Summary Embryonic development is a series of tightly regulated events that build a single cell into a complex organism. A key milestone in early embryonic development is gastrulation, when the body plan is first shaped by the behaviors of thousands of individual cells. However, how these cells communicate to ensure proper morphogenesis is not completely understood. Mesoderm and neuroectoderm (NE, future neural tube) tissue morphogenesis is driven by convergence and extension (C&E), when cells intercalate to simultaneously narrow and elongate the body axis and bring the neural folds together to ensure proper neural tube closure. Disruptions in these highly conserved cell movements can result in neural tube defects that affect many pregnancies worldwide. Nodal is a well conserved signaling pathway that is active during gastrulation and most well-known for the role it plays in mesoderm and endoderm specification in vertebrates. However, little is understood about the role of Nodal in C&E morphogenesis independent of mesoderm formation, particularly in NE. By utilizing zebrafish ex vivo and in vivo models, I will study the role of Nodal specifically in morphogenesis independent of its better-known function during tissue specification. To examine the role of Nodal underlying NE morphogenesis, I can independently analyze NE extension using ex vivo zebrafish blastoderm explants, naïve clusters of embryonic cells that form all three germ layers and undergo C&E morphogenesis in response to exogenous Nodal signaling. An early peak of Nodal signaling in explants promotes mesoderm extension and specification. However, preliminary data from our lab demonstrated that a relatively delayed peak of Nodal signaling promotes NE- specific C&E morphogenesis that is independent of its role in mesoderm specification. This suggests that the temporal regulation of Nodal regulates NE-specific morphogenesis. Based on my promising preliminary data, I hypothesize that Nodal signaling activates a novel, temporally regulated, NE-specific transcriptional program that drives C&E morphogenesis independent of mesoderm specification. To test this hypothesis, I propose 3 aims: Aim 1 will investigate the role of temporal Nodal signaling dynamics in NE-specific morphogenesis using optogenetic Nodal receptors to precisely manipulate signaling in embryonic explants. In Aim 2, I will restore Nodal signaling specifically within the NE of Nodal-deficient zebrafish embryos using innovative transgenic lines to determine whether Nodal can drive in vivo NE extension in the absence of mesoderm. Finally, in Aim 3 I will perform over expression and knock-down of temporally regulated candidate genes to determine if they are drivers of NE-specific extension both explants and in vivo. This proposal will define the foundational knowledge on the primary role of Nodal in NE-specific morphogenesis, disruptions in which may contribute to neural tube defect prevalence and severity.