Early determination of the dorsal-ventral axis in endochondral ossification in mice

Endochondral ossification is a highly coordinated process involving distinct progenitor cell populations within the mesenchymal condensation and subsequent cartilage anlage and perichondrium, all of which drive skeletal formation. Cell-type specific lineage tracing conducted to understand fetal bone development has revealed various fates of early skeletal cells. However, the underlying continuous and precise cellular dynamics of fetal skeletal cells, particularly along the dorsoventral axis, remain unclear. Here, we show that spatiotemporally specific skeletal progenitor cells in the early developmental stage contribute to the dorsal-ventral axis in a manner that is strictly determined during initial developmental stages. Lineage-tracing experiments using Fgfr3-creER and Dlx5-creER lines revealed that Fgfr3+ cells in mesenchymal condensation exclusively contributed to hypertrophic chondrocytes and the dorsal side of the resting and proliferating zones within the cartilage anlage. These cells made dorsal-restricted contributions to skeletal development, including growth plate chondrocytes, trabecular and cortical osteoblasts, and bone marrow stromal cells. Functional ablation of Fgfr3+ cells using the Rosa26iDTA (inducible diphtheria toxin fragment A) allele during the mesenchymal condensation stage caused severe disruption in long-bone development, underscoring its indispensable role in initiating skeletal growth. Collectively, these findings suggest that the condensation stage is pivotal for the formation of skeletal progenitors and dorsoventral patterning during bone development. Understanding these mechanisms will provide insight into skeletal growth disorders and therapeutic strategies for bone regeneration.