Fig. 3: Mkx⁺ cells contribute to calvarial bone repair.

A–H Single-cell RNA sequencing of calvarial defects within Mkx^tdT reporter mice in relation to intact calvaria. A UMAP plot of cell clusters all time points (top) and across time points after defect creation (bottom). N = 3 mice/time point. Intact: 8321 cells (data also shown in Fig. 2), Day 7: 6699 cells, Day 28: 7065 cells. B UMAP plot of Mkx gene expression (top) and tdTomato reporter activity (bottom) across time. C Pseudotemporal cell ordering along differentiation trajectories. Pseudotime is depicted from red to purple. The tdT negative state (appearing in gray) was not included for downstream analyses. D Mkx^tdT cell distribution over pseudotime. E Mkx gene expression over pseudotime. F GO terms enriched in Mkx^tdT cells on day 7. G Module score of cell migration, osteogenesis, and tenocyte-associated genes in Mkx^tdT cells across time points. A Kruskal–Wallis test with Dunn’s multiple comparison was used to determine differences between groups. H Schematic for calvarial bone defect with or without BMP2 treatment in Mkx^tdT reporter mice. I μCT reconstructions of the defect area without or with BMP2 treatment. The margins of original defect are indicated by dashed white. J–M Representative tile scans and high-magnification images of Mkx reporter activity in the calvarial defect site J, K without or L, M with BMP2 treatment at 28 d post-defect. Reporter activity appears red and green, nuclear counterstain appears blue. Dashed white lines at high-magnification images indicate healing bone edge. Scale bar: 100 μm (J, L) or 20 μm (K, M). N Osteocalcin (OCN) immunostaining and O CD31 immunostaining within the healing area on day 28 after defect. GFP reporter activity not shown in (M, O). Scale bar: 20 μm. n = 3 for (I–O).