Retinoic acid receptors and the control of positional information in the regenerating axolotl limb

We know little about the control of positional information (PI) during axolotl limb regeneration which ensures that the limb regenerates exactly what was amputated and the work reported here investigates this phenomenon. Retinoic acid administration changes the PI in a proximal direction so that a complete limb can be regenerated from a hand. Rather that identifying all the genes altered by RA treatment of the limb, we have eliminated many off-target effects by using retinoic acid receptor selective agonists. We firstly identify the receptor involved in this respecification process as RARα and secondly identify the genes involved by RNA sequencing of the RARα-treated blastemal mesenchyme. We find 1177 up-regulated genes and 1403 down-regulated genes which could be identified using the axolotl genome. These include several genes known to be involved in retinoic acid metabolism and in patterning. Since positional in-formation is thought to be a property of the cell surface of blastemal cells when we examine our dataset with an emphasis on this aspect, we find the top canonical pathway is integrin signaling. In the extracellular matrix compartment, we find an MMP and several collagens are up-regulated and several cell membrane genes and secretory factors are also up-regulated. This provides data for future testing of the function of these candidates in the control of PI during limb regeneration. Gene expression analysis of axolotl forelimb blastema mesenchyme tissue following 25 nM systemic Am580 treatment

目前我们对保障肢体精准再生为截肢前形态的墨西哥钝口螈（axolotl）肢体再生过程中位置信息（positional information，PI）的调控机制所知甚少，本研究针对这一现象展开了探究。视黄酸（retinoic acid，RA）处理可使PI向近端方向发生改变，从而使肢体可从手部完整再生。相较于直接筛选经RA处理的肢体中发生表达改变的全部基因，本研究通过采用视黄酸受体选择性激动剂，有效排除了大量脱靶效应。本研究首先确定了参与该重特化过程的受体为视黄酸受体α（RARα），其次通过对经RARα处理的芽基间充质（blastemal mesenchyme）组织进行RNA测序，鉴定出相关差异基因。基于墨西哥钝口螈基因组注释信息，本研究共筛选得到1177个上调基因与1403个下调基因，其中包含若干已知参与视黄酸代谢与模式建成的基因。鉴于学界普遍认为位置信息是芽基细胞细胞表面的属性，本研究在重点关注该维度的前提下对数据集进行分析，结果显示排名首位的经典通路为整合素信号通路（integrin signaling）。在细胞外基质组分中，本研究发现1种基质金属蛋白酶（matrix metalloproteinase，MMP）与若干胶原蛋白的表达量上调，同时还有若干细胞膜基因及分泌因子的表达量亦出现上调。本数据集为后续验证这些候选基因在肢体再生过程中PI调控中的功能提供了数据支撑。本数据集来源于经25纳摩尔全身给药Am580处理后的墨西哥钝口螈前肢芽基间充质组织的基因表达分析。