Molecular mechanism of Hedgehog signal transduction

Hedgehog（Hh）信号传导是动物的主要发育途径。在腹侧神经管模式中，Sonic hedgehog（Shh）作为一种形态发生素，在特定的背腹侧位置编程形成不同的细胞类型。分级的Shh信号传导如何有助于不同细胞命运的规范在分子方面知之甚少。本实验室一直在研究Hh信号转导的分子机制。我们发现，三个Gli蛋白的功能作为转录激活因子和/或抑制剂的Hh信号在小鼠中。我们还确定了Kif 7和Sufu作为Gli激活剂和抑制剂的关键调节剂，并且它们在腹侧神经管图案形成期间合作定义分级Hh信号传导。初级纤毛是Hh信号转导所必需的基于微管的细胞器。在通路激活后，Kif 7与Gli蛋白相互作用，并一起运输到纤毛，然后Kif 7促进细胞质Gli-Sufu复合物的解离，导致Gli激活剂的核转位。然而，这些事件背后的分子机制仍然难以捉摸。我们最近的研究表明，非磷酸化形式的Kif 7（Kif 7S 1337 A）定位于纤毛的尖端，并促进Hh通路的活性，而磷酸化形式的Kif 7（Kif 7S 1337 D）定位于初级纤毛的基部，并阻断Hh通路的激活。因此，这些突变体是有用的工具，以确定在分级的Shh信号转导在体内的纤毛定位的Kif 7的作用。尽管是Gli蛋白的关键调节剂，但Sufu在细胞核和细胞质中的功能尚不清楚。我们最近证明了Sufu是由核输出调节的，并鉴定了Sufu的两种突变形式，SufumNES和SufuS 342 A，它们分别主要定位于细胞核和细胞质。这些新的腐乳突变体可用于探测腐乳的核输出在体外和体内Hh信号转导中的生理意义。* 在本提案中，我们将在小鼠受精卵中使用CRISPR/Cas9介导的点突变产生表达Kif 7S 1337 A、Kif 7S 1337 D、SufumNES或SufuS 342 A的小鼠，并检查这些突变如何影响腹侧神经管模式。突变胚胎和小鼠胚胎成纤维细胞的分子分析将用于确定Kif 7的纤毛定位和Sufu的核输出如何影响Gli活性和Hh信号传导。免疫共沉淀质谱法将用于鉴定与细胞核和细胞质中的腐乳相互作用的特定蛋白质伴侣。拟议的研究将为发育细胞生物学和信号转导领域的研究生和博士后研究员提供一个独特的研究培训机会。这项研究计划将揭示与Kif 7和Sufu调控作用相关的新机制细节，并推进我们对Hh信号转导的分子理解。*****

Hedgehog (Hh) signalling is a major developmental pathway in animals. In ventral neural tube patterning, Sonic hedgehog (Shh) acts as a morphogen programming the formation of diverse cell types at specific dorsoventral positions. How graded Shh signalling contributes to the specification of distinct cell fates is poorly understood in molecular terms. My laboratory has been studying the molecular mechanism of Hh signal transduction. We showed that three Gli proteins function as transcriptional activator and/or repressor of Hh signalling in mice. We also identified Kif7 and Sufu as key regulators of both Gli activators and repressors, and that they cooperate to define graded Hh signalling during ventral neural tube patterning. Primary cilia are microtubule-based organelles essential for Hh signal transduction. Upon pathway activation, Kif7 interacts with Gli proteins and together they traffic to the cilia, and then Kif7 promotes the dissociation of cytoplasmic Gli-Sufu complexes, resulting in nuclear translocation of Gli activator. However, the molecular mechanism underlying these events remains elusive.***Our recent studies showed that a nonphosphorylated form of Kif7 (Kif7S1337A) localizes to the tips of cilia and promotes Hh pathway activity, whereas a phosphomimetic form of Kif7 (Kif7S1337D) is localized to the base of primary cilia and blocks Hh pathway activation. These mutants are therefore useful tools to determine the role of ciliary localization of Kif7 in graded Shh signalling in vivo. Despite being the critical regulator of Gli proteins, how Sufu functions in the nucleus versus cytoplasm is unclear. We recently demonstrated that Sufu is regulated by nuclear export, and identified two mutant forms of Sufu, SufumNES and SufuS342A, which are predominantly localized to the nucleus and cytoplasm, respectively. These novel Sufu mutants can be utilized to probe the physiological significance of Sufu's nuclear export in Hh signalling in vitro and in vivo. ***In this proposal, we will generate mice that express Kif7S1337A, Kif7S1337D, SufumNES or SufuS342A using CRISPR/Cas9-mediated point mutagenesis in mouse zygotes and examine how these mutations affect ventral neural tube patterning. Molecular analysis of mutant embryos and mouse embryonic fibroblasts will be employed to determine how ciliary localization of Kif7 and nuclear export of Sufu affect Gli activities and Hh signalling. Co-immunoprecipitation mass spectrometry will be used to identify specific protein partners that interact with Sufu in the nucleus and cytoplasm. The proposed studies will provide a unique research training opportunity for graduate students and postdoctoral fellows in developmental cell biology and signal transduction. This research program should unveil novel mechanistic detail related to the regulatory actions of Kif7 and Sufu, and advance our molecular understanding of Hh signal transduction. *****