The salamander has the remarkable ability to regenerate its limb after amputation. Cells at the site of amputation form a blastema and then proliferate and differentiate to regrow the limb. To better understand this process, we performed deep RNA sequencing of the blastema over a time course in the axolotl, a species whose genome has not been sequenced. Using a novel comparative approach to analyzing RNA-seq data, we characterized the transcriptional dynamics of the regenerating axolotl limb with respect to the human gene set. This approach involved de novo assembly of axolotl transcripts, RNA-seq transcript quantification without a reference genome, and transformation of abundances from axolotl contigs to human genes. We found a prominent burst in oncogene expression during the first day and blastemal/limb bud genes peaking at 7 to 14 days. In addition, we found that limb patterning genes, SALL genes, and genes involved in angiogenesis, wound healing, defense/immunity, and bone development are enriched during blastema formation and development. Finally, we identified a category of genes with no prior literature support for limb regeneration that are candidates for further evaluation based on their expression pattern during the regenerative process.

Understanding how the limb blastema is established after the initial wound healing response is an important aspect of regeneration research. Here we performed parallel expression profile time courses of healing lateral wounds versus amputated limbs in axolotl. This comparison between wound healing and regeneration allowed us to identify amputation-specific genes. By clustering the expression profiles of these samples, we could detect three distinguishable phases of gene expression – early wound healing followed by a transition-phase leading to establishment of the limb development program, which correspond to the three phases of limb regeneration that had been defined by morphological criteria. By focusing on the transition-phase, we identified 93 strictly amputation-associated genes many of which are implicated in oxidative-stress response, chromatin modification, epithelial development or limb development. We further classified the genes based on whether they were or were not significantly expressed in the developing limb bud. The specific localization of 53 selected candidates within the blastema was investigated by in situ hybridization. In summary, we identified a set of genes that are expressed specifically during regeneration and are therefore, likely candidates for the regulation of blastema formation.

The evolutionary origins of the gene network underlying cellular pluripotency, a central theme in developmental biology, have yet to be elucidated. In mammals, Oct4 is a factor crucial in the reprogramming of differentiated cells into induced pluripotent stem cells. The Oct4 and Pou2 genes evolved from a POU class V gene ancestor, but it is unknown whether pluripotency induced by Oct4 gene activity is a feature specific to mammals or was already present in ancestral vertebrates. Here we report that different vertebrate Pou2 and Oct4 homologues can induce pluripotency in mouse and human fibroblasts and that the inability of zebrafish Pou2 to establish pluripotency is not representative of all Pou2 genes, as medaka Pou2 and axolotl Pou2 are able to reprogram somatic cells into pluripotent cells. Therefore, our results indicate that induction of pluripotency is not a feature specific to mammals, but existed in the Oct4/Pou2 common ancestral vertebrate.

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