Project Summary The Fibroblast Growth Factor (FGF) signaling pathway consists of eighteen FGF ligands and four FGF receptors (FGFRs), members of the receptor tyrosine kinase (RTK) family. FGF signaling is integral to mammalian development and is active throughout numerous tissues and stages in the developing embryo. Additionally, FGF signaling is a major coordinator of craniofacial morphogenesis. Misregulation of FGF is implicated in a wide range of congenital disorders, such as cleft lip or palate (CL/P). Numerous studies have examined the role of individual ligand-receptor combinations necessary for different aspects of development, as well as the various intracellular signaling pathways that are activated downstream of FGF signaling. FGFRs have been shown to recruit various adaptor proteins that facilitate the propagation of intracellular signals such as the ERK1/2 and PI3K pathways. Previous work by the Soriano lab has shown that FGF controls craniofacial development through the combinatorial recruitment of various effectors that activate multiple downstream signaling pathways. However, there are still gaps in the complete picture of how FGF signaling is relayed downstream of receptor activation, as abrogating the binding of known effectors does not replicate the phenotype of Fgfr null mutations. The aim of this proposal is to identify these missing effectors. I will utilize two complementary approaches to identify novel effectors of FGF signaling: a hypothesis-driven candidate approach and an unbiased protein screen through stable isotope labeling by amino acids in cell culture (SILAC) coupled with mass spectrometry (MS). To analyze the potential roles of these effectors, I will utilize in vivo and in vitro techniques to assay genetic interactions and cellular functions. I will quantify cellular proliferation, death, and migration, and changes in downstream signaling dynamics, both in the presence of wild-type or signaling mutant backgrounds. From those targets that exhibit positive evidence of downstream interaction with FGFRs, we will select critical genes for further biochemical and genetic analysis. Utilizing a neural crest specific Cre line, Wnt1Cre2, we will examine the role of each novel effector in craniofacial development on their own, or in conjunction with Fgfr null or signaling mutant backgrounds to verify in vivo genetic interaction. The innovative studies proposed here aim to provide novel insight into the mechanism by which FGFR activation propagates intracellular signals to coordinate the complexity of craniofacial development. Our research strategy will utilize an array of existing reagents, alongside newly developed strains, to uncover missing aspects of FGF signaling and provide new avenues for therapeutic intervention and prevention of congenital craniofacial disorders.