The vertebrate central nervous system (CNS) is patterned by environmental signals that regulate neuronal phenotype and function. Understanding the mechanism of cell differentiation in the nervous system is vital for our ability to diagnose disease and design regenerative therapeutic treatments. One important signal in the developing CNS is produced by Wnts, which regulate transcription through the downstream effectors ?-catenin and Lef/Tcf. We have identified a specific role for Wnt signaling, acting through Lef1, in the development of zebrafish GABAergic hypothalamic neurons. Our data indicate that Lef1 continues to be expressed in these neurons after they become postmitotic, suggesting that Wnt signaling pathway may control the expression of particular genes critical for their function. In this study we will test the hypothesis that Lef1 regulates the development of posterior hypothalamic neurons by activating genes that regulate their specification, differentiation, and survival. In the process we will establish the zebrafish hypothalamus as a model for vertebrate neurogenesis, and we will discover new targets of Lef1 function in the developing CNS. First, we will test whether Wnt signaling and Lef/Tcf-mediated transcription are required for the proper differentiation of postmitotic GABAergic neurons in the hypothalamus. Using transgenically-expressed pathway inhibitors we will assess the temporal and spatial requirements for Wnts and Lef/Tcf factors during the period of neurogenesis. These experiments will determine whether target gene regulation by these molecules participates in a conserved pathway of GABAergic neurogenesis and determines the phenotype of a specific hypothalamic cell population. Second, candidate target genes will be tested as direct transcriptional targets of Lef1 signaling by chromatin immunoprecipitation analysis. We will also screen for novel Lef1 targets by constructing a library of precipitated DNA fragments from hypothalamic tissue, followed by secondary confirmation of specificity. The expression of potential target genes will then be tested for in vivo regulation by Lef1 using in situ hybridization. These experiments will yield a picture of Lef1 targets in vivo in the hypothalamus and will help characterize the role of the ?-catenin signaling pathway in hypothalamic neurogenesis. Overall, these studies will allow us to understand how genes are regulated by environmental signals during hypothalamic neurogenesis, ultimately resulting in the correct function of this structure. The mechanisms we uncover will help in the treatment of CNS disorders and diseases. PUBLIC HEALTH RELEVANCE: The hypothalamus is an important regulatory center in the brain that controls hormone release and behavior. Little is understood about the developmental signals governing the production of hypothalamic neurons. In this proposal we will determine the role of the Wnt signaling pathway in hypothalamic neurogenesis, and identify target genes of the Wnt effector Lef1 that underlie this process.