During embryonic development, mesenchymal cell populations from the cranial neural crest diversify into a wide range of connective tissues including teeth, cartilage, bone, dermis, tendon, and perivascular tissue. During this process, cells coordinate rates of cell division and differentiation in order to form these structures with appropriate shape and sizes. Disruptions to this process can result in craniofacial anomalies, which rank among the most common birth defects, and represent a significant social burden. These include common birth defects like facial clefts, craniosynostoses, and skeletal dysplasias (together affecting ~1/600 newborns) are caused by improper development of bones and cartilage in the embryo. Such diseases are incurable in part because of a limited understanding of molecular mechanisms disrupted in congenital disease states. As a result, etiological explanations are missing for most disease-related single nucleotide polymorphisms (SNPs), especially those in noncoding regions. Using a novel mouse model to perform high-throughput clonal lineage tracing combined with multiplexed gene perturbations, we are investigating the genetic and epigenetic bases of cell fate decisions underlying mesenchymal tissue shaping in normal and altered craniofacial development. We will test the hypothesis that orphan SNPs affect gene regulatory cascades that balance cell proliferation with cell differentiation tempo. Specifically we will investigate known single-gene diseases to identify face-related SNPs within regulatory elements both upstream and downstream of each gene in facial mesenchyme. As an example, we will study mutations in a known enhancer sequence upstream of SOX9, which has previously been determined to be responsible for the Pierre Robin Sequence disorder. Further, we will utilize recently-published epigenetic atlases of embryonic development across model organisms to identify conserved patterns of predicted enhancer activity, in order to guide future experimental validation studies. Overall, the project will reveal the structure of a regulatory feedback network involving chromatin architecture and cell signaling to balance proliferation with fate decisions of stem cells in vivo.