tBid mediated genetic ablation of connective tissue cells reveal their key regulatory function during limb regeneration in axolotls

Limb regeneration in adulthood is a fascinating phenomenon unique to salamanders, requiring precise coordination and interaction of various cell types. Connective tissue (CT) constitutes a major component of the limb and serves as the primary reservoir of positional information during regeneration. However, the direct evidence and the extent of CT in positional contribution remain limited. Here we develop an inducible Cre-LoxP-mediated tBid cell ablation system in axolotls and demonstrate efficient elimination of targeted muscle tissue and muscle stem cells through transient electroporation or genetic approach. Ablation of CT cells at early stages of regeneration results in delayed regeneration and loss of proximal limb segments (e.g., upper and lower limb), with minimal impact on hand differentiation. CT ablation during development yields similar defects as observed during regeneration, indicating an essential role for CT cells in determining the positional identity of developing and regenerating limb structures. Further scRNA-seq reveal a progressive proximal-to-distal transition among CT cells and identify distinct CT subtypes that contribute to proximal and distal segments. CT ablation results in reduction of the differentiated CT1--a subpopulation that maintains proximal identity throughout regeneration, and a delay of distal transformation in the proliferative CT1--a major CT subtypes showing progenitor property. These cellular shifts likely lead to the observed morphological deficits in regenerating limbs after CT ablation. Moreover, differentiated CT1 cells enhanced their interactions with surrounding cell types after CT ablation, to modulate adaptive proliferation in other populations, e.g., muscle stem cells. Our work establishes a tBid-based ablation strategy for functional studies of CT and other cell types in axolotls, and provides direct evidence demonstrating pivotal roles of CT cells in guiding positional memory and segmental patterning during organ regeneration.