Fig. 4: Dlx5-creER⁺ early perichondrial cells regulate marrow formation in response to Hedgehog signaling.

a–f CellChat intercellular communication analysis of the E13.5 Col2a1-cre-marked chondrocyte and perichondrial cell scRNA-seq dataset. a Outgoing and incoming interaction strength of signaling pathways. Each circle represents the number of interactions a cluster participates in. (Left): Cluster 1 has a low tendency to send signals but a high tendency to receive signals in overall pathways. (Right): Cluster 1 exhibits a high propensity to both send and receive signals via the Hh pathways. Dotted box: UMAP-based plot and cluster labels of the E13.5 scRNA-seq data. b Feature plots of Ihh and Ptch1. Violet: high expression, yellow: low expression. c Violin plots displaying the expression distribution of Ihh and Ptch1 in each cluster. d Chord diagram shows the signals linking Cluster 1 and other clusters via the Hh pathway. e Heatmap represents the relative importance of each cluster. Dark green: higher importance, white: lower importance. Cluster 1 is scored as the dominant sender, receiver, and influencer of the Ihh-Ptch1 signaling pathway. Cluster 5 is measured as a dominant receiver. f Hierarchical plot showing the intercellular interactions of the chondrocyte-perichondrial cell lineage via the Ihh-Ptch1 signaling pathway. g Functional analysis of Hedgehog signaling in Dlx5-creER⁺ early perichondrial cells and Fgfr3-creER⁺ fetal chondrocytes in bone marrow development. Left panels: Histological images of Dlx5-creER; Ptch1^fl/+; R26R^tdTomato (Dlx5-Control), Dlx5-creER; Ptch1^fl/fl; R26R^tdTomato (Dlx5-Ptch1 cKO), Fgfr3-creER; Ptch1^fl/+; R26R^tdTomato (Fgfr3-Control), Fgfr3-creER; Ptch1^fl/fl; R26R^tdTomato (Fgfr3-Ptch1 cKO), femur at E18.5 (pulsed at E12.5). Scale bar: 500 μm. n = 8 (Dlx5-Control), n = 11 (Dlx5-Ptch1 cKO), n = 10 (Fgfr3-Control), n = 8 (Dlx5-Ptch1 cKO) mice per each group. Right panels: quantification of whole femur length (top left), bone marrow length per total bone length (middle left), distal cartilage length per total bone length (bottom left), Col1a1-GFP⁺ trabecular area per total bone marrow area (top right), number of chondrocytes per column (middle right) and total number of columns in growth plate (bottom right). n = 8 (Dlx5-Control), n = 11 (Dlx5-Ptch1 cKO), n = 10 (Fgfr3-Control), n = 8 (Dlx5-Ptch1 cKO) mice per group. Two-tailed, Mann–Whitney’s U test. Data are presented as mean ± s.d. Exact P value is indicated in the figures. Source data are provided as a Source Data file.