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

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.

探明初始伤口愈合反应后肢体芽基（limb blastema）的建立机制，是再生研究的重要方向之一。本研究以墨西哥钝口螈（axolotl）为模型，对愈合中的侧部伤口与截肢肢体开展平行的表达谱（expression profile）时间序列检测。通过对比伤口愈合与肢体再生的过程，我们成功鉴定出截肢特异性基因。通过对上述样本的表达谱进行聚类分析，我们检测到三个可明确区分的基因表达阶段：早期伤口愈合阶段，随后是导向肢体发育程序建立的过渡阶段，这三个阶段与此前通过形态学标准定义的肢体再生三阶段完全对应。聚焦过渡阶段的基因表达特征，我们鉴定出93个严格的截肢相关基因，其中诸多基因参与氧化应激反应、染色质修饰、上皮发育或肢体发育过程。我们进一步根据这些基因是否在发育中的肢芽（limb bud）中显著表达，对其进行了分类。通过原位杂交（in situ hybridization）技术，我们对筛选出的53个候选基因在芽基内的特异性定位进行了研究。综上，本研究鉴定出一组仅在再生过程中特异性表达的基因，因此它们可作为调控芽基形成的潜在候选靶点。