The essential role of connective-tissue cells during axolotl limb regeneration

Abstract

Axolotls (Ambystoma mexicanum) are known for their remarkable limb-regeneration abilities, which involve the formation of the blastema, a specialized structure consisting of progenitor cells contributed by all major tissues of the limb. Lateral plate mesoderm (LPM)-derived connective tissue (CT) cells dedifferentiate and play a critical role in blastema formation and subsequent limb regeneration. However, the complexity of the blastema cellular composition and the extent of CT participation and necessity have not been rigorously explored. To address this gap, we conducted spatial transcriptomics using a select array of probes, revealing that CT cells constitute up to 75% of the blastema cells. Genetic ablation of CT cells significantly delays or truncates limb regeneration, underscoring their necessity during this process. Analysis of the molecular profile during blastema formation identifies a temporally ordered gene expression pattern reflecting sequential phases of CT cell dedifferentiation, proliferation, and redifferentiation. Finally, a cell-cell communication inference identifies a CT-cell-autonomous FGFR1/AKT1 signaling axis as a cell-intrinsic molecular requirement for limb regeneration. Together, our work reaffirms the central role of CT cells in axolotl limb regeneration and lays the foundation for identifying molecular mechanisms that govern blastema formation during the initial phases of limb regeneration.