Single-cell level transcriptomic anaylsis of the homeostatic and regenerating axolotl limb

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, studying the contributions of these tissues is hindered due to limited information regarding the molecular identity of the cell types in regenerating limbs. By performing unbiased single-cell RNA-sequencing on over 25,000 cells from axolotl limbs, we 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 and origin of fibroblast-derived blastema progenitor cells residing in homeostatic limbs. This work will enable application of molecular techniques to assess the contribution of these populations to limb regeneration. It will also facilitate work aimed at identifying transcripts and cells critical for limb regeneration. Overall design: A timecourse of single-cell transcriptomic data for the regenerating axolotl limb was obtained. Samples were collected at 3 days post amputation (dpa) (wound healing, 3 samples), 14 dpa (early-bud blastema, 2 samples), and 23 dpa (medium-bud blastema, 6 samples). Additonally, two sets of transcriptomic data was collected from 2 samples of intact (homeostatic) limb and 3 samples of intact limb contralateral to an early-bud blastema (14dpa) (not the same early-bud blastema as samples S3-14 and S5-14). All samples were collected on inDrops.