In preprints: retinoic acid degradation and tissue stiffness coordinate axolotl limb regeneration

The axolotl limb has long served as a model in which to study complex appendage regeneration. From classical grafting experiments to cutting-edge transcriptomics, this system continues to yield fresh insights into the rules that govern tissue patterning and growth. Two recent preprints by Duerr et al. and Edwards-Jorquera et al. highlight complementary mechanisms that underlie this remarkable regenerative capacity (Duerr et al., 2024 preprint; Edwards-Jorquera et al., 2025 preprint). Duerr and colleagues took a new look at an old question, how retinoic acid regulates the establishment of proximodistal identity, while Edwards-Jorquera and colleagues explored the role of mechanical regulation of growth during axolotl limb regeneration. While these are two seemingly complementary mechanisms, together they suggest a unified framework in which positional identity and biophysical context are co-regulators of regenerative patterning.Retinoic acid (RA), a metabolite of vitamin A, has been implicated as a key regulator of proximodistal positional identity during limb regeneration. Early studies demonstrated that exogenous RA administration could induce distal blastemal cells to adopt a more proximal fate, effectively transforming a regenerating wrist into an upper arm (Maden, 1982; Niazi and Saxena, 1978). These observations led to the hypothesis that RA serves as a proximalizing morphogen. Duerr and colleagues revisit this classic story with a crucial twist: rather than focusing on RA supplementation, they investigated what happens when RA breakdown is impaired. Using the CYP26 inhibitor talarozole (TAL), Duerr and colleagues show that impaired RA degradation leads to proximodistal patterning defects reminiscent of RA overexposure. This result supports a model in which spatially restricted RA clearance is essential for proper limb patterning – an elegant counterpoint to the long-standing focus on RA concentration alone. Their work suggests RA not as a static gradient but as a dynamically regulated signal, interpretation of which depends on finely tuned catabolism.Importantly, Duerr and colleagues identified Shox, a transcription factor previously linked to upper limb development in mammals (Ye et al., 2016), as an RA-responsive gene enriched in proximal blastemas. Functional assays using F0 CRISPR mutants revealed that Shox is required for specifying stylopod and zeugopod (proximal) identity, but not autopod (distal) elements in both limb development as well as regeneration. These findings suggest that proximodistal identity is encoded not simply by RA levels, but by their clearance and the context-specific sensitivity of target genes.Whereas Duerr and colleagues highlight the biochemical orchestration of patterning, Edwards-Jorquera and colleagues shift attention to a less visible but equally powerful regulator: tissue mechanics. Their study begins with a long-recognized but poorly understood phenomenon – axolotl limbs take the same amount of time to regenerate regardless of the amputation plane. That is, between animals of identical size, a digit and an entire upper arm regenerate in the same timeframe. They show that proximally amputated limbs grow faster and exhibit shorter cell-cycle durations than their distal counterparts.What drives this? Through a combination of atomic force microscopy and Brillouin microscopy, they reveal that distal blastemas are significantly stiffer than proximal ones, suggesting that tissue stiffness regulates cell cycle length. Removing either upper or lower arm blastema cells from their context-specific environment and culturing them in vitro equalized their cell cycle rates, but culturing AL1 cells (an axolotl limb cell line) in PEG hydrogels of different stiffnesses affected their cell cycle. Consistent with the in vivo observations, increasing the stiffness of the PEG hydrogel decreased the proliferation of AL1 cells. Finally, using a newly developed axolotl YAP/TAZ activity reporter, the authors could show differences in YAP/TAZ activity in upper and lower arm blastemas in vivo, which correlates with expression of classic YAP/TAZ target genes, such as Cyr61 and Ctgf. These data point to a model in which stiffness-dependent YAP activity tunes growth rate according to positional context.Although Duerr et al. and Edwards-Jorquera et al. explore distinct molecular systems – retinoid signaling versus mechanotransduction – their findings are deeply complementary. RA degradation modulates the expression of positional genes, such as Shox, while tissue stiffness differentially regulates blastema proliferation through YAP/TAZ.One tantalizing bridge between these studies lies in emerging evidence that RA and YAP/Hippo signaling may be interconnected. Duerr and colleagues’ manipulation of RA metabolism may indirectly shape the mechanical properties of the regenerating tissue. Conversely, mechanical forces could affect RA responsiveness through changes in chromatin accessibility or RA receptor dynamics. Indeed, the broader literature has drawn links where, for example, RA affects Hippo signaling via retinoic acid-induced protein 14 (RAI14), as well as the reverse where YAP activation affects RA receptors and target gene expression levels (Jeong et al., 2024; Bauzone et al., 2021).The implication is that positional identity and proliferation rate are not independently determined but may be co-regulated through biochemical–mechanical feedback loops. This integrated view aligns with broader trends in developmental biology, whereby morphogen signaling, epigenetic states and mechanical inputs increasingly appear as interdependent rather than modular.These insights might have far-reaching implications. Efforts to promote human limb regeneration or engineer patterned tissues in vitro will need to consider not only which signals to supply, but also how those signals are spatially and temporally cleared, and how the mechanical landscape of the tissue may shape the cellular response. This work could inform biomaterials design for regenerative scaffolds. Conversely, manipulating RA catabolism might offer a more nuanced means of guiding tissue identity than traditional supplementation approaches.Duerr et al. and Edwards-Jorquera et al. present two elegant and complementary views of regeneration, one from the vantage point of morphogen clearance and the other from the landscape of tissue mechanics. Their integration suggests that position is not merely a coordinate but an interplay of chemical and physical information. As regenerative biology moves forward, we may need to recalibrate our models to include morphogens, genetic and epigenetic factors, but also moduli and gradients of stiffness.