Cardiocraniofacial syndromes, including Velocardiofacial Syndrome and DiGeorge Syndrome, are among the most common congenital multiple anomaly syndromes. The signs and symptoms of cardiocraniofacial syndromes are highly variable, but are frequently characterized by congenital heart disease, especially tetralogy of Fallot, and cleft palate. The co-occurrence of birth defects of the heart, face, and structures of the pharynx reflects not only their common embryological origin from a cardiocraniofacial field, but also that the normal development of these structures depends heavily on the neural crest cells (NCCs). The NCCs are a transient, migratory cell population that gives rise to diverse cell types. The cardiac NCCs, a subpopulation of neural crest, is required for proper remodeling of the arch arteries and correct alignment of the arterial pole of the heart. In addition to outflow shortening and abnormal connections of the outflow with the caudal aortic arch arteries, neural crest ablated (NCA) embryos show an excess of FGF8 signaling in the pharynx, and treatment with FGF8b blocking antibody or an FGF receptor blocker rescues arterial alignment. Notably defects in the outflow of the heart occur well before the cardiac NCCs reach the outflow, and therefore the alignment defects appear to be indirectly caused by NCA. In the head the cranial NCCs contribute to cartilage, bone, fascia and tendons associated with craniofacial muscles. Cranial NCCs are known to regulate head muscle patterning and differentiation, even though the NCCs do not contribute to the head muscles themselves. In NCA embryos myogenic cell proliferation is increased resulting in delayed and reduced differentiation of the branchiomeric musculature. It is hypothesized that the increase in myogenic cell proliferation is due to overexpression of Fgf8 in the pharyngeal arches. Altogether this evidence suggests that the NCCs modulate signaling factors, particularly FGF8, in the pharynx. One way in which this could be accomplished is through endocytosis of excess signaling factors. It has been shown that endocytosis and subsequent degradation of FGF8 by target cells can limit the availability of FGF8 ligand to other target cells. The proposed aims will test the hypothesis that the NCCs modulate levels of FGF8 in the pharynx via endocytosis, which is required for normal cardiocraniofacial development. While the NCCs have been found to physically contribute to diverse structures during development, their role in the regulation of signaling is not well understood. Endocytosis of excess signaling factors would be an entirely new described function of the NCCs, and could therefore contribute to our understanding of the many neural crest-related developmental disorders. This project aims to 1) establish if the cardiac NCCs endocytose and degrade FGF8 in the pharynx, 2) determine if endocytosis changes the availability/levels of FGF8 in the vicinity of the cardiac NCCs, 3) determine if blocking endocytosis by the NCCs has an effect on the development of structures in the vicinity of the migrating crest streams, and 4) determine if other signaling molecules are endocytosed by the neural crest. PUBLIC HEALTH RELEVANCE: Birth defects of the heart and face commonly occur together and often require medical intervention. The proposed studies will help determine the mechanism of such birth defects.