Molecular mechanism of Hedgehog signal transduction

Hhedgehog(HH)信号是动物的一条主要发育途径。在腹侧神经管构型中，Sonic hedgehog(Shh)作为一种形态原，编程在特定的背腹位置形成不同类型的细胞。分级Shh信号如何有助于明确不同的细胞命运，在分子方面还知之甚少。我的实验室一直在研究HH信号转导的分子机制。我们发现有三种Gli蛋白在小鼠体内作为HH信号的转录激活和/或抑制因子发挥作用。我们还发现Kif7和Sufu是Gli激活物和抑制物的关键调节者，它们在腹侧神经管模式形成过程中合作定义分级的HH信号。初级纤毛是HH信号转导所必需的微管细胞器。在途径激活后，Kif7与Gli蛋白相互作用，共同运输到纤毛，然后Kif7促进细胞质Gli-Sufu复合体的解离，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Kif7(Kif7S1337A)定位于纤毛末端，促进HH途径的活性，而拟磷酸型Kif7(Kif7S1337D)定位于初级纤毛的底部，阻断HH途径的激活。因此，这些突变体是确定Kif7纤毛定位在体内分级Shh信号中的作用的有用工具。尽管Sufu是Gli蛋白的关键调节因子，但它在细胞核和细胞质中的功能尚不清楚。我们最近证实了Sufu受核输出的调控，并鉴定了两种突变形式的Sufu，SufumNES和SufuS342a，它们分别主要定位于细胞核和细胞质。这些新的Sufu突变体可以用来探讨Sufu核输出在体内和体外HH信号转导中的生理意义。 在这项提议中，我们将使用CRISPR/Cas9介导点突变在小鼠受精卵中产生表达Kif7S1337A、Kif7S1337D、SufumNES或SufuS342A的小鼠，并检测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被用来确定Kif7的纤毛定位和Sufu的核输出如何影响Gli活性和HH信号。免疫共沉淀质谱仪将被用来鉴定在细胞核和细胞质中与腐乳相互作用的特定蛋白质伙伴。拟议的研究将为研究生和博士后在发育细胞生物学和信号转导方面提供一个独特的研究培训机会。这一研究计划将揭示与Kif7和Sufu调节作用相关的新的机制细节，并促进我们对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.