Osteoclast-mediated resorption primes the skeleton for successful integration during axolotl limb regeneration

Abstract

Early events during axolotl limb regeneration include an immune response and the formation of a wound epithelium. These events are linked to a clearance of damaged tissue prior to blastema formation and regeneration of the missing structures. Here, we report the resorption of calcified skeletal tissue as an active, cell-driven and highly regulated event. This process, carried out by osteoclasts, is essential for a successful integration of the newly formed skeleton. Indeed, the extent of resorption is directly correlated with the integration efficiency and treatment with zoledronic acid resulted in osteoclast function inhibition and failed tissue integration. Moreover, we identified the wound epithelium as a regulator of skeletal resorption, likely releasing signals involved in recruitment/differentiation of osteoclasts. Finally, we reported a correlation between resorption and blastema formation, particularly, a coordination of resorption with cartilage condensation. In sum, our results identify resorption as a major event upon amputation, playing a critical role in the overall process of skeletal regeneration.

Axolotl mandible regeneration occurs through mechanical gap closure and a shared regenerative program with the limb

“…In situ hybridization was used to assess gene expression in sectioned tissues. Probes for the axolotl Kazald2 and Krt17 were generated as previously described (Riquelme-Guzmán et al, 2022). Primer sequences for probe amplification were taken from Leigh et al, 2018( Krt17 ) and Bryant et al, 2017 ( Kazald1 ).…”

The salamander limb: a perfect model to understand imperfect integration during skeletal regeneration

“…In axolotls, several highly expressed transcripts were identified in perichondrial cells based on the expression of Col8a2 ( Gerber et al, 2018 ). Additionally, lineage tracing using transgenic reporter lines identified that Col1a2 + perichondrial cells contribute to the regenerated structure ( Gerber et al, 2018 ), and that some perichondrial cells express Ctsk upon amputation ( Riquelme-Guzmán et al, 2022a ). Moreover, the expression of PTHrP and Gli1 was reported in the perichondrium of newts ( Kaucka et al, 2022 ).…”

“…Tissue histolysis is the clearance of debris and non-functional tissue prior to blastema formation ( Stocum, 2017 ). In the skeleton, a rapid and substantial resorption of the calcified tissue (in both cartilage and bone) occurs starting at 5 days post amputation (dpa) ( Riquelme-Guzmán et al, 2022a ). This resorption is driven by multinucleated Ctsk + /Trap + resorbing cells.…”

“…The WE appears to be linked to resorption induction, as blocking its formation by full skin flap surgery downregulates genes associated with ECM remodelling (e.g. Mmp2 , Mmp13 , Ctsk , among others), inhibits skeletal resorption and reduces number of Ctsk + cells ( Riquelme-Guzmán et al, 2022a ; Tsai et al, 2020 ).…”

“…Various studies identified these regeneration-induced resorbing cells as osteoclasts by their multinucleation and/or their expression of Ctsk and Trap ( Fischman and Hay, 1962 ; Riquelme-Guzmán et al, 2022a ; Tank et al, 1976 ). Osteoclasts are myeloid-derived cells specialised in the degradation of skeletal matrices.…”