Axolotls possess a remarkable ability to regenerate complete limbs with precise spatial patterning. One key aspect of this process is the establishment of proximodistal (PD) identity, which distinguishes distal structures from proximal structures, including the humerus. Retinoic acid (RA) is known to promote proximal identity; however, the changes in RA levels during limb regeneration remain unclear.
This study focuses on the role of the CYP26B1 enzyme, a RA-degrading enzyme that contributes to the formation of RA signaling gradients, and examines its role in patterning during axolotl limb regeneration. The scientists explained how limb patterning during regeneration is influenced by RA degradation using pharmacological inhibition, gene expression analysis, and genetic manipulation in axolotls. RA is produced endogenously by damaged blastema tissue. The researchers found that proximal blastemas (PBs) show 3.5 times more RA signaling than distal blastemas (DBs). Notably, Cyp26b1 is identified as the primary RA-degrading enzyme responsible for  RA degradation in axolotl blastemas.
The findings suggest that the PD signalling gradient is established by the spatial regulation of RA degradation rather than its synthesis. To copy the results of direct RA application, proximal limb structures were regenerated in distal limbs treated with talarozole (TAL). Due to high concentrations, regeneration is completely prevented, whereas treatment with one ÎĽM TAL caused the formation of zeugopod and stylopod elements in DBs. Shox and Shox2 genes concentrated in proximal areas, exhibited differential expression along the PD axis. The loss of Shox in axolotls resulted in shorter limbs due to poor endochondral ossification in the zeugopod and stylopod regions. These findings align with the French Flag Model, which suggests that segment identity is determined by morphogen (RA) thresholds, thereby determining segmental identity.
RNA-seq data observed no significant differences between PBs and DBs in the expression of FGF genes. Interestingly, Hoxa13 and Cyp26b1 showed overlapping spatial expression. In DBs at 10- and 14-day post-amputation (DPA), Shox expression was limited to a specific region. Stage 47 limb buds showed Shox expression only in proximal mesenchyme, not in Hoxa13+ distal mesenchyme. A transitional gene expression profile was observed in DBs treated with 0.1-1 µM TAL, represented by cluster four, which contains genes that are strongly expressed in DBs.
The Northeastern University Institutional Animal Care and Use Committee approved all surgeries, which were performed under anaesthesia with 0.01% benzocaine. The limb segment morphology was modified by using blastema cells to generate intra-segmental positioning values based on RA signalling levels. A one-way analysis of variance (ANOVA) with post-hoc Tukey’s honestly significant difference (HSD) post-hoc test on ΔCt values or a two-tailed Student’s t-test was used to assess statistical significance across groups.
This study concluded that Shox expression is regulated by RA signaling, which in turn coordinates the endochondral ossification of proximal skeletal structures. Overall, these findings underscore that graded clearance of morphogen, rather than production alone, is essential for spatial patterning during vertebrate limb regeneration.
Reference: Duerr TJ, Miller M, Kumar S, et al. Retinoic acid breakdown is required for proximodistal positional identity during axolotl limb regeneration. Nat Commun. 2025;16:4798. doi:10.1038/s41467-025-59497-5
