Fibroblast Dedifferentiation as a Determinant of Successful Regeneration. Fibroblast Dedifferentiation as a Determinant of Successful Regeneration

Limb regeneration, while observed lifelong in salamanders, is restricted to pre-metamorphic stages in Xenopus laevis frogs. After amputation, post-metamorphic frogs form a blastema that grows only an unsegmented cartilage rod. Whether this loss is due to systemic factors such as the immune system or due to an intrinsic incapability of cells to form competent stem cells has been unclear. Here, we use genetic fate mapping to establish that, as in axolotl, connective tissue (CT) cells form the post-metamorphic frog blastema. Using heterochronic transplantation into the limb bud and single-cell transcriptomic profiling, we show that axolotl CT cells fully dedifferentiate and integrate to form lineages including cartilage in the developing limb. In contrast, frog blastema CT cells do not fully re-express the limb bud progenitor program, even when transplanted into the limb bud. Correspondingly, transplanted cells contribute to extraskeletal CT but not to developing cartilage. Further, using single-cell RNA-seq analysis we find that the embryonic and the adult frog cartilage differentiation programs are molecularly distinct. This work defines intrinsic restrictions in CT dedifferentiation as a limitation in adult regeneration. Overall design: Tissue grafting, single-cell transcriptomics, reporter lineage tracking and cell transplantions were used to deconstruct cell compositions and to reconstruct lineage relationships aiming to find determinants of the successful regeneration in axolotl compared to the incomplete regenration in frogs.

肢体再生在蝾螈中可终生发生，但在非洲爪蟾（Xenopus laevis）中仅局限于变态前阶段。截肢后，变态后的非洲爪蟾会形成芽基（blastema），但仅能生长出未分节的软骨杆。这种再生能力的丧失究竟是源于免疫系统等系统性因素，还是细胞本身无法形成具有功能的干细胞，此前尚不明确。本研究通过遗传命运图谱（genetic fate mapping）技术证实，与美西钝口螈（axolotl）一样，非洲爪蟾变态后的结缔组织（CT）细胞参与形成了其肢体芽基。本研究通过异时移植（heterochronic transplantation）至肢芽（limb bud）以及单细胞转录组图谱分析（single-cell transcriptomic profiling），证实美西钝口螈的CT细胞可完全去分化，并整合至发育中的肢体中，形成包括软骨在内的多种细胞谱系。与之相反，即便被移植至肢芽中，非洲爪蟾芽基的CT细胞也无法完全重表达肢芽祖细胞程序。相应地，移植后的细胞仅能参与骨外结缔组织的形成，而无法参与发育中的软骨形成。此外，通过单细胞RNA测序（single-cell RNA-seq）分析，本研究发现非洲爪蟾胚胎与成体软骨分化程序在分子层面存在显著差异。本研究明确，结缔组织细胞去分化的内在限制是成年非洲爪蟾肢体再生能力不足的关键原因。整体实验设计：本研究采用组织移植、单细胞转录组学、报告基因谱系追踪以及细胞移植技术，解析细胞组成并重构细胞谱系关系，旨在探寻美西钝口螈实现完美肢体再生，而非洲爪蟾仅能实现不完全再生的关键决定因素。