Control of Shh Activity and Signaling in the Neural Tube

描述（由申请人提供）：在此应用程序中，我们的目标是定义 胆固醇和Gli3在控制Shh活性和信号传导中的作用 在神经管中。Shh基因编码的分泌分子已被 在脊椎动物中枢神经系统的发育中起着关键作用 系统Shh的活性被认为是由胆固醇控制的 在其N-末端共价连接，但这种脂质的确切功能 神经元模式化的修饰尚不清楚。对Shh的回应是 已知依赖于Gli家族的转录因子，但详细的 机制不被理解。我们的遗传学研究表明， 在Shh中，Gli3以剂量依赖性方式抑制腹侧神经元细胞命运。 而Shh突变胚胎显示几类中间神经元减少， 和运动神经元的完全缺失，这些细胞类型被拯救， Shh/Gli3双突变体。这些观察结果表明，Shh需要 拮抗Gli3，否则会抑制腹侧神经元细胞的命运。的 Shh/Gli3双突变体产生运动神经元和中间神经元的能力 强烈表明，除了Shh之外，因子（s）也参与了 这些腹侧神经元的生成。我们的建议旨在测试 以下假设：（1）胆固醇修饰的Shh是正常的 CNS中腹侧神经元细胞类型的分化。(2)嘘抵消 Gli3阻遏物在腹侧神经元前体细胞生成中的作用 通过控制Gli3的加工(3)Gli3阻遏物干扰RA信号传导 在腹侧神经管中。为了实现这些目标，我们制定了五个 具有改变的内源性Shh基因座的强壮嵌合小鼠，其设计为表达 在Cre重组酶依赖性细胞中没有胆固醇加合物的Shh（Shh-N） 方式Shh-N的细胞分布和神经元图形化活性 将取决于生殖系传播。我们还将确定Gli3 使用我们生成的N-末端Gli3特异性抗体进行处理， 分析功能丧失和获得突变体的基因表达模式 在嘘的信号。我们还将利用RARE-lacZ报告小鼠，RAR拮抗剂， 和神经组织块来确定Gli3阻遏物与RA的关系。 这些研究将加深我们对Shh在神经管中功能的理解， 并为神经管相关异常的病因学提供了新的线索， 人类

DESCRIPTION (provided by applicant): In this application, our goal is to define the role of cholesterol and Gli3 in the control of Shh activity and signaling in the neural tube. The secreted molecule encoded by the Shh gene has been shown to play a key role in the development of the vertebrate central nervous system. The activity of Shh is thought to be controlled by cholesterol covalently linked at its N-terminus, but the precise function of this lipid modification in neuronal patterning is not understood. The response to Shh is known to depend on transcription factors of the Gli family, but the detailed mechanism is not understood. Our genetic studies indicated that in the absence of Shh, Gli3 represses ventral neuronal cell fates in a dose-dependent manner. Whereas Shh mutant embryos show reduction in several classes of interneurons and a complete absence of motor neurons, these cell types are rescued in Shh/Gli3 double mutants. These observations indicate that Shh is required to antagonize Gli3, which would otherwise repress ventral neuronal cell fates. The ability of Shh/Gli3 double mutants to generate motor neurons and interneurons strongly suggests that factor(s), in addition to Shh, is involved in the generation of these ventral neurons. Our proposal is aimed at testing the following hypotheses:(l) Cholesterol-modified Shh is essential for normal differentiation of ventral neuronal cell types in the CNS. (2) Shh counteracts Gli3 repressor function in the generation of ventral neuronal progenitor cells by controlling Gli3 processing. (3) Gli3 repressor interferes with RA signaling in the ventral neural tube. To accomplish these aims, we have generated five strong chimeric mice with an altered endogenous Shh locus, designed to express Shh without the cholesterol adduct (Shh-N) in a Cre recombinase-dependent manner. The cellular distribution and neuronal patterning activities of Shh-N will be determined upon germline transmission. We will also determine Gli3 processing using N-terminal Gli3-specific antibody that we generated and analyze patterns of gene expression in mutants with loss-and gain-of-function in Shh signaling. We will also utilize RARE-lacZ reporter mice, RAR antagonist and neural explants to define the relationship between Gli3 repressor and RA. These studies will deepen our understanding of Shh function in the neural tube, and shed new light on the etiology of neural tube-associated anomalies in humans.