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，被认为是由肾上皮细胞过度增殖引起的。PKD影响了60多万美国人，其中一半的患者在60岁时会发展为终末期肾脏疾病。目前，这种毁灭性的疾病还没有治愈方法。最近的PKD研究进展表明，脊椎动物的纤毛从上皮细胞的顶端表面伸出到管腔内，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可导致细胞过度增殖并最终形成囊肿。目前，人们对纤毛组装的理解最好的方面是纤毛内运输，这是一种基于微管的运动，对纤毛组装的货物运输至关重要。然而，这种运动是如何调节的，人们知之甚少。这个项目的重点是海马，我们在斑马鱼的遗传筛选中分离出一种囊肾突变体和三个IFT基因，海马突变体表现出与IFT突变体几乎相同的表型，表明海马蛋白可能与IFT蛋白参与相同的途径。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。在本项目中，我们将首先通过原位，免疫染色和eGFP标记详细表征海马基因和基因产物。在Aim 2中，我们将通过检测海马突变体的细胞增殖、凋亡和纤毛形成来分析海马表型的细胞基础。在Aim 3中，我们将详细分析该函数

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.