The enteric nervous system (ENS) is the intrinsic nervous system within the muscle walls of the entire gastrointestinal (GI) tract. The ENS consists of a series of interconnected enteric ganglia and is responsible for mediating peristalsis, water balance, and regulation of local GI hormonal secretions. While much recent progress has been made in understanding how the ENS functions, far less is known about its formation during early embryological development. During development, neural crest cells (NCCs) that contribute to the ENS immigrate into the primitive foregut and navigate caudally along the gut length to its distal end, during which time they are referred to as enteric neural crest cells (ENCCs). A failure of ENCCs to fully populate the gut and differentiate into neurons leads to improper ENS development and results in the congenital disease, Hirschsprung Disease, which is characterized by a lack of enteric ganglia along variable regions of the GI. While ENCCs migrate along the primitive gut, they receive various extrinsic signals—such as GDNF, Retinoic Acid and Sonic Hedgehog—from the surrounding gut tissues and neighboring ENCCs that promotes their proliferation, migration, differentiation and multipotency. Indeed, while the field has characterized some of the signaling pathways that are important for colonization of the gut by the ENCCs, the gene regulatory and molecular mechanisms that link extrinsic signals to ENCC migration and neuronal specification is poorly elucidated. My strong in vivo imaging-based and transcriptomic preliminary data using zebrafish embryos indicates that the Bone Morphogenetic Protein (BMP) signaling pathway is highly enriched in migrating ENCCs and is required in a temporally-specific manner for their colonization and neuronal differentiation along the gut. Zebrafish embryos afford the unique opportunity to readily study development of the ENS due to their transparent, external, and rapid development. This proposal tests the hypothesis that BMP signaling directly regulates colonization of the gut by ENCCs and their timely differentiation to dictate the establishment of ENS. The specific aims of this proposal are 1. To map the spatiotemporal expression of BMP pathway members and BMP pathway activity in the developing zebrafish ENS, 2. Uncover the cellular mechanisms by which BMP signaling controls ENCC gut colonization. The results of these experiments will elucidate the molecular mechanism driving ENS development and expand the gene regulatory network that modulates the colonization of the GI tract by neural crest cells.