GENETIC ANALYSIS OF EARLY DEVELOPMENT AND DISEASES IN ZEBRAFISH

多囊肾病（PKD）的特点是形成多个肾囊肿，被认为是由于肾上皮细胞过度增殖。PKD影响超过60万美国人，其中一半的患者在60岁时将进展为终末期肾病。目前，这种毁灭性的疾病还没有治愈方法。近年来PKD的研究表明，在脊椎动物中，从上皮细胞顶端表面突出到管腔中的纤毛可能作为环境抗增殖信号的传感器。因此，纤毛形成和功能的缺陷可导致细胞过度增殖和最终囊肿形成。目前，纤毛组装的最好理解的方面是IFT（鞭毛内运输），基于微管的运动性对于运送纤毛组装的货物是必需的。然而，这种运动是如何调节的知之甚少。本项目的重点是海马，我们在斑马鱼的遗传筛选中分离到一种囊性肾突变体，其中有沿着三个IFT基因，海马突变体表现出与IFT突变体几乎相同的表型，表明海马蛋白可能参与与IFT蛋白相同的途径。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，表明它可能参与多蛋白复合物。在这个项目中，我们将开始详细描述海马基因和基因产物的原位，免疫染色和eGFP标记。在目标2中，我们将通过检查海马突变体中的细胞增殖、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 海马首先通过测试其与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伴侣。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，这些实验将揭示海马，一个非IFT基因，在纤毛组装和囊肿形成的功能。

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.