Wild-type Axolotl Research Articles

Eya2 promotes cell cycle progression by regulating DNA damage response during vertebrate limb regeneration

How salamanders accomplish progenitor cell proliferation while faithfully maintaining genomic integrity and regenerative potential remains elusive. Here we found an innate DNA damage response mechanism that is evident during blastema proliferation (early- to late-bud) and studied its role during tissue regeneration by ablating the function of one of its components, Eyes absent 2. In eya2 mutant axolotls, we found that DNA damage signaling through the H2AX histone variant was deregulated, especially within the proliferating progenitors during limb regeneration. Ultimately, cell cycle progression was impaired at the G1/S and G2/M transitions and regeneration rate was reduced. Similar data were acquired using acute pharmacological inhibition of the Eya2 phosphatase activity and the DNA damage checkpoint kinases Chk1 and Chk2 in wild-type axolotls. Together, our data indicate that highly-regenerative animals employ a robust DNA damage response pathway which involves regulation of H2AX phosphorylation via Eya2 to facilitate proper cell cycle progression upon injury.

Identification of a human mitochondrial RNA that promotes tropomyosin synthesis and myocardial differentiation.

Heart disease is the number one killer in the USA, making cardiogenesis and its related pathways a relevant area of study for improving health and life expectancy. The Mexican salamander (axolotl), Ambystoma mexicanum, provides an excellent vertebrate animal model for studying myofibrillogenesis due to its naturally occurring cardiac nonfunction mutation. Homozygous recessive embryos do not develop normal hearts due to a lack of myofibril formation. In previous studies, myofibril-inducing ribonucleic acid (MIR) from the normal wild-type axolotl genome was found to rescue mutant nonfunctioning hearts through restoration of tropomyosin levels followed by normal myofibril formation. Our purpose in this study is to identify and characterize functional homologs for the MIR from human fetal heart ribonucleic acid (RNA). After randomized cloning of human fetal heart RNA, 396 clones were analyzed for rescuing ability by using mutant heart rescue bioassays and confocal microscopy. By these analyses, we discovered a functional homolog of MIR from human fetal heart RNA, which is associated with the mitochondrial cytochrome c oxidase subunit II gene. This RNA came from our clone #30 and induces tropomyosin synthesis and myofibrillogenesis in mutant axolotl hearts which ordinarily do not synthesize tropomyosin or form organized myofibrils. Clone #30, a mitochondrial RNA molecule associated with human cytochrome c oxidase, serves as a functional homolog of MIR, leading to tropomyosin production, organized myofibrils, and beating cardiac tissue in mutant hearts. These findings hold great potential for the treatment and repair of damaged hearts in patients who have suffered from myocardial infarctions and other heart diseases.

Matrix metalloproteinase expression during blastema formation in regeneration‐competent versus regeneration‐deficient amphibian limbs

We used an antibody array to compare the protein expression of matrix metalloproteinases (MMPs)-1, -2, -3, -8, -9, -10, and -13, as well as the tissue inhibitors of metalloproteinases (TIMPs)-1, -2, and -4 during blastema formation in amputated hindlimbs of regeneration-competent wild-type axolotls and stage-54 Xenopus, and regeneration-deficient short-toes axolotls and Xenopus froglets. Expression of MMP-9 and -2 was also compared by zymography. Both short-toes and froglet failed to up-regulate MMPs in a pattern comparable to the wild-type axolotl, suggesting that subnormal histolysis is at least in part responsible for the poor blastema formation characteristic of both short-toes and froglet. MMP levels were much lower in amputated stage-54 Xenopus limb buds than in the other animals, suggesting that blastema formation in these limb buds requires much less extracellular matrix degradation than in fully differentiated limbs. TIMP expression patterns followed the same trends as the MMP's in each group of animals.

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.

Vertebrate non-retinal pigment cells are derived from neural crest (NC) cells, and several mutations have been identified in the Mexican axolotl Ambystoma mexicanum (Ambystomatidae) that affect the development of these cell lineages. In "white" (d) mutant axolotls, premigratory NC cells differentiate as pigment cells, yet fail to disperse, survive, or both, and this leads to a nearly complete absence of pigment cells in the skin. Previous studies revealed that d affects pigment cell development non-autonomously, and have reported differences between white and wild-type axolotls in the structure and composition of the extracellular matrix through which NC and pigment cells migrate. Here we test the correspondence of d and two candidate genes: steel and AxPG. In amniotes, Steel encodes the cytokine Steel factor (mast cell growth factor; stem cell factor; kit ligand), which is expressed along the migratory pathways of melanocyte precursors and is required by these cells for their migration and survival; mammalian Steel mutants resemble white mutant axolotls in having a deficit or complete absence of pigment cells. In contrast, AxPG encodes a PG-M/versican-like proteoglycan that may promote the migration of A. mexicanum pigment cells, and AxPG expression is reduced in white mutant axolotls. We cloned a salamander orthologue of steel and used a partial genetic linkage map of Ambystoma to determine the genomic locations of steel, AxPG, and d. We show that the three genes map to different linkage groups, excluding steel and AxPG as candidates for d.

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 or 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 meet 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.

The pigmentary system of developing axolotls: III. An analysis of the albino phenotype

ABSTRACT The albino mutant in the Mexican axolotl (Ambystoma mexicanum) is analysed with respect to the differentiation of pigment cells. Pigment cells were observed with the transmission electron microscope in order to determine any unusual structural characteristics and to determine what happens to each of the cell types as development proceeds. Chemical analyses of pteridine pigments were also carried out, and the pattern of pteridines in albino animals was found to be more complex than, and quantitatively enhanced (at all developmental stages examined) over, the pattern observed in comparable wild-type axolotls. The golden colour of albino axolotls is due primarily to sepiapterin (a yellow pteridine) and secondarily to riboflavin (and other flavins). Coincident with enhanced levels of yellow pigments, xanthophore pigment organelles (pterinosomes) in albino skin reach a mature state earlier than they do in wild-type axolotl skin. This morphology is conserved throughout development in albino animals whereas it is gradually lost in the wild type. Unpigmented melanophores from albino axolotls are illustrated for the first time, and in larval albino axolotls the morphology of these cells is shown to be very similar to xanthophore morphology. In older albino animals xanthophores are easily distinguished from unpigmented melanophores. Iridophores seem to appear in albino skin at an earlier stage than they have been observed in wild-type skin. Morphologically, wild-type and albino iridophores are identical.

The pigmentary system of developing axolotls

ABSTRACT The melanoid mutant in the Mexican axolotl (Ambystoma mexicanum) is analysed with respect to the differentiation of pigment cells. Pigment cells were observed with the transmission electron microscope in order to determine any unusual structural characteristics and to determine what happens to each of the cell types as development proceeds. Chemical analysis of pteridine pigments was also carried out, and changes in pteridine biosynthesis were found to correlate well with changes in xanthophore morphology and number. In melanoid axolotls, as development proceeds, melanophore numbers increase, xanthophores decrease, and iridophores fail to differentiate at all. This is considered to result from: (a) conversion of xanthophores (that are present in young larvae) to melanophores; (b) the gradual programming of the majority of chromatoblasts to become, exclusively, melanophores, and (c) the failure of some chromatoblasts (possibly iridoblasts) to differentiate altogether. The ultrastructural and chemical evidence presented in this study is compared to similar data for wild-type axolotls, and a mechanism regarding how the melanoid gene might act is suggested.

Spastic mutant axolotl: identification of a phenocopy pathway with implications for the control of axolotl swimming by the vestibulocerebellum.

The spastic mutant axolotl shows abnormal swimming behavior, which includes a preponderance of "embryonic" swimming elements (coils) versus mature swimming elements (sinusoids) and a failure to entrain sinusoids into a prolonged swimming sequence. The mutant also shows anatomical disorganization in the area acousticolateralis and cerebellar auricle, but it is unclear (1) to what extent the behavioral abnormalities are traceable to the vestibulocerebellar defect or (2) how the vestibulocerebellar pathway modulates swimming behavior in the normal axolotl. We have performed quantitative cine analysis of electric shock-induced swimming bouts in normal axolotls, spastic mutants, and a variety of neurosurgically altered wild-type axolotls. We scored the incidence of coil elements (25% in controls, 70-90% in spastics) versus sinusoid elements, as well as length distributions of coilfree intervals (short to long trains of sinusoidal swimming) and of sinusoidfree intervals (of brief of prolonged coiling). We found that bilateral VIIIth nerve lesions or surgical undercutting of the cerebellar auricle in wild-type axolotls almost exactly reproduced the behavioral deficit seen in spastic (75-81% coils, loss of long sinusoid trains, and appearance of prolonged coiling intervals at least some of which coupled several coils into trains of thrashing behavior). By contrast, neither complete transection of the CNS at low midbrain levels nor section of cranial nerves V, VII, or X (lateral line) resulted in an increased incidence of coil elements beyond 26% nor significantly altered the length distributions of S-intervals and C-intervals. Nor did any of the latter lesions disrupt the spasticlike swimming patterns of axolotls already subjected to auricle or VIIIth nerve lesions.(ABSTRACT TRUNCATED AT 250 WORDS)

Experimental studies on a mutant gene, a, causing albinism in the axolotl, Ambystoma mexicanum

Axolotls which are homozygous for the gene, a, are true albinos, i.e., they lack completely the ability to synthesize melanin. Previous investigations (C. P. Benjamin, 1970; Develop. Biol. 23, 62–85) showed that homogenates of albino embryos or larvae, unlike those of normally pigmented axolotls, are unable to carry out melanin synthesis when supplied with l-dopa. This result showed that tyrosine-dopa oxidase (TDO), the only known enzyme in the melanin pathway, is inactive in albinos. The present investigation has shown that, though the albino lacks TDO activity, the enzyme itself is present and can be activated by proteolytic enzymes. Immune precipitation of TDO from fresh crude skin extracts and comparison of the precipitates on SDS-polyacrylamide gels reveals that the inactive subunits from albino samples are some 8000 daltons larger than the active subunits from wild-type samples. Activation apparently consists of the cleavage of a covalently bound fragment(s) from the proenzyme. Albino skin homogenates also contain a TDO inhibitory factor specific for axolotl TDO. Evidence is presented that suggests that the inhibitor interacts reversibly with the proenzyme and irreversibly with the active form. A model is presented that proposes that, in wild-type axolotls, the A gene is responsible for the normal activity of a TDO activator. In the albino the activator does not function properly and this results in the continued presence of the inhibitor and of the latent form of the enzyme.

A lethal mutant gene in the Mexican axolotl.

Gene ph was discovered in a wild-type axolotl male received from Mexico City. Larvae homozygous for this gene become recognizable by their lighter color at hatching or shortly after. The development of their forelimbs is retarded, and all limbs are of subnormal length because of the reduction in length of their long bones. Many affected larvae die without feeding, and very few survive beyond their third month. At death, older larvae usually show abnormalities of the renal system, edema, ascites, or adhesions of the viscera. The gene is apparently a simple recessive with full penetrance.

Patterns of purine synthesis related to iridophore development in the wild type, melanoid, and axanthic strains of the Mexican axolotl, Ambystoma mexicanum shaw

Differentiation of the iridophore in the axolotl is inhibited by each of two nonlinked autosomal recessive genes, melanoid ( m) and axanthic ( ax). Purines were extracted from wild type ( M-Ax-), melanoid ( mm), axanthic ( axax), and melanoid-axanthic ( mm axax) somatopleur. This tissue is rich in iridophores in wild-type axolotl larvae. Purines in ethanol and in water extracts from each genotype were separated by paper chromatography. Patterns with respect to the kinds of purines present were distinct for each genotype. Two compounds present in wild type were missing in both melanoid and axanthic mutants, which lack iridophores. One compound was present in wild type and melanoid, which have xanthophores, but was lacking in the axanthic mutant, which does not. The double homozygote ( mm axax) lacked a purine found in wild type and each of the mutants singly. Possibly in this case two biosynthetic pathways may produce the same intermediate product. Thus the actions of the melanoid and axanthic genes in the axolotl are demonstrable chemically as differences in the kinds of purines present in the various genotypes.

The biochemical effects of the d, m, and a genes on pigment cell differentiation in the axolotl

Summary Three mutations alter the basic pigmentation of the wild-type axolotl. In this study an attempt has been made to describe the biochemical alterations associated with these mutations and to correlate them with the cellular changes observed by other investigators and verified by this author. The a gene has no effect on either pteridine or purine synthesis or on xanthophore or iridiophore differentiation. Rather, it inactivates tyrosinase completely, resulting in the absence of melanin in albino axolotls. The effects of the m gene are primarily on the production of propigment cells by the neural crest. Transplants indicate that more melanophores and fewer xanthophores (than in the wild-type), and no iridiophores are produced in axolotls homozygous for m. Correlated with these observations are a 35% increase in tyrosinase activity and a 40% decrease in pteridine synthesis in melanoid animals. The former may, in part, be due to an increased amount of tyrosinase in each melanophore. Moreover, an accumulation of IDP + ITP, AMP, and an unidentified purine derivative suggest that a somewhat “leaky” enzymatic block in the conversion of IMP into XMP (or of XMP into GMP) may contribute to the reduction and absence, respectively, of fully differentiated xanthophores and iridiophores in melanoid skin. The extent of these contributions remains to be determined. Transplantation experiments indicate that the d gene does not alter the production of propigment cells by the neural crest. The 70% decrease in melanin synthesis (and melanophores) in white axolotls appears to be the result of decreased tyrosinase activity rather than of inhibited melanoblast migration. Likewise, the 50% decrease in pteridine synthesis (and xanthophores) appears to be the result of both decreased (40%) glucose-6-phosphate dehydrogenase activity during embryonic stages and of a “leaky” enzymatic block in the conversion of GMP into biopterin, rather than a result of inhibited xanthoblast migration. Since the former block is operative only during embryonic stages, and since the latter block results in a shunting of GMP into guanine synthesis, normal iridiophore differentiation occurs in axolotls homozygous for d.