Regeneration of complex multi-tissue structures, such as limbs, requires the coordinated effort of multiple cell types. In axolotl limb regeneration, the wound epidermis and blastema have been extensively studied via histology, grafting, and bulk-tissue RNA-sequencing. However, defining the contributions of these tissues is hindered due to limited information regarding the molecular identity of the cell types in regenerating limbs. Here we report unbiased single-cell RNA-sequencing on over 25,000 cells from axolotl limbs and identify a plethora of cellular diversity within epidermal, mesenchymal, and hematopoietic lineages in homeostatic and regenerating limbs. We identify regeneration-induced genes, develop putative trajectories for blastema cell differentiation, and propose the molecular identity of fibroblast-like blastema progenitor cells. This work will enable application of molecular techniques to assess the contribution of these populations to limb regeneration. Overall, these data allow for establishment of a putative framework for adult axolotl limb regeneration.

Activation of progenitor cells is crucial to promote tissue repair following injury in adult animals. In the context of successful limb regeneration following amputation, progenitor cells residing within the stump must re-enter the cell cycle to promote regrowth of the missing limb. We demonstrate that in axolotls, amputation is sufficient to induce cell-cycle activation in both the amputated limb and the intact, uninjured contralateral limb. Activated cells were found throughout all major tissue populations of the intact contralateral limb, with internal cellular populations (bone and soft tissue) the most affected. Further, activated cells were additionally found within the heart, liver, and spinal cord, suggesting that amputation induces a common global activation signal throughout the body. Among two other injury models, limb crush and skin excisional wound, only limb crush injuries were capable of inducing cellular responses in contralateral uninjured limbs but did not achieve activation levels seen following limb loss. We found this systemic activation response to injury is independent of formation of a wound epidermis over the amputation plane, suggesting that injury-induced signals alone can promote cellular activation. In mammals, mTOR signaling has been shown to promote activation of quiescent cells following injury, and we confirmed a subset of activated contralateral cells is positive for mTOR signaling within axolotl limbs. These findings suggest that conservation of an early systemic response to injury exists between mammals and axolotls, and propose that a distinguishing feature in species capable of full regeneration is converting this initial activation into sustained and productive growth at the site of regeneration.