Wnt targets in hypothalamic neurogenesis

描述(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于我们诊断疾病和设计再生治疗方法至关重要。中枢神经系统发育中的一个重要信号是由WNTS产生的，它通过下游的效应因子-连环蛋白和Lef/Tcf来调节转录。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，Lef1在这些神经元分裂后继续表达，这表明Wnt信号通路可能控制着对其功能至关重要的特定基因的表达。在这项研究中，我们将测试Lef1通过激活调控其特定、分化和存活的基因来调节下丘脑后部神经元的发育的假设。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，并将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Wnt信号和Lef/Tcf介导的转录是否是下丘脑有丝分裂后GABA能神经元正确分化所必需的。使用转基因表达的途径抑制物，我们将评估在神经发生期间对Wnts和Lef/Tcf因子的时间和空间需求。这些实验将确定这些分子对靶基因的调控是否参与了GABA能神经发生的保守途径，并决定了特定下丘脑细胞群体的表型。其次，通过染色质免疫沉淀分析，将候选靶基因作为Lef1信号的直接转录靶基因进行测试。我们还将通过构建下丘脑组织中沉淀的DNA片段的文库，然后二次确认特异性来筛选新的Lef1靶点。潜在的靶基因的表达将通过原位杂交来测试Lef1对体内的调控。这些实验将提供下丘脑中Lef1靶标的活体图像，并将有助于表征β-连环蛋白信号通路在下丘脑神经发生中的作用。总体而言，这些研究将使我们了解在下丘脑神经发生过程中，基因是如何受到环境信号调控的，最终导致这种结构的正确功能。我们揭示的机制将有助于治疗中枢神经系统疾病和疾病。 与公共健康相关：下丘脑是大脑中控制荷尔蒙释放和行为的重要调节中心。人们对控制下丘脑神经元产生的发育信号知之甚少。在这项提案中，我们将确定Wnt信号通路在下丘脑神经发生中的作用，并确定Wnt效应器Lef1的靶基因，该基因是这一过程的基础。

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.