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.