TRPV4 Inhibition Reduces Cartilage Growth During Axolotl Limb Regeneration

BackgroundAxolotls can regenerate entire limbs, recapitulating mammalian developmental processes. During development, mechanosensitive ion channels such as TRPV4, PIEZO1, and PIEZO2 regulate tissue morphogenesis by transducing mechanical signals. Their roles in regeneration, however, have yet to be thoroughly explored. To investigate this, we assessed the expression of these channels during limb regeneration using single-cell RNA sequencing dataset, hybridization chain reaction fluorescence in-situ hybridization, and immunofluorescence. Additionally, functional relevance was tested by pharmacological inhibition of TRPV4 and PIEZO1/2 mechanosensitive ion channels during limb regeneration. ResultsWhile PIEZO1 expression was undetected, we observed TRPV4 and PIEZO2 expression in uninjured cartilage and at both the mid-bud and palette blastemal stages of limb regeneration. TRPV4 and PIEZO2 were highly expressed in chondrocytes, with PIEZO2 enriched in fibroblasts. Inhibition with GSK205, a TRPV4 antagonist, significantly reduced calcium influx and humeral length without inhibiting cartilage differentiation. Treatment with gadolinium chloride, a broad spectrum mechanosensitive ion channel inhibitor, had no significant morphological impact. ConclusionsTRPV4 and PIEZO2 are dynamically regulated during axolotl limb regeneration. Selective TRPV4 inhibition altered final limb morphology, but chondrogenesis was unaffected. This suggests a role for these genes in shaping tissue architecture during limb regeneration. These findings underscore the importance of ion channel-mediated mechanotransduction in regenerative patterning. Key findingsO_LITRPV4 and PIEZO2 show distinct, stage- and cell type-specific expression patterns during axolotl limb regeneration C_LIO_LISelective pharmacological inhibition of TRPV4 significantly reduces calcium signaling in chondrocytes and leads to shorter limb and humeral lengths in vivo C_LI