Type E endothelial cells orchestrate embryonic skeletogenesis via chondrogenic priming of skeletal progenitors

Skeletal stem/progenitor cells (SSPCs) are pivotal orchestrators of embryonic skeletogenesis, underlying chondrogenesis and osteogenesis, however, the molecular determinants governing SSPC functionality remain elusive. We performed single-cell RNA sequencing (scRNA-seq) to delineate cellular heterogeneity within the developing limb bud and to identify candidate cellular markers. The spatiotemporal distribution of target cell populations was further examined via immunofluorescence staining. Fluorescence-activated cell sorting (FACS) was used to isolate endothelial cells and SSPCs based on surface markers. Functional properties of these cells were assessed using in vitro assays, and direct co-culture experiments were conducted to evaluate intercellular communication and underlying molecular pathways. We delineate a CD140a + PDPN + SSPC population characterized by robust self-renewal and multipotency, peaking in abundance at embryonic day 14.5. Concomitantly, we resolved the heterogeneity within endothelial cell (EC) populations during embryonic angiogenesis, identifying a distinct subpopulation exhibiting high Pecam1 and low Emcn expression (recapitulating type E). Critically, SSPCs manifested enhanced chondrogenic differentiation potential and type II collagen synthesis when co-cultured specifically with type E ECs, highlighting an indispensable role in endochondral ossification during long bone formation. Mechanistic intercellular crosstalk analyses demonstrated that BMP signaling plays an essential role in modulating type E endothelial cell-mediated regulation of SSPC potency through the coordinated actions of transcription factors ID1, MSX2, and SOX9. Notably, pharmacological inhibition of BMP signaling abolished the pro-chondrogenic and pro-osteogenic enhancement conferred by type E ECs upon SSPCs. These findings uncover a fundamental mechanism of long bone development mediated by CD140a + PDPN + SSPCs and modulated by type E ECs. This reciprocal, bipotent coupling between skeletogenesis and angiogenesis provides a conceptual framework for understanding skeletal development and homeostasis, proffering novel therapeutic avenues for associated pathologies. By elucidating the regulatory function of type E endothelial cells in orchestrating chondrogenic priming during embryonic skeletogenesis, this study unveils potential therapeutic strategies for bone regeneration through targeting the vascular network.