A Single-Cell Transcriptomic Atlas Identifies Hierarchical Skeletal Progenitors and Zonal Patterning in Mouse Mandibular Cartilage

Skeletal and synovial joint development involves distinct skeletal stem/progenitor populations. However, in neural crest-derived temporomandibular joint (TMJ), skeletal stem/progenitor lineage dynamics and ossification programs remain poorly defined. Here, we performed single-cell RNA sequencing (scRNA-seq) on mouse mandibular cartilage at embryonic day 16.5 (E16.5), postnatal day 0 (P0), and 13 weeks to construct a high-resolution atlas of skeletal stem/progenitor populations. The outer perichondrium lining the mandibular cartilage contained Lgr5?Pthlh? cells expressing Dlx5, suggesting intramembranous ossification. In contrast, a previously uncharacterized Crabp1? subpopulation within the inner perichondrium expressed Nrg1, Lsamp, Tac1, and Igfbp5, consistent with early cartilage progenitors biased toward endochondral ossification. These results reveal a spatial division of ossification modes with both intramembranous and endochondral growth and clarify the transcriptional regulation underlying TMJ development. Our findings provide a framework for understanding neural crest–derived skeletal stem/progenitor cell regulation and may inform cartilage regenerative strategies. Overall design: To characterize the cellular composition of the TMJ during development, we performed scRNA-seq on mandibular condylar cartilage from C57BL6 mice at three developmental stages: embryonic day (E) 16.5 (E16.5, formation of superior joint cavity), post-natal day (P) 0 (P0, initial use of TMJ), and 13-week-old (13w, adults).