Hedgehog signaling and intracellular transport

描述(申请人提供)：胚胎细胞使用Hedgehog信号通路来传递控制细胞命运、增殖和存活的信息。这一途径的错误调控会导致人类的出生缺陷和各种癌症。最近的数据表明，哺乳动物中控制Hedgehog途径的关键机制在无脊椎动物模式生物中似乎不是进化保守的。这项拟议的研究的主要目标是以小鼠为模型系统，了解刺猬信号转导是如何在机械水平上发挥作用的。具体地说，两种新的哺乳动物途径拮抗剂Wdr10和Tulp3的作用将使用3个特定目标中概述的遗传、细胞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发挥作用，这是一种用于构建纤毛和鞭毛的过程。目标1中提出的实验将确认Wdr10通过靶向突变和转基因拯救方法调控Hedgehog途径的必要性。其他实验将解决Wdr10的亚细胞定位问题，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发挥作用。Wdr10在Hedgehog途径中的作用机制将在目标2中阐述。Wdr10自主作用于细胞的假说将通过嵌合体分析来验证，Wdr10在Hedgehog途径中的作用步骤将通过上位性分析来阐明。此外，Wdr10直接或间接作用于控制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来进行。最后，目标3中的实验将检验关于Tulp3在Hedgehog信号中的功能的两个假说。首先，通过在体内阻断肌球蛋白VB的活性来检验Tulp3和肌球蛋白VB相互作用的重要性。其次，将在组织培养细胞中检测调节Tulp3核转位在Hedgehog信号中的意义。

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.