Hedgehog signaling and intracellular transport

描述（由申请人提供）：胚胎细胞使用Hedgehog信号通路传递控制细胞命运、增殖和存活的信息。这种途径的失调会导致人类出生缺陷和多种癌症。最近的数据指出，在哺乳动物中控制刺猬通路的关键机制在无脊椎动物模式生物中似乎并不具有进化保守性。本研究的主要目的是利用小鼠作为模型系统，了解Hedgehog信号转导如何在机制水平上发挥作用。具体来说，哺乳动物通路的两种新型拮抗剂Wdr10和Tulp3的作用将通过遗传学、细胞学和生化方法进行研究。Wdr10蛋白及其无脊椎动物同源物在鞭毛内运输中起作用，这是一个用于构建纤毛和鞭毛的过程。在Aim 1中提出的实验将通过靶向诱变和转基因救援等途径证实Wdr10在调控Hedgehog通路中的必要性。其他实验将解决Wdr10的亚细胞定位，并使用免疫细胞化学和活细胞成像技术测试它是否在逆行运输中起作用。Wdr10在Hedgehog通路中的作用机制将在Aim 2中讨论。我们将通过嵌合体分析来验证Wdr10细胞自主作用的假设，并通过上位分析来阐明Wdr10在Hedgehog通路中的作用步骤。此外，Wdr10直接或间接控制Gli转录因子的核定位的假设将通过免疫组织化学和生化方法进行探讨。最后，Aim 3中的实验将验证关于Tulp3在Hedgehog信号传导中的功能的两个假设。首先，将通过在体内阻断肌凝蛋白Vb活性来测试Tulp3与肌凝蛋白Vb相互作用的重要性。其次，将在组织培养细胞中检测受调控的Tulp3核易位在Hedgehog信号传导中的意义。

DESCRIPTION (provided by applicant): Embryonic cells use the Hedgehog signaling pathway to convey information that controls cell fate, proliferation, and survival. Misregulation of this pathway leads to birth defects and a variety of cancers in humans. Recent data point to key mechanisms controlling the Hedgehog pathway in mammals that do not appear to be evolutionary conserved in invertebrate model organisms. The primary objective of the proposed research is to understand how Hedgehog signal transduction functions at the mechanistic level using the mouse as a model system. Specifically, the roles of 2 novel antagonists of the mammalian pathway, Wdr10 and Tulp3, will be studied using genetic, cellular, and biochemical approaches outlined in 3 specific aims. The Wdr10 protein, as well as its invertebrate homologs, functions in intraflagellar transport, a process used for building cilia and flagella. The experiments proposed in Aim 1 will confirm the requirement for Wdr10 in regulating the Hedgehog pathway through targeted mutagenesis and transgenic rescue approaches. Other experiments will address the subcellular localization of Wdr10 and test whether it acts in retrograde transport using immunocytochemistry and live cell imaging techniques. The mechanism of Wdr10 function in the Hedgehog pathway will be addressed in Aim 2. The hypothesis that Wdr10 acts cell autonomously will be tested through chimera analysis and the step in the Hedgehog pathway at which Wdr10 acts will be clarified through epistasis analysis. In addition, the hypothesis that Wdr10 acts directly or indirectly in controlling nuclear localization of the Gli transcription factors will be pursued using immunohistochemical and biochemical methods. Finally, the experiments in Aim 3 will test 2 hypotheses regarding the function of Tulp3 in Hedgehog signaling. First, the importance of the interaction between Tulp3 and myosin Vb will be functionally tested by blocking myosin Vb activity in vivo. Second, the significance of regulated Tulp3 nuclear translocation in Hedgehog signaling will be examined in tissue culture cells.