Single-cell analysis uncovers convergence of cell identities during axolotl limb regeneration

INTRODUCTION Axolotls ( Ambystoma mexicanum ) and other salamanders are the only tetrapods that can regenerate whole limbs. During this complex process, changes in gene expression regulate the outgrowth of a new appendage, but how injury induces limb cells to form regenerative progenitors that differentiate into diverse cell fates is poorly understood. Tracking and molecular profiling of individual cells during limb regeneration would resolve distinct differentiation pathways and provide clues for how cells convert from a mature resting state into regenerative cell lineages. RATIONALE Axolotl limbs are composed of many different cell types originating from neural, myogenic, epidermal, and connective tissue (CT) lineages. Upon limb amputation, cells from nearby the amputation plane accumulate in a distinctive tissue called the blastema, which serves as a progenitor cell source to build the new limb. Transgenic axolotl strains in which descendants of distinct adult cell types can be labeled, tracked, and isolated during the regenerative process provide an opportunity to understand how particular cell lineages progress during blastema formation and subsequent limb regrowth. Combining transgenic axolotl strains with single-cell RNA sequencing (scRNA-seq) enables the tracking of individual cell types, as well as the reconstruction of the molecular steps underlying the regeneration process for these particular cell lineages. CT cells, descendants of lateral plate mesoderm, are the most abundant lineage contributing to the blastema and encompass bone and cartilage, tendons, periskeleton, and dermal and interstitial fibroblasts. These cells detect the position of the amputation site, leading to the regeneration of appropriate limb parts and making the CT a key cell lineage for deciphering and understanding molecular programs of regeneration. RESULTS We used an inducible Cre-loxP fluorescence system to establish genetically marked transgenic axolotl strains for isolating CT cells from adult limb tissue as well as CT descendants in the blastema. We used scRNA-seq to molecularly profile CT cells along a dense time course of blastema formation and the outgrowth of the regenerated arm, as well as stages of embryonic limb development. This profiling indicated that CT cells express adult phenotypes that are lost upon the induction of regeneration. The heterogeneous population of CT-derived cells converges into a homogeneous and transient blastema progenitor state that at later stages recapitulates an embryonic limb bud–like program. Notably, we did not find evidence of CT stem cells or blastema-like precursors in the mature arm. We found that CT subtypes have spatially restricted contributions to proximal and distal compartments in the regenerated limb. Specifically, a particular CT subtype—periskeletal cells—extended the severed skeleton at the amputation site whereas fibroblastic CT cells de novo regenerated distal skeletal segments. By using high-throughput single-cell transcriptomics and Brainbow axolotl-based clonal lineage tracing, we could follow the redifferentiation trajectories of CT lineages during the final stages of regeneration. These findings established the formation of a multipotent skeletal progenitor cell that contributed to tendons, ligaments, skeleton, periskeleton, and fibroblasts. CONCLUSION CT cells are a key cell type for understanding regeneration because they form the patterned limb skeleton that guides the regeneration of the other limb tissues, such as muscle. Because of the cells’ heterogeneity and intermingling with other cell types, it had been difficult to study how CT forms regenerative blastema cells. The use of these newly generated transgenic reporter strains combined with single-cell transcriptomic analysis and clonal tracing have allowed us to determine that CT cells with diverse molecular features traverse through a distinctive molecular state as they dedifferentiate into a common, multipotent progenitor resembling an embryonic limb bud cell. In the future, it will be important to test which components of the transition state are required for the dedifferentiation process. Furthermore, this work opens the possibility to examine how regeneration-associated genes and their associated chromatin structure are regulated during this transition. Lastly, the work raises the possibility that the limited regeneration seen among mammals is due to an inability to reprogram CT to such embryonic states.