研究Hh信号通路中哪一类Gα参与到该通路信号传导的问题，对于回答 Smo激活信号通路的分子机制有重要的意义。本研究拟应用模式生物斑马鱼为研究载体，借助CRISPR-Cas9基因组编辑技术制备相应的Gα突变体，通过对这些突变体的遗传、生化和细胞学研究，细致、综合、全面的分析不同类型Gα在脊椎动物Hh信号传导过程中发挥的功能。同时，结合初级纤毛定位基因 NPHP3的N末端以及 PKA活性报告基因AKAR2构建初级纤毛PKA活性报告基因的转基因材料。借助这些材料，分析Hh信号通路抑制和激活状态下，PKA活性的变化。结合Gα的功能分析，深入探索Hh信号通路信号传导的分子机制。该研究一方面为理解Hh信号通路的分子机制提供新的证据，另一方面为分离克隆Hh信号通路相关的组织特异性G蛋白偶联受体提供积极的线索，为进一步筛选Hh信号通路相关药物提供理论依据和新的筛选手段。

Whether Smoothened (Smo), Frizzled family G protein coupled receptor (GPCR), key component of Hedgehog (Hh) signaling pathway, activates downstream signal transduction directly through small Gα protein is still in dispute. Interestingly, several new orphan GPCRs, including Gpr161, have been identified as regulators of Hedgehog signaling pathway, however, through different Gα proteins. These studies raise the questions that which Gα plays major role, and how these Gα protein with different effects work together in Hh signal transduction? To address these questions, CRISPR-Cas9 DNA editing tool will be employed to generate null mutant of different Gα proteins in both zebrafish and mouse tissue culture. Intensively genetic, biochemical and cellular analyses will be performed to assay the Hh signaling pathway activity in all single mutants and possible double mutants to dissect Gα protein function in regulating Hh activity. In addition, in order to effectively examine the correlation between PKA activity and Hh pathway activity, stable cell lines and transgenic zebrafish carrying primary cilium (PC) compartment PKA activity reporter construct will be generated by combining the nephronophthisis 3 N terminal and PKA reporter substrate AKAR2. Using these transgenic lines, molecular mechanism of the Hh pathway will be substantially examined. These works will provide: 1) important evidence for understanding of the mechanism of Hh signal transduction; 2) new clues for identification of potential GPCRs involved in Hh pathway in different tissues and cells.