Retinoic acid breakdown is required for proximodistal positional identity during axolotl limb regeneration. Retinoic acid breakdown is required for proximodistal positional identity during axolotl limb regeneration

Regenerating limbs retain their proximodistal (PD) positional identity following amputation. This positional identity is encoded genetically by PD patterning genes, which instruct blastema cells to regenerate the appropriate PD limb segment. Retinoic acid (RA) is known to specify proximal limb identity, but how RA concentration is established in the blastema is unknown. Here, we show that RA breakdown via CYP26B1 is essential for determining the RA concentration within blastemas. CYP26B1 inhibition molecularly reprograms distal blastemas into a proximal identity, phenocopying the effects of administering excess RA. We identify Shox as an RA responsive gene that is differentially expressed between proximally and distally amputated blastemas. Ablation of Shox results in shortened limbs with proximal skeletal elements that fail to undergo endochondral ossification. These results suggest that PD positional identity is determined by RA degradation and that targets of RA have a critical role in skeletal element formation during limb regeneration. Overall design: To understand the impact of excess endogenous retinoic acid on proximodistal patterning of the regenerating salmander forelimb, distal forelimb blastemas (mid-carpal amputation) were treated with increasing doses of talarozole (TAL) suspended in DMSO. Control distal and proximal (upper humerus amputation) forelimb blastemas were treated with vehicle (DMSO). Pools of 3 blastemas per sample (3 samples per condition) were collected at 14 days post amputation and immediately flash frozen in liquid nitrogen. Samples were stored at -80C until sent for sequencing.

肢体截肢再生后仍保留其近远轴（proximodistal, PD）位置同一性。该位置同一性由PD模式基因通过遗传方式编码，可指导芽基（blastema）细胞再生出对应节段的肢体。视黄酸（Retinoic acid, RA）已被证实可指定肢体近端身份，但芽基内RA浓度的调控机制尚不明确。本研究证实，经CYP26B1介导的RA降解对维持芽基内RA浓度至关重要。抑制CYP26B1可在分子层面将远端芽基重编程为近端身份，其表型与过量施加RA的效果一致。本研究鉴定出Shox为RA响应基因，其在近端截肢与远端截肢的芽基中存在差异表达。敲除Shox会导致肢体缩短，且其近端骨骼结构无法完成软骨内骨化（endochondral ossification）。上述结果表明，肢体再生过程中的近远轴位置同一性由RA降解调控，且RA靶基因在骨骼结构形成中发挥关键作用。
实验设计：为探究内源性过量RA对再生蝾螈前肢近远轴模式建成的影响，研究人员对腕中部截肢获得的远端前肢芽基施加梯度浓度的他拉唑罗（talarozole, TAL），该试剂悬浮于二甲基亚砜（Dimethyl sulfoxide, DMSO）中。对照组的远端与肱骨上段截肢获得的近端前肢芽基则仅接受溶剂（DMSO）处理。于截肢后14天收集样本，每个样本包含3个芽基，每组设置3个生物学重复，样本收集后立即置于液氮中快速冷冻。所有样本保存于-80℃环境，直至送检测序。