PROJECT SUMMARY To enable deformation of the skull during birth and allow for the rapid growth of the brain during early life stages, the bones of the skull roof are connected through fibrous tissue, called sutures. These sutures only fully fuse later in life to form a stable brain case. Early fusion of these fibrous connections is called craniosynostosis and leads to deformations of the skull, which in turn can lead to increased cranial pressure and restricted brain growth. Craniosynostosis is the second most common craniofacial birth defect, occurring with a frequency of 1 in 2100-2500 live birth. Craniosynostosis often occur isolated, without additional malformations, but in 20-25% of cases patients show additional changes like midface hypoplasia and limb abnormalities (syndromic craniosynostosis). Despite the relatively high incidence, the pathomechanisms leading to craniosynostosis are still incompletely understood and surgical removal of the fused tissue is the current standard of care. These surgical procedures bear a risk of high blood loss, and the sutures often re-fuse requiring repeated surgeries. Therefore, there is a critical need to better understand the pathomechanisms leading to craniosynostosis to enable the development of alternative, non-surgical treatment strategies. The zebrafish has become a popular animal model to study human disease and it has been shown that the patterning of the skull roof as well as the regulatory pathways controlling craniofacial development are highly conserved. In a genetic screen for mutants with skeletal phenotypes, we have identified a unique mutant that exhibits features of a severe form of syndromic craniosynostosis with coronal suture fusion, midface hypoplasia, open fontanelles, fewer teeth, deformed and shorter fin rays and mis-pattered scales. To establish a correlation of the observed mutant phenotype to the human disease, we will perform comprehensive phenotypic, mechanistic and genetic evaluation of our novel mutant. In Aim1 we will perform skeletal staining on developmental time series to identify which stages and skeletal elements are affected in combination with analyses of shape and bone density changes through micro computed tomography scanning. In a second set of experiments, we will use in vivo labeling of the growing bones to determine bone formation dynamics of forming skull bones. Finally, we will determine the mechanical properties of the developing sutures and calvarial bones. In the second aim, we will define the cellular and molecular mechanisms underlying the CRS phenotype using immunohistochemistry, transcriptomic and phospho-protein analyses. The third aim will identify the genetic changes responsible for these severe skeletal phenotypes. We will use CRISPR mediated genome editing to functionally test previously identified candidate genes and determine their expression pattern. A detailed and broad description of the phenotypic changes occurring in our craniosynostosis mu...