Comparative Transcriptional Profiling of the Axolotl Limb Identifies a Tripartite Regeneration-Specific Gene Program

Abstract

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 axolotl genome and the evolution of key tissue formation regulators

“…Functional analysis of axolotl development, physiology and regeneration is facilitated by the availability of tissue-and time-dependent gene expression profiles [28][29][30]36 . The axolotl genome provides a foundation for applying methods such as chromatin immunoprecipitation with sequencing (ChIP-seq) or assay for transposase-accessible chromatin using sequencing (ATAC-seq) to investigate the genomic basis of gene regulation during regeneration.…”

“…Recent functional work has highlighted the role of diverged gene or protein function during regeneration [25][26][27] . Analysis of published tissue-enriched datasets 28 , combined with regeneration time courses 29,30 and our own transcriptional profiling of 22 tissues, identified five transcripts that are upregulated in the limb blastema (the mass of proliferating cells involved in regenerating the limb) with orthology limited to non-amniote vertebrates (Supplementary Information section 7). One of these protein sequences shows a weak similarity to tectorin, a basement membrane component normally found in the inner ear, consistent with studies that implicate extracellular matrix components with having an important role in limb regeneration 31,32 .…”

A microRNA-mRNA expression network during oral siphon regeneration in Ciona

“…Appendage regeneration is thought to occur in three morphologically distinct stages; wound healing, transition and redevelopment (Knapp et al, 2013). The wound healing stage is primarily defined by a localized immune response, closure of the epithelium and initiation of blastema formation (Murawala et al, 2012).…”

“…The ability to detect transcript level changes in a comprehensive and unbiased manner can identify previously unknown coordination between signaling pathways, transcription factors and effector genes required for regeneration. There have been several transcriptome-wide studies of gene expression of both mRNA (Campbell et al, 2011;Hamada et al, 2015;Knapp et al, 2013;Love et al, 2011;Monaghan et al, 2009;Schebesta et al, 2006;Stewart et al, 2013;Wu et al, 2013) and microRNA (miRNA) (Gearhart et al, 2015;Holman et al, 2012;Hutchins et al, 2016;Thatcher et al, 2008) during appendage regeneration. However, none of these studies connected the expression profiles of miRNA to those of mRNA.…”

Temporal Expression Pattern of Peptides in the Regenerating Caudal Fin of Teleost Fish Poecilia latipinna With Special Emphasis on krt15 and myl-1

“…There are reports on proteomic profile in regeneration models to unravel the conserved and diverse mechanisms amongst them [16][17][18][19]. It includes the regeneration in axololtl [20,21], Xenopus laevis froglet limb-bud [22,23] and zebrafish caudal fin regeneration [24].…”