GENETIC ANALYSIS /EARLY DEVELOPMENT /DISEASES IN ZEBRAFI

PKD(多囊肾病)的特征是形成多个肾囊肿，被认为是由于肾上皮细胞过度增殖所致。PKD影响着60多万美国人，其中一半的患者将在60岁前进展为终末期肾病。目前，还没有治愈这种毁灭性疾病的方法。PKD研究的最新进展表明，在脊椎动物中，纤毛从上皮细胞的顶端突起进入管腔，可能是环境抗增殖信号的传感器。因此，纤毛形成和功能的缺陷会导致细胞过度增殖，并最终形成包囊。目前，对纤毛装配最了解的方面是IFT(鞭毛内转运)，这是一种基于微管的运动性物质，对于纤毛装配的货物运输是必不可少的。然而，人们对这种能动性是如何调控的知之甚少。本研究以我们在斑马鱼中分离到的囊性肾突变体海马为研究对象，该突变体具有3个IFT基因，表现出与IFT突变体几乎相同的表型，提示海马蛋白可能与IFT蛋白具有相同的作用途径。有趣的是，海马编码一种高度保守的新型非IFT蛋白。此外，海马蛋白含有富含亮氨酸的重复序列，这表明它可能参与了多蛋白质复合体。在本项目中，我们将首先通过原位、免疫染色和EGFP标记对海马基因和基因产物进行详细的鉴定。在目标2中，我们将通过检测海马突变体的细胞增殖、细胞凋亡和纤毛形成来分析海马表型的细胞基础。在目标3中，我们将剖析函数 首先测试它与IFT基因的相互作用。然后，我们将使用酵母双杂交筛选和串联亲和纯化来鉴定海马的结合伙伴。最后，在目标4中，我们将与Somlo实验室合作，测试海马的功能是否在哺乳动物细胞中保守。总之，这些实验将阐明海马--一种非IFT基因--在纤毛组装和包囊形成中的作用。

PKD (polycystic kidney disease) is characterized by the formation of multiple kidney cysts that are thought to result from over-proliferation of renal epithelial cells. PKD affects more than 600,000 Americans and half of the patients will progress into end stage renal disease by the age of 60. Presently, no cure is available for this devastating illness. Recent progresses in PKD research suggest that in vertebrates, the cilium, protruding from apical surface of epithelial cells into tube lumen, may act as a sensor for environmental antiproliferative signals. Defects in cilia formation and function can therefore lead to cell over-proliferation and eventual cyst formation. Presently, the best understood aspect of cilia assembly is IFT (intraflagellar transport), microtubule based motility essential for transporting cargoes for cilia assembly. However, how this motility is regulated is poorly understood. This project focuses on seahorse, a cystic kidney mutant we isolated in a genetic screen in zebrafish along with three IFT genes, seahorse mutant show almost identical phenotypes as IFT mutants, indicating that Seahorse protein may be involved in the same pathway as IFT proteins. Interesting, seahorse encodes a highly conserved novel non-IFT protein. In addition, Seahorse protein contains leucine-rich repeats, suggesting that it may be involved in multi-protein complexes. In this project, we will start by characterizing seahorse gene and gene product in detail with in situ, immuno-staining and eGFP tagging. In Aim 2, we will analyze the cellular basis of seahorse phenotype by examining cell proliferation, apoptosis and cilia formation in seahorse mutants. In Aim 3, we will dissect the function seahorse first by testing its interaction with IFT genes. We will then use yeast two-hybrid screen and tandem affinity purification to identify binding partners of Seahorse. Finally, in Aim 4, we will collaborate with Somlo lab to test whether the function of seahorse is conserved in mammalian cells. Together, these experiments will shed light on the function of seahorse, a non-IFT gene, in cilia assembly and cyst formation.