Project Summary/Abstract. Neural crest cells (NC) are unique to vertebrates, arise early in development, emigrate from the dorsal aspect of the neural tube, and differentiate into a plethora of derivatives throughout the body, contributing neurons and glia of the peripheral nervous system, melanocytes, and craniofacial bone and cartilage amongst other derivatives. Failed NC development is associated with a large number of conditions known as Neurocristopathies, which include common orofacial clefts, aggressive cancers and rare syndromes. The long term goal of this research program is to advance our understanding of the earliest signaling events responsible for the formation of the NC. Currently signaling by WNT, FGF and BMP are amongst a few of key inputs recognized to be critical for the formation of NC, yet our understanding of the molecular mechanisms by which they execute their effects remain limited. To improve our knowledge of this events, it is critical to identify the effectors modulated by these pathways and to characterize their effects. The objectives of this proposal are: 1) to establish and characterize for the first time the role of TGFb /SMAD2-SMAD3 signaling during early facets of NC formation, prior to their allocation into the neural tube, migration or differentiation; and 2) to identify the effectors of the WNT and/or TGFb and characterize their role during early NC formation. Our Hypotheses are that the TGFb /SMAD2-SMAD3 signaling is required to launch the earliest events in NC formation, and that a combination of TGFb and WNT responsive molecules direct the specific acquisition of the NC lineage. To address these hypotheses our proposal is guided by 3 specific aims: 1) to demonstrate the requirement and contribution of the TGFb /SMAD2-SMAD3 signaling during early facets of NC formation, 2) to identify novel candidate effectors of WNT and/or TGFb and characterize their specific contributions to the acquisition of the NC fate, and 3) to assess their function in vivo in a vertebrate embryo. This innovative proposal takes advantage of a robust model of human NC formation based in pluripotent stem cells and classic chick embryology to, for the first time address the possible contribution of TGFb signaling (not BMPs) during this process and pursues an integrative approach addressing the combinatorial signaling of WNT and TGFb. The proposal is significant because the gains in this area will have impact in key aspects of fate acquisition and differentiation applicable to other cell types, and should improve our capacity to manage the many pathologies associated with NC.