Sco, A Zebrafish Model Links Cilia and Kidney Cysts

描述（由申请人提供）：常染色体显性PKD （ADPKD）是最常见的单基因疾病之一，发病率为千分之一。一半的患者在60岁时会发展到终末期肾病。最近的研究结果表明，纤毛是环境抗增殖信号的传感器，纤毛缺陷可导致囊肿的形成。鉴于纤毛在PKD中的重要性，解剖纤毛的形成和维持对于了解PKD的发病机制至关重要。在斑马鱼囊肾突变体的插入突变筛选中，一个新的基因Sco （Scorpion）与Pkd2和三个IFT（鞭毛内运输）基因一起被鉴定出来，这些基因被认为是纤毛形成和维持所必需的。Sco编码的蛋白在n端有一个小的GTPase结构域，在c端有一个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，并且Sco蛋白位于纤毛上。本项目旨在验证Sco是一种在肾脏发育过程中调节纤毛组装的信号分子的假设。首先，我们将详细研究sco突变体的表型和sco的亚细胞定位，以解决sco是否直接参与调节纤毛形成的问题。然后通过生化分析和过表达、缺失分析、显性活性结构体和阴性结构体的生物学试验，详细研究Sco的GTPase小结构域和c端区域的功能。此外，通过酵母双杂交筛选和串联亲和纯化，将鉴定Sco不同区域的结合伙伴。这些实验将揭示Sco如何在斑马鱼肾脏发育过程中作为信号传感器调节纤毛形成的遗传网络中发挥作用。最后，将通过检测Sco在小鼠肾脏中的表达和亚细胞定位，并通过RNAi分析其在小鼠细胞中的功能和优势构建体来分析Sco的功能保护。

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.