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

Dunja Knapp,Eugen Nacu,Herbert Schulz,Elly M Tanaka,Juliane Scholz,Cynthia Alexander Rascon,Michael Volkmer,Tina Jacob,Stephanie Protze,Mu Le,Bianca Habermann,Akira Tazaki,Sergey Novozhilov,Norbert Hubner,Mike O Karl

doi:10.1371/journal.pone.0061352

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.

Highlights

In salamander, limb amputation initiates a wound-healing response followed by the emergence of a proliferative zone of cells, called the blastema, that consists of mesenchymal progenitor cells covered by an epithelium [1].Injuries trigger a wound-healing response as the first step in regeneration, but simple wounding is not sufficient to launch a full regeneration response
As an initial assessment of the time course dataset, we examined the profile of cell cycle regulators to determine if we could detect the onset of cell proliferation associated with injury and blastema formation
Based on morphological observations and expression analysis of selected genes in the injured limbs, it has been established that early wound healing lasting about 24 hours is comparable for both regenerating and lateral wounds [40,44]

Summary

Introduction

Limb amputation initiates a wound-healing response followed by the emergence of a proliferative zone of cells, called the blastema, that consists of mesenchymal progenitor cells covered by an epithelium [1].Injuries trigger a wound-healing response as the first step in regeneration, but simple wounding is not sufficient to launch a full regeneration response. Limb amputation initiates a wound-healing response followed by the emergence of a proliferative zone of cells, called the blastema, that consists of mesenchymal progenitor cells covered by an epithelium [1]. A number of axolotl limb studies have indicated that limb wounds in the absence of full amputation are repaired imperfectly, as in mammals (for review see [2]). Critical size bone defects are not repaired in the axolotl limb, similar to mammals [3,4,5]. The specific conditions related to amputating the limb are critical to the accumulation of mesenchymal blastema cells that will regenerate the limb. An important question is what are the molecular factors that determine the establishment of a blastema only after amputation, in contrast to other injuries

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