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 Lgr5Pthlh 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 crestderived skeletal stem/progenitor cell regulation and may inform cartilage regenerative strategies. 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).骨骼与滑膜关节的发育涉及不同的骨骼干/祖细胞群。然而，神经嵴源性颞下颌关节（temporomandibular joint, TMJ）的骨骼干/祖细胞谱系动态及成骨程序仍尚不明确。本研究对胚胎第16.5天（E16.5）、出生后第0天（P0）及13周龄的小鼠下颌软骨开展单细胞RNA测序（single-cell RNA sequencing, scRNA-seq），以构建骨骼干/祖细胞群的高分辨率图谱。覆盖下颌软骨的软骨外膜中存在表达Dlx5的Lgr5Pthlh细胞，提示该区域存在膜内成骨过程。与之相对，软骨内膜中存在此前未被表征的Crabp1亚群，该亚群表达Nrg1、Lsamp、Tac1与Igfbp5，其分子特征与倾向于软骨内成骨的早期软骨祖细胞相符。上述结果揭示了兼具膜内成骨与软骨内成骨的成骨方式空间分区，并阐明了颞下颌关节发育背后的转录调控机制。本研究的发现为理解神经嵴源性骨骼干/祖细胞的调控机制提供了理论框架，或可为软骨再生策略的研发提供参考。为表征发育过程中颞下颌关节的细胞组成，本研究对三个发育阶段的C57BL/6小鼠下颌髁突软骨开展了单细胞RNA测序：胚胎第16.5天（E16.5，上关节腔形成阶段）、出生后第0天（P0，颞下颌关节初始功能行使阶段）及13周龄（13w，成年阶段）。