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

Amputation of the axolotl forelimb results in the formation of a blastema, a transient tissue where progenitor cells accumulate prior to limb regeneration. Connective tissue (CT) – skeleton, periskeleton, tendon, dermis, interstitial cells – contributes the vast majority of cells that populate the blastema, however it is unclear how individual CT cells may reprogram their fate in order to rebuild the tetrapod limb. Here we use a combination of Cre-loxP reporter lineage tracking and single-cell (sc) RNA-seq to molecularly track, for the first time, adult CT cell heterogeneity and its transition to a limb blastema state. We uncover a multi-phasic molecular program where CT cell types found in the uninjured adult limb revert to a relatively homogenous progenitor state that participates in inflammation and extracellular matrix disassembly prior to proliferation, establishment of positional information, and ultimately re-differentiation. While the early regeneration transcriptome states are unique to the blastema, the later stages recapitulate embryonic limb development. Notably, we do not find evidence of a pre-existing blastema-like precursor nor limb bud-like progenitors in the uninjured adult tissue. However, we find that distinct CT subpopulations in the adult limb differentially contribute to proximal and distal portions of the regenerated limb. Together, our data illuminates molecular and cellular reprogramming during complex organ regeneration in a vertebrate.

美西螈前肢截肢后会形成芽基（blastema）——一种短暂存在的组织，祖细胞会在其中积聚，随后启动肢体再生过程。结缔组织（CT）——包括骨骼、骨膜、肌腱、真皮及间质细胞——是构成芽基的绝大多数细胞的来源，但目前尚不清楚单个CT细胞如何重编程其细胞命运，以重建四足动物肢体。本研究结合Cre-loxP报告基因谱系示踪技术与单细胞（single-cell, sc）RNA测序，首次在分子层面追踪了成年CT细胞的异质性及其向肢体芽基状态的转变过程。本研究揭示了一套多阶段分子程序：未损伤成年肢体中的CT细胞类型会先回复至相对均一的祖细胞状态，该状态会在细胞增殖、位置信息建立及最终再分化之前，参与炎症反应与细胞外基质降解过程。尽管再生早期的转录组状态为芽基所特有，但后期阶段则重现了胚胎肢体发育的进程。值得注意的是，本研究未在未损伤的成年组织中发现预先存在的芽基样前体细胞或肢芽样祖细胞。但本研究发现，成年肢体中不同的CT亚群会差异性地贡献于再生肢体的近端与远端部分。综上，本研究的数据阐明了脊椎动物复杂器官再生过程中的分子与细胞重编程机制。