Sco, A Zebrafish Model Links Cilia and Kidney Cysts

描述（申请人提供）：常染色体显性遗传性PKD（ADPKD）是最常见的单基因遗传性疾病之一，每1000例活产婴儿中就有一例。一半的患者在60岁时将进展为终末期肾病。最近的研究结果表明，纤毛的功能作为一个传感器的环境抗增殖信号和纤毛缺陷可能会导致囊肿的形成。鉴于纤毛在PKD中的重要性，解剖纤毛的形成和维持对于理解PKD发病机制至关重要。在斑马鱼囊性肾突变体的插入诱变筛选中，鉴定了一个新基因Sco（Scorpion）沿着Pkd 2和三个IFT（鞭毛内转运）基因，这些基因被认为是纤毛形成和维持所需的。Sco编码的蛋白质在N-末端有一个小的GT3结构域，在C-末端有一个卷曲螺旋结构域和一个富含脯氨酸的区域。初步分析表明，Sco是肾纤毛形成所必需的，Sco蛋白位于纤毛上。这个项目的目的是测试的假设，Sco是一个信号分子，调节纤毛组装在肾脏发育。首先，sco突变体的表型和Sco的亚细胞定位将被详细检查，以解决Sco是否直接参与调节纤毛形成的问题。然后通过生物化学分析和生物学测试，利用过表达、缺失分析、显性活性和阴性构建体，详细研究Sco的小GT3结构域和C末端区域的功能。此外，结合伴侣的不同区域的Sco将确定与酵母双杂交筛选和串联亲和纯化。这些实验将揭示在斑马鱼肾脏发育过程中，Sco如何作为调节纤毛形成的遗传网络中的信号转导子发挥作用。最后，将通过检查其在小鼠肾脏中的表达和亚细胞定位以及通过用RNAi和小鼠细胞中的显性构建体分析其功能来分析Sco的功能保守性。

DESCRIPTION (provided by applicant): Striking one in 1000 live births, autosomal dominant PKD (ADPKD) is among the most common monogenetic disorders. Half of the patients will progress to end stage renal disease by age 60. Recent results suggest that the cilium functions as a sensor for environmental anti-proliferation signals and that cilial defects can cause cyst formation. Given the importance of cilia in PKD, dissecting cilia formation and maintenance is critically important for understanding PKD pathogenesis. In an insertional mutagenesis screen for cystic kidney mutants in zebrafish, a novel gene Sco (Scorpion) was identified along with Pkd2 and three IFT (intraflagellar transport) genes, which are thought to be required for the formation and maintenance of cilia. Sco encodes a protein with a small GTPase domain at the N-terminus and a coiled-coil domain and a proline-rich region in the C-terminal half. Preliminary analysis showed that Sco is required for renal cilia formation and that Sco protein is located on cilia. This project is designed to test the hypothesis that Sco is a signaling molecule that regulates cilia assembly during kidney development. First, the phenotypes of sco mutant and the subcellular localization of Sco will be examine in detail to address the question whether Sco is directly involved in regulating cilia formation. Then the functions of the small GTPase domain and the C-terminal region of Sco will be studied in detail via biochemical assays and biological tests with over-expression, deletion analysis, dominant active and negative constructs. Furthermore, binding partners for different regions of Sco will be identified with yeast two-hybrid screen and tandem affinity purification. These experiments will reveal how Sco functions as a signal transducer in the genetic network that regulates cilia formation during zebrafish kidney development. Finally, the functional conservation of Sco will be analyzed by examining its expression and subcellular localization in mouse kidney and by analyzing its function with RNAi and dominant constructs in mouse cells.