Wnt targets in hypothalamic neurogenesis

描述（由申请人提供）：脊椎动物中枢神经系统（CNS）是由调节神经元表型和功能的环境信号形成的。了解神经系统中细胞分化的机制对我们诊断疾病和设计再生治疗方法的能力至关重要。发育中的中枢神经系统中的一个重要信号是由wnt产生的，wnt通过下游效应物调节转录。-catenin和left /Tcf。我们已经确定了Wnt信号通过Lef1在斑马鱼gaba能下丘脑神经元发育中的特定作用。我们的数据表明，在这些神经元进入有丝分裂后，Lef1继续在这些神经元中表达，这表明Wnt信号通路可能控制对其功能至关重要的特定基因的表达。在本研究中，我们将验证Lef1通过激活调节后下丘脑神经元的规范、分化和存活的基因来调节后下丘脑神经元发育的假设。在此过程中，我们将建立斑马鱼下丘脑作为脊椎动物神经发生的模型，并在发育中的中枢神经系统中发现Lef1功能的新靶点。首先，我们将测试下丘脑有丝分裂后gaba能神经元的适当分化是否需要Wnt信号和left / tcf介导的转录。使用转基因表达的通路抑制剂，我们将评估在神经发生期间Wnts和left /Tcf因子的时间和空间需求。这些实验将确定这些分子的靶基因调控是否参与gaba能神经发生的保守途径，并决定特定下丘脑细胞群的表型。其次，候选靶基因将通过染色质免疫沉淀分析作为Lef1信号的直接转录靶点进行测试。我们还将通过构建下丘脑组织沉淀DNA片段文库来筛选新的Lef1靶点，然后进行二次特异性确认。潜在靶基因的表达将通过原位杂交检测Lef1在体内的调控作用。这些实验将产生下丘脑体内Lef1靶点的图像，并将有助于表征？下丘脑神经发生中的-catenin信号通路。总的来说，这些研究将使我们了解在下丘脑神经发生过程中基因是如何被环境信号调节的，最终导致该结构的正确功能。我们发现的机制将有助于治疗中枢神经系统紊乱和疾病。

DESCRIPTION (provided by applicant): 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.