventually those cell divisions gave rise to progeny that differentiated as hair cells, replacing those that had been killed. Macrophages were active in these epithelia, and their phagocytic activity had a significant influence on the standing population of cells. The first new hair cells appeared 3-5 d after the treatments, and additional hair cells usually appeared every 1-2 d for at least 2 weeks. We conclude that the fate of the progeny produced by supporting cell divisions is plastic to a degree, in that these progeny can differentiate either as supporting cells or as hair cells in epithelia where hair cells are missing or depleted.

Development of the mechanoreceptive lateral-line system in the axolotl: placode specification, guidance of migration, and the origin of neuromast polarity.

The mechanosensory lateral-line system offers a unique opportunity to study a wide variety of developmental phenomena, including cell migration, the origin of polarity, and pattern formation. In this study, we use a series of transplantation experiments to examine some of the factors affecting the origin of the lateral-line placodes, the establishment of sensory organ polarity and placement, and the guidance of cell migration in the Mexican axolotl (Ambystoma mexicanum). We find that placode-forming ectoderm is at least partially specified as early as the beginning of neurulation, and we suggest that this may be a result of early processes involved in neural induction. Furthermore, we find that the migration of the primordia on the body depends on the presence of both the ectoderm and the subjacent mesoderm for guidance. Sensory organ polarity on the body appears to be the result of an interaction between the primordia, which deposit organs of set polarity relative to the direction of migration, and the substrate, which determines the direction of migration. Spacing of the organs is independent of the substrate, and may be due to an intrinsic property of either the primordia or the emerging organs themselves. Finally, we suggest that the lateral-line primordia are guided, as they migrate, by a contact guidance mechanism.