Orofacial clefting is a common disease that causes significant morbidity for the affected individual and a major burden to families and society. The etiology of orofacial clefting is complex, but genetic factors contribute significant risk. To date, scores of genes have been identified where rare variants cause rare syndromic forms of orofacial clefting, and DNA variants at 24 loci have been associated with the common non-syndromic form of orofacial clefting. Despite these efforts, less than half of the heritable risk has been identified. In addition, the pathogenic variant has yet to be determined at most of the 24 associated loci. New approaches are needed to identify these genetic risk factors. Since most of the associated loci are located between genes, it is hypothesized that the pathogenic allele is in a regulatory element. Thus, the new approaches should not only help find new candidate genes but they should also find the elements that regulate the expression of those genes. In this proposal, we will use a triumvirate approach to find new candidate genes, the DNA elements that regulate their expression and the transcription factors that bind to those regulatory elements. In total, analysis of these three datasets will yield a gene regulatory network for a target tissue. The target tissue for this proposal is periderm. Periderm is the most superficial layer of squamous epithelial cells that covers the entire embryo, including the oral cavity. Recent studies from human and animal models identified 19 genes that are essential for periderm development, and of these, 10 have DNA variation that causes or increases risk for orofacial clefting. Despite its significant role in palate development, little is known about periderm at the molecular level. The immediate goals of this proposal are to i) determine the gene regulatory network for periderm and ii) apply that new knowledge to identify alleles within the 24 associated loci that perturb the gene regulatory network for periderm. The longterm goals of this proposal are to identify genetic pathways that are essential for periderm differentiation and to identify the actual pathogenic DNA variants that perturb these pathways. This new knowledge will help determine recurrence risk and provide opportunities to develop new rational therapies. In particular, since periderm only exists in the embryo and since it is the most superficial layer of the embryo, it is an ideal target for in utero therapies, when and where orofacial clefting actually occurs. We have two parallel specific aims. In Aim 1 we will determine the gene regulatory network for periderm differentiation in zebrafish by systematically perturbing the system. In Aim 2, we will use a parallel approach to determine the gene regulatory network for mouse periderm. It is essential to use both animal models because while studies of zebrafish periderm led to the identification of genes required for development of the lip and palate, the ?periderm? between these two clades has a different embryonic origin and it is possible aspects of the gene regulatory network are different. Our project thus exploits the power and tractability of the zebrafish, and the human-like origin of periderm in the mouse.