ABSTRACT Skull development requires the proper function of sutures, comprised of the edges of adjacent bones (osteogenic fronts, OFs) and intervening suture mesenchyme (SM). Suturogenesis is a complex and intriguing process, particularly for the coronal suture between the frontal and parietal bones of the calvaria. Coronal suturogenesis involves the migration of osteoprogenitors through ectocranial mesenchyme to the OFs at early embryonic stages and maintenance of the SM to preserve an open suture that develops a stem cell niche postnatally. Misregulation of suturogenesis is a significant source of human pathology, such as wider sutures or craniosynostosis (CRS), the premature fusion of sutures. CRS can adversely affect neurological development and requires corrective surgery. The coronal suture is the most frequently fused suture in syndromic CRS, which is generally thought to result from an imbalance between osteoprogenitor induction, proliferation, and differentiation. The cell populations involved are poorly defined in terms of their transcriptional signatures and spatial organization, hindering understanding of the effects of genes mutated in coronal CRS. Understanding the pathogenesis of CRS has positive implications for human health in providing insight toward treating dysgenesis. In this proposal we will analyze murine coronal suturogenesis in three increasingly focused Aims: 1) define the organization of transcriptionally-characterized cell populations; 2) establish the roles of a newly identified SM population; and 3) investigate the role of the hedgehog (HH) signaling pathway that when altered can result in CRS in humans and mice. For Aim 1, we will generate and integrate spatial transcriptomics and single-cell (sc)RNA-seq analyses between embryonic day (E)12.5-E18.5 in wild type (WT) mice and two models of syndromic coronal CRS. From this, we will generate a detailed 4D representation of the transcriptional signatures and spatial organization of cell populations and identify potential mechanisms of dysgenesis in coronal CRS. For Aim 2, by scRNA-seq analysis we have identified a discreet population within the SM that separates the overlapping frontal and parietal bones of the coronal suture. We will use targeted genetic manipulations to ablate and fate map this population, and perform lineage differentiation and calvarial defect assays. These experiments will determine the function of this SM population as a barrier to prevent fusion of adjacent bones and their potential contribution to suture stem cells. For Aim 3, we have identified a novel coronal suture phenotype in mutants of Hhip, a HH inhibitor highly expressed in the SM population studied in Aim 2. We will characterize the Hhip-/- phenotype and combine targeted genetic manipulations of the HH pathway in the SM population with RNA-seq analysis to comprehensively determine the roles of HH signaling at the level of both transcriptional programs and cell populations. We will fate m...