Fig. 4: Molecular basis of human iliac perichondral ossification.

a, RNA velocity maps overlaid on UMAPs of human ilium and adjacent soft tissue cells at E54, E57 and E67. Arrows depict cell trajectories. Colour scheme as in Fig. 2. b, Expression of selected marker genes (log-transformed): RUNX2 (perichondrium/osteoblasts), SP7 (osteoblasts), and FOXP1, VEGFA and VEGFB (blood vessels) from Visium spatial analysis. Red dashed lines in the segmented model on the left depict the anatomical position of the transverse section. c, Single-cell multiomics data (accessibility is depicted as peaks; gene expression is depicted as violin plots on the right) at E54 and E57, focusing on the RUNX2–SUPT3H locus. The blue band highlights a putative enhancer region intersecting with a HAR. d, LacZ reporter assay in transgenic mice, showing the anatomical location of RUNX2 enhancer expression in iliac perichondrium at E14.5 (blue staining highlighted by arrow), and no detectable expression in embryos carrying the orthologous chimpanzee RUNX2 enhancer sequence or in the negative control (wild-type) pelvic girdle. Fe, femoral head; Pe, perichondrium. e, Enhancer-driven GRN for human chondrocyte populations at E53, inferred using SCENIC+ and visualized with Cytoscape. Hexagons (orange) represent eRegulons (transcription factors) and circular nodes represent genes containing transcription factor binding sites. Green circles highlight genes identified through GO enrichment analysis as involved in human pelvic girdle abnormalities. Nodes outlined in red indicate genes associated with HAR intersections.