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Connective tissue (CT) constitutes a major component of the limb and serves as the primary reservoir of positional information during regeneration. However, the role and extent of CT cell contribution remain largely undefined. Here we develop an inducible Cre\u2010LoxP\u2010mediated\n                    <jats:italic>tBid<\/jats:italic>\n                    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 regeneration stages 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, supporting an essential role for CT cells in determining the positional identity of developing and regenerating limb structures. Further single\u2010cell RNA\u2010sequencing (scRNA\u2010seq) reveals a progressive proximal\u2010to\u2010distal transition among CT cells and identifies distinct CT subtypes contributing to proximal and distal segments. CT ablation reduces differentiated CT1\u2014a subpopulation maintaining proximal identity throughout regeneration, and delays distal transformation in proliferative CT1 progenitors. These cellular shifts likely explain the observed morphological deficits. Moreover, differentiated CT1 cells enhance interactions with surrounding cells after CT ablation to modulate adaptive proliferation in other populations (e.g., muscle stem cells). Our work establishes a\n                    <jats:italic>tBid<\/jats:italic>\n                    \u2010based ablation strategy for functional studies of CT and other cell types in axolotls and demonstrates pivotal roles of CT cells in limb regeneration.\n                  <\/jats:p>","DOI":"10.1002\/advs.202524339","type":"journal-article","created":{"date-parts":[[2026,5,27]],"date-time":"2026-05-27T15:05:36Z","timestamp":1779894336000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["tBid\u2010Mediated Genetic Ablation of Connective Tissue Cells Reveals Their Key Regulatory Function During Limb Regeneration in Axolotls"],"prefix":"10.1002","volume":"13","author":[{"given":"Yan","family":"Hu","sequence":"first","affiliation":[{"name":"Guangdong Cardiovascular Institute Guangdong Provincial People's Hospital Guangdong Academy of Medical Sciences  Guangzhou China"},{"name":"Department of Pathology Guangdong 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