Fig. 5: scRNA-seq captures various subtypes of osteoblasts at the coronal suture.

a Lineage analysis and (b) pseudotime analysis of the osteoblast subset using Monocle 3. The gray lines in (a) indicate the lineage relationship between cells in the trajectory. In (b) cells further along the single-cell trajectory are indicated by progressively lighter colors. c Feature plot of selected markers within the osteogenic subset. d Expression plots of selected genes across pseudotime. e, j Feature plots of genes validated by in situ experiments. f–i, k Combinatorial in situ analysis of coronal sutures for indicated markers at E15.5. Sp7 marks the frontal (F) and parietal (P) bones. f Ifitm5 and Sost. Arrows indciate Sp7⁺ expression in presumptive newly formed osteoblasts, and the arrowhead Sost expression. g Lef1 and Inhba. Arrowheads mark Lef1⁺/Inhba⁺ expression near bone tips. h Podnl1 and Mmp13. Arrowheads mark Podnl1⁺/Mmp13⁺ expression in periosteum distant from suture. i Erg and Pthlh. Arrowhead marks Erg⁺/Pthlh⁺ expression in suture mesenchyme. k Six2 and Erg. Arrowhead marks Six2⁺/Erg⁺ expression in suture mesenchyme. l, m Immunostaining for Sp7 and tdTomato in a Six2:Cre;Ai9 coronal suture at E16.5 (n = 3) and DAPI staining of a Six2:Cre;Ai9 coronal suture at 3 weeks (n = 4). Asterisk, labeled cells above the frontal bone. Arrows, coronal suture. Arrowheads, osteocytes. Dashed lines outline bones. n Images of the coronal suture (cs, arrowhead) and adjacent bones from a P7 Six2:CreERT2; R26-CAG-LSL-Sun1-sfGFP-myc mouse treated with tamoxifen at P0 (n = 3). Converted cells display strong nuclear GFP; the diffuse green signal in the bones is autofluorescence. Top views are from above the cranium, with digital cross-sections through the suture shown below. o Model summarizing gene expression patterns within the developing bones and coronal suture mesenchyme. In situs were performed in biological triplicate. Scale bars = 50 µm; images (f–i) are at the same magnification.