Early gene expression during natural spinal cord regeneration in the salamander Ambystoma mexicanum

In contrast to mammals, salamanders have a remarkable ability to regenerate their spinal cord and recover full movement and function after tail amputation. To identify genes that may be associated with this greater regenerative ability, we designed an oligonucleotide microarray and profiled early gene expression during natural spinal cord regeneration in Ambystoma mexicanum. We sampled tissue at five early time points after tail amputation and identified genes that registered significant changes in mRNA abundance during the first 7 days of regeneration. A list of 1036 statistically significant genes was identified. Additional statistical and fold change criteria were applied to identify a smaller list of 360 genes that were used to describe predominant expression patterns and gene functions. Our results show that a diverse injury response is activated in concert with extracellular matrix remodeling mechanisms during the early acute phase of natural spinal cord regeneration. We also report gene expression similarities and differences between our study and studies that have profiled gene expression after spinal cord injury in rat. Our study illustrates the utility of a salamander model for identifying genes and gene functions that may enhance regenerative ability in mammals.     

Conserved vertebrate chromosome segments in the large salamander genome

Urodele amphibians (salamanders) are important models for embryological, physiological, and natural history research and are also a biomedically important group because they are the only vertebrates capable of regenerating entire organ systems. To enhance the utility of salamanders for biomedical research and for understanding genome evolution, genetic linkage analysis was used to identify chromosome segments that are homologous between ambystomatid salamanders and distantly related vertebrate model organisms. A total of 347 loci (AFLPs, RAPDs, and protein-coding loci) were mapped using an interspecific meiotic mapping panel (Ambystoma mexicanum and A. tigrinum tigrinum; family Ambystomatidae). Genome size in Ambystoma was estimated to be 7291 cM, the largest linkage map estimate reported for any organism. However, the relatively large size of the salamander genome did not hinder efforts to map and identify conserved syntenies from a small sample of 24 protein-coding loci. Chromosomal segments that are conserved between fishes and mammals are also conserved in these salamanders. Thus, comparative gene mapping appears to be an efficient strategy for identifying orthologous loci between ambystomatid salamanders and genomically well-characterized vertebrate model organisms.     

Candidate gene analysis of metamorphic timing in ambystomatid salamanders

Although much is known about the ecological significance of metamorphosis and metamorphic timing, few studies have examined the underlying genetic architecture of these traits, and no study has attempted to associate phenotypic variation to molecular variation in specific genes. Here we report on a candidate gene approach (CGA) to test specific loci for a statistical contribution to variation in metamorphic timing. Three segregating populations (SP1, SP2 and SP3) were constructed utilizing three species of paedomorphic Mexican ambystomatid salamander, including the axolotl, Ambystoma mexicanum. We used these replicated species to test the hypothesis that inheritance of alternate genotypes at two thyroid hormone receptor loci (TRalpha, TRbeta) affects metamorphic timing in ambystomatid salamanders. A significant TRalpha*SP effect indicated that variation in metamorphic timing may be influenced by TRalpha genotype, however, the effect was not a simple one, as both the magnitude and direction of the phenotypic effect depended upon the genetic background. These are the first data to implicate a specific gene in contributing to variation in metamorphic timing. In general, candidate gene approaches can be extended to any number of loci and to any organism where simple genetic crosses can be performed to create segregating populations. The approach is thus of particular value in ecological studies where target genes have been identified but the study organism is not one of the few well-characterized model systems that dominate genetic research.     

Evolutionary genetics of metamorphic failure using wild-caught vs. laboratory axolotls (Ambystoma mexicanum)

In many organisms metamorphosis allows for an ecologically important habitat-shift from water to land. However, in some salamanders an adaptive life cycle mode has evolved that is characterized by metamorphic failure (paedomorphosis); these species remain in the aquatic habitat throughout the life cycle. Perhaps the most famous example of metamorphic failure is the Mexican axolotl (Ambystoma mexicanum), which has become a focal species for developmental biology since it was introduced into laboratory culture in the 1800s. Our previous genetic linkage mapping analysis, using an interspecific crossing design, demonstrated that a major gene effect underlies the expression of metamorphic failure in laboratory stocks of the Mexican axolotl. Here, we repeated this experiment using A. mexicanum that were sampled directly from their natural habitat at Lake Xochimilco, Mexico. We found no significant association between the major gene and metamorphic failure when wild-caught axolotls were used in the experimental design, although there is evidence of a smaller genetic effect. Thus, there appears to be genetic variation among Mexican axolotls (and possibly A. tigrinum tigrinum) at loci that contribute to metamorphic failure. This result suggests a role for more than one mutation and possibly artificial selection in the evolution of the major gene effect in the laboratory Mexican axolotl.     

Amiloride blocks salt taste transduction of the glossopharyngeal nerve in metamorphosed salamanders

Studies in the last two decades have shown that amiloride-sensitive Na(+) channels play a role in NaCl transduction in rat taste receptors. However, this role is not readily generalized for salt taste transduction in vertebrates, because functional expression of these channels varies across species and also in development in a species. Glossopharyngeal nerve responses to sodium and potassium salts were recorded in larval and metamorphosed salamanders and compared before and after the oral floor was exposed to amiloride, a blocker of Na(+) channels known to be responsible for epithelial ion transport. Pre-exposure to amiloride (100 microM) did not affect salt taste responses in both axolotls (Ambystoma mexicanum) and larval Ezo salamanders (Hynobius retardatus). In contrast, in metamorphosed Ezo salamanders the nerve responses to NaCl were significantly reduced by amiloride. In amphibians amiloride-sensitive components in salt taste transduction seem to develop during metamorphosis.     

Thyroid hormone responsive QTL and the evolution of paedomorphic salamanders

The transformation of ancestral phenotypes into novel traits is poorly understood for many examples of evolutionary novelty. Ancestrally, salamanders have a biphasic life cycle with an aquatic larval stage, a brief and pronounced metamorphosis, followed by a terrestrial adult stage. Repeatedly during evolution, metamorphic timing has been delayed to exploit growth-permissive environments, resulting in paedomorphic salamanders that retain larval traits as adults. We used thyroid hormone (TH) to rescue metamorphic phenotypes in paedomorphic salamanders and then identified quantitative trait loci (QTL) for life history traits that are associated with amphibian life cycle evolution: metamorphic timing and adult body size. We demonstrate that paedomorphic tiger salamanders (Ambystoma tigrinum complex) carry alleles at three moderate effect QTL (met1–3) that vary in responsiveness to TH and additively affect metamorphic timing. Salamanders that delay metamorphosis attain significantly larger body sizes as adults and met2 explains a significant portion of this variation. Thus, substitution of alleles at TH-responsive loci suggests an adaptive pleiotropic basis for two key life-history traits in amphibians: body size and metamorphic timing. Our study demonstrates a likely pathway for the evolution of novel paedomorphic species from metamorphic ancestors via selection of TH-response alleles that delay metamorphic timing and increase adult body size.     

Identification of differentially expressed genes in 4-day axolotl limb blastema by suppression subtractive hybridization

The goal of our study was the identification of up-regulated genes during axolotl (Ambystoma mexicanum) hindlimb regeneration 4 days after amputation using suppression subtractive hybridization (SSH). Approximately 400 clones that harbored upregulated genes in regenerating blastema tissue were selected for sequence analysis. A BLAST homology search against NCBI non-redundant database and an ambystoma EST database revealed 102 clones that showed homology to known sequences in GenBank with annotated function, 31 were known genes without known function, 74 were novel and 72 belonged to mitochondrial sequences. Differential expression of Hmox1, Orc4L, Pls3, Fen-1, Mcm7 and Mmp3/10a was confirmed using qRT-PCR analysis. Among all genes, only Mmp3/10a has been previously described as involved in limb regeneration. Other important identified genes belong to the group of cell cycle regulators (Orc4L, Nasp, Skp1A and Mcm7, the latter being a possible proliferative marker), those involved in protein synthesis and transport (Sec63, Srp72, Sara2) and V- ATPase pump. The novel genes we identified might be important for the process of blastema formation and the onset of cell proliferation in a regenerating limb.     

Regulation of lactate dehydrogenase (LDH) and alcohol dehydrogenase (ADH) synthesis in liver nuclei,following their transfer into oocytes

The regulatory effect of oocyte cytoplasm on the synthetic activity of transferred somatic cell nuclei was studied using an interspecific hybrid combination of Ambystoma texanum and Ambystoma mexicanum (axolotl). The enzymes lactate dehydrogenase (LDH) and alcohol dehydrogenase (ADH) were used as markers of gene activity. In both species of salamanders, LDH is synthesized in the liver and oocytes, while ADH is tissue-specific being synthesized in the liver but not oocytes. Both LDH and ADH show species-specific patterns on starch gels which permit detection of enzymes synthesized by texanum liver nuclei following their transfer into axolotl oocytes. Analysis of recipient oocytes after 1-3 weeks in culture reveals the presence of newly synthesized texanum LDH but not ADH. These results indicate that the transferred texanum liver cell nuclei continue to synthesize a product (LDH) found in both liver cells and oocytes, but fail to synthesize the liver-specific product (ADH) which is normally absent in oocytes. Thus, in the case of ADH and LDH the oocyte cytoplasm appears to be able to regulate the synthetic activity of the transferred somatic cell nuclei so as to conform to the oocytes' normal synthetic output.     

Limb chondrogenesis of the seepage salamander, Desmognathus aeneus (amphibia: plethodontidae)

Salamanders are infrequently mentioned in analyses of tetrapod limb formation, as their development varies considerably from that of amniotes. However, urodeles provide an opportunity to study how limb ontogeny varies with major differences in life history. Here we assess limb development in Desmognathus aeneus, a direct-developing salamander, and compare it to patterns seen in salamanders with larval stages (e.g., Ambystoma mexicanum). Both modes of development result in a limb that is morphologically indistinct from an amniote limb. Developmental series of A. mexicanum and D. aeneus were investigated using Type II collagen immunochemistry, Alcian Blue staining, and whole-mount TUNEL staining. In A. mexicanum, as each digit bud extends from the limb palette Type II collagen and proteoglycan secretion occur almost simultaneously with mesenchyme condensation. Conversely, collagen and proteoglycan secretion in digits of D. aeneus occur only after the formation of an amniote-like paddle. Within each species, Type II collagen expression patterns resemble those of proteoglycans. In both, distal structures form before more proximal structures. This observation is contrary to the proximodistal developmental pattern of other tetrapods and may be unique to urodeles. In support of previous findings, no cell death was observed during limb development in A. mexicanum. However, apoptotic cells that may play a role in digit ontogeny occur in the limbs of D. aeneus, thereby suggesting that programmed cell death has evolved as a developmental mechanism at least twice in tetrapod limb evolution.     

Microarray Analysis of microRNA Expression during Axolotl Limb Regeneration

Among vertebrates, salamanders stand out for their remarkable capacity to quickly regrow a myriad of tissues and organs after injury or amputation. The limb regeneration process in axolotls (Ambystoma mexicanum) has been well studied for decades at the cell-tissue level. While several developmental genes are known to be reactivated during this epimorphic process, less is known about the role of microRNAs in urodele amphibian limb regeneration. Given the compelling evidence that many microRNAs tightly regulate cell fate and morphogenetic processes through development and adulthood by modulating the expression (or re-expression) of developmental genes, we investigated the possibility that microRNA levels change during limb regeneration. Using two different microarray platforms to compare the axolotl microRNA expression between mid-bud limb regenerating blastemas and non-regenerating stump tissues, we found that miR-21 was overexpressed in mid-bud blastemas compared to stump tissue. Mature A. mexicanum (“Amex”) miR-21 was detected in axolotl RNA by Northern blot and differential expression of Amex-miR-21 in blastema versus stump was confirmed by quantitative RT-PCR. We identified the Amex Jagged1 as a putative target gene for miR-21 during salamander limb regeneration. We cloned the full length 3′UTR of Amex-Jag1, and our in vitro assays demonstrated that its single miR-21 target recognition site is functional and essential for the response of the Jagged1 gene to miR-21 levels. Our findings pave the road for advanced in vivo functional assays aimed to clarify how microRNAs such as miR-21, often linked to pathogenic cell growth, might be modulating the redeployment of developmental genes such as Jagged1 during regenerative processes.     

Thyroid Hormone Receptor Genes of Neotenic Amphibians

Since thyroid hormones play a pivotal role in amphibian metamorphosis we used PCR to amplify DNA fragments corresponding to a portion of the ligand-binding domain of the thyroid hormone receptor (TR) genes in several neotenic amphibians: the obligatory neotenic members of the family Proteidea the mudpuppy Necturus maculosus and Proteus anguinus as well as two members of the facultative neotenic Ambystoma genus: the axolotl Ambystoma mexicanum and the tiger salamander Ambystoma tigrinum. In addition, we looked for TR genes in the genome of an apode Typhlonectes compressicaudus. TR genes were found in all these species including the obligatory neotenic ones. The PCR fragments obtained encompass both the C and E domains and correspond to α and β genes. Their sequences appear to be normal, suggesting that there is no acceleration of evolutionary rates in the TR genes of neotenic amphibians. This result is not surprising for Ambystomatidae, which are known to respond to T3 (3,3′,5-triiodothyronine) but is not in agreement with biochemical and biological data showing that Proteidea cannot respond to thyroid hormones. Interestingly, by RT-PCR analysis we observed a high expression levels of TRα in gills, intestine, and muscles of Necturus as well as in the liver of Ambystoma mexicanum, whereas TRβ expression was only detected in Ambystoma mexicanum but not in Necturus. Such a differential expression pattern of TRα and TRβ may explain the neoteny in Proteidea. The cloning of thyroid-hormone-receptor gene fragments from these species will allow the molecular study of their failure to undergo metamorphosis. Received: 23 April 1996 / Accepted: 20 January 1997     

Fibroblast growth factors in regenerating limbs of Ambystoma: cloning and semi-quantitative RT-PCR expression studies

Urodele amphibians (newts and salamanders) have the ability to regenerate amputated limbs throughout their life span. Because fibroblast growth factors (Fgfs) play important roles in developing limbs, we initiated studies to investigate these growth factors in regenerating limbs. Partial cDNAs of Fgf4, 8, and 10 were cloned from both the Mexican axolotl, Ambystoma mexicanum, and locally collected spotted salamander, Ambystoma maculatum, two salamanders well recognized for their regenerative capabilities. cDNAs from the two Ambystoma species were virtually identical, ranging from 97-100% nucleotide identity. Axolotl Fgf4, 8, and 10 showed nucleotide sequence identity with chick Fgf4, 8, and 10 of 79%, 83%, and 72%, respectively. RT-PCR showed that these growth factors are expressed in regenerating axolotl limbs as well as in developing salamander larvae at the three-digit forelimb stage. Fgf8 and 10 are upregulated during regeneration and thus may be involved in distal signaling similar to that of the developing chick limb. Fgf4, however, was undetectable by RT-PCR in the distal tips of regenerates, suggesting that it does not play the same role in limb regeneration that it does in limb development. We also investigated the role these Fgfs may have in the nerve-dependence of regeneration. They were expressed similarly in aneurogenic and innervated limbs, suggesting that they are not the neurotrophic factors responsible for nerve-dependence. Denervation prevented Fgf8 and 10 upregulation, suggesting Fgf pathways are downstream of nerve-dependence. These data highlight important similarities and differences in Fgf expression between limb development and limb regeneration. J. Exp. Zool. 290:529-540, 2001.