During fetal development and early childhood, growth of the bony skull accommodates a rapid expansion of the underlying brain. This is accomplished first by progenitors that grow the individual skull bones, and then by stem cells residing in the flexible bony joints called sutures. In a common birth defect called craniosynostosis (1 in 2000 live births), loss of the cranial sutures results in bony fusions that impede brain growth, thus leading to cognitive impairment if left untreated. Surgical correction involves invasive and risky surgeries on infants to break apart the fused bones. Unfortunately, the skull bones often re-fuse, necessitating repeated surgeries. There is thus a critical need to better understand the causes of craniosynostosis, such that we can develop therapies that minimize repeated surgical interventions. In the previous funding cycle, we generated and characterized the first zebrafish model of Saethre-Chotzen Syndrome, which preferentially affects the coronal suture. In so doing, we pinpointed early changes in the growth rates of the embryonic skull bones as a major cause of suture fusions. In this renewal we address three outstanding questions in the field of craniosynostosis. In Aim 1, we investigate the embryonic origins of the suture stem cells that grow and maintain the skull. While suture stem cells have been studied at postnatal stages, whether they arise from progenitors at the tips of growing bones, or alternatively from migrating cells, remains debated. By generating the first single-cell transcriptomes of the developing mouse and zebrafish coronal sutures, we have uncovered conserved embryonic cell types and molecular markers for suture progenitors. Using new lineage tracing tools in mouse and fish, we will test that bone front progenitors expressing ETS-family transcription factors are the origin of suture-resident stem cells. In Aim 2, we investigate how the Saethre-Chotzen genes Twist1 and Tcf12 regulate the transition from bone front progenitors to suture stem cells. Preliminary data reveal that Twist1 and Tcf12 upregulate the Bmp antagonists Grem1 and Noggin during suture formation, suggesting that tighter regulation of Bmp signaling is essential to slow bone growth and prevent fusions. Using mouse conditional genetics and new zebrafish mutants, we will test that direct regulation of Grem1 and Noggin expression by Twist1 and Tcf12 is necessary and sufficient for regulated bone growth and normal suture formation. In Aim 3, we address a central mystery of the craniosynostosis field – why do particular mutations tend to affect only particular sutures? By generating and contrasting new zebrafish models for 11 coronal and 7 midline craniosynostosis genes, we will test whether coronal suture formation is particularly sensitive to mutations that perturb the rate of bone growth. To do so, we will make use of new zebrafish transgenic reporters that allow quantitative in vivo measurements of osteoblast addition and sutu...