Single-cell analysis identifies a key role for Hhip in murine coronal suture development

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. We performed single-cell RNA-seq analysis of the embryonic, murine coronal suture. Seven populations at E16.5 and nine at E18.5 comprised the suture mesenchyme, osteogenic cells, and associated populations. Expression of Hhip, an inhibitor of hedgehog (HH) signaling, marked a mesenchymal population distinct from other neurocranial sutures. At E18.5, Hhip-/- coronal osteogenic fronts were closely apposed and HH signaling was increased throughout the depleted suture mesenchyme compared to WT, demonstrating that Hhip is required for normal coronal suture development. Tracing of the neonatal Hhip-expressing population showed that descendant cells persisted in the coronal suture and contributed to calvarial bone growth. Our transcriptomic approach provides a rich resource for insight into normal and abnormal development. Laser capture microdissection (LCM) was performed on fresh frozen sections of the coronal (C), lambdoid (L), sagittal (S), and frontal (IF) sutures of C57BL/6J mice at embryonic stages E16.5 and E18.5. Five biological replicates of the suture mesenchyme (SM) and osteogenic front regions were obtained from each suture. RNA was isolated from each LCM sample, converted to RNA-Seq libraries, and sequenced on the Illumina HiSeq 2500 platform. For each suture and time point, pairwise comparisons were then performed for each osteogenic front region compared to suture mesenchyme to identify differentially expressed genes between front and mesenchymal regions.