Craniofacial development depends on formation and maintenance of sutures between bones of the skull. In sutures, growth occurs at osteogenic fronts along the edge of each bone, and suture mesenchyme separates adjacent bones. Here, we perform single-cell RNA-seq analysis of the embryonic, wild type murine coronal suture to define its population structure. Seven populations at E16.5 and nine at E18.5 comprise the suture mesenchyme, osteogenic cells, and associated populations. Expression of Hhip, an inhibitor of hedgehog signaling, marks a mesenchymal population distinct from those of other neurocranial sutures. Tracing of the neonatal Hhip-expressing population shows that descendant cells persist in the coronal suture and contribute to calvarial bone growth. In Hhip−/− coronal sutures at E18.5, the osteogenic fronts are closely apposed and the suture mesenchyme is depleted with increased hedgehog signaling compared to those of the wild type. Collectively, these data demonstrate that Hhip is required for normal coronal suture development.

Craniofacial development depends on proper formation and maintenance of sutures between adjacent bones of the skull. Within sutures, bone growth occurs through proliferation and differentiation of osteoprogenitors to osteoblasts within the osteogenic front at the edge of each bone, and suture mesenchyme maintains the separation between them. Many genes have been found that cause suture dysgenesis when mutated, particularly of the coronal suture. Such genes are largely studied in isolation, and there is little understanding of the overall transcriptional programs within the suture or of the cell populations in which these programs are organized. We performed single-cell and bulk RNA-seq analyses of the murine coronal suture during embryonic development. Replicate libraries at E16.5 and E18.5 were analyzed. We identified 14 cell type populations when both ages were analyzed together. Seven populations at E16.5 and nine at E18.5 comprised the suture mesenchyme, osteogenic fronts, osteoblasts, and associated cell types. We found a distinct coronal suture mesenchyme population at both ages compared to other neurocranial sutures, which was marked by expression of Hhip, an inhibitor of hedgehog signaling. We examined Hhip-/- mice and found a previously unrecognized coronal suture phenotype. At E18.5, Hhip-/- frontal and parietal bones lack the overlap typical of the WT coronal suture. The osteogenic fronts are closely apposed and the suture mesenchyme is depleted, demonstrating that Hhip function is required for normal coronal suture development. Our transcriptomic approach provides a rich resource for further discoveries of potential normal and abnormal developmental relevance.