Molecular mechanism of Hedgehog signal transduction

Hedgehog （Hh）信号传导是动物发育的主要途径。在腹侧神经管形成过程中，Sonic hedgehog基因（Shh）作为一种形态因子，在特定的背腹侧位置对不同类型细胞的形成进行编程。从分子的角度来看，分级的Shh信号如何对不同的细胞命运做出贡献尚不清楚。我的实验室一直在研究Hh信号转导的分子机制。我们发现三种Gli蛋白在小鼠中作为Hh信号的转录激活因子和/或抑制因子。我们还发现Kif7和Sufu是Gli激活因子和抑制因子的关键调节因子，并且它们在腹侧神经管模式中合作定义分级Hh信号传导。初级纤毛是基于微管的细胞器，对Hh信号转导至关重要。途径激活后，Kif7与Gli蛋白相互作用，并共同运输到纤毛，然后Kif7促进细胞质Gli- sufu复合物的解离，导致Gli激活物的核易位。然而，这些事件背后的分子机制仍然难以捉摸。

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.