Fig. 2: Single-cell RNA sequencing of the intact calvaria within Mkx reporter animals.

Mkx^tdT animals (male and female, 8-week old, n = 3) were administered TM for three consecutive days, and subjected to sacrifice after 5 days from the last dosage. The frontal and parietal bone including coronal and sagittal sutures was micro-dissected, dissociated, and subjected to scRNA-seq. A Uniform Manifold Approximation and Projection (UMAP) plot of total cells isolated from intact calvaria. Hemato hematopoietic cell, EC endothelial cell, Peri pericyte. B UMAP of mesenchymal lineage cells. C Violin plots of marker gene expression for subclusters. D UMAP plot of Mkx expression and tdTomato reporter activity. E UMAP plot of subclusters of suture cells only. F UMAP plot of Mkx expression and tdTomato reporter activity across subclusters. G Selected GO terms and KEGG pathways enriched in each subcluster. H Module score of progenitor cell markers and tenocyte markers across 4 subclusters (top) and in Mkx⁻ and Mkx⁺ cells from all subclusters (bottom). The tdTomato expression >2.5 was considered as Mkx positive. I Heatmap showing selected tenocyte-associated genes among tdT⁻ and tdT⁺ cells in suture cells. J Trajectory analyses of cell clusters showing the distribution of identified 4 subclusters and pseudotemporal cell ordering along tdTomato trajectories. K Tenocyte-associated genes and Mkx gene expression over pseudotime. L Immunostaining of TNMD and tdTomato reporter activity in coronal and sagittal suture (n = 4). GFP reporter activity not shown. Scale bar: 20 μm.