Genetic analysis of zebrafish kidney development

描述(申请人提供)：纤毛在肾囊性疾病、视黄素变性、脑积水和左右不对称缺陷的病理中是一个重要的细胞器。现在，大量的蛋白质已经被鉴定为纤毛蛋白质组的一部分，我们提出作为这一应用的中心假设，相当数量的人类疾病基因将与目前在纤毛蛋白质组中代表的但尚未表征的新基因相关联。我们利用斑马鱼来识别参与纤毛形成的新基因，并确定纤毛在肾脏、大脑和左右不对称产生中的生理作用。斑马鱼特别适合于纤毛蛋白质组的高通量分析，因为1)纤毛功能可以在多器官系统的活胚胎中研究，2)感觉纤毛和运动纤毛都可以研究，3)与纤毛缺陷相关的表型已经得到很好的表征，4)基因功能的正向和反向遗传方法已经建立。在目标1中，我们计划解决缺乏有效的表型工具来高通量分析纤毛；通过建立新的表位标记的表达纤毛蛋白的斑马鱼转基因系来成像纤毛、基体和IFT蛋白在活胚胎中的定位。我们还将制作报告感觉纤毛活动的转基因产品(钙指示剂转基因产品)。蛋白质组学和基因组学方法已经鉴定出数百种与纤毛相关的蛋白质，每一种都是人类疾病的候选基因。在目标2中，我们将对高度保守但完全没有特征的纤毛蛋白质组基因进行系统分析。新型纤毛基因的斑马鱼同源物将与反义吗啉寡核苷酸和形态胚胎一起进行一系列分析，包括实时成像、纤毛和基础体标记物的共聚焦免疫荧光以及纤毛感觉信号。在目标3中，我们将定位克隆SchMalhans基因，这是一个与纤毛运动相关的新基因，也是人类原发纤毛运动障碍的候选基因。这些研究的总体目标是1)识别新的纤毛形成基因，2)产生研究纤毛形成的新工具，3)根据纤毛相关蛋白的功能对其进行分类(S)。

DESCRIPTION (provided by applicant): The cilium has emerged as a critical cellular organelle in the pathology of kidney cystic disease, retin degeneration, hydrocephalus, and left-right asymmetry defects. Now that a large number of proteins have been identified as part of the "cilia proteome", we propose as the central hypothesis of this application that a significant number of human disease genes will be linked to novel genes that are currently represented in the cilia proteome but are yet uncharacterized. We have exploited the zebrafish to identify novel genes involved in cilia formation and to determine the physiological roles of cilia in kidney, brain, and the generation of left- right asymmetry. Zebrafish are particularly well-suited for high-throughput analysis of the cilia proteome because 1) cilia function can be studied in living embryos in multiple organ systems, 2) both sensory and motile cilia can be studied, 3) phenotypes related to cilia defects have been well characterized, and 4) forward and reverse genetic approaches to gene function are well established. In Aim 1 we plan to address the lack of useful phenotyping tools for high-throughput analysis of the cilia; proteome by generating new epitope-tagged cilia protein-expressing transgenic lines of zebrafish to image cilia, basal bodies and the localization of IFT proteins in living embryos. We will also make transgenics that report that activity of sensory cilia (calcium indicator transgenics). Proteomic and genomic approaches have identified hundreds of proteins associated with cilia and each o these is a candidate human disease gene. In Aim 2 we will conduct a systematic analysis of cilia proteome genes that are highly conserved but completely uncharacterized. Zebrafish homologs of novel cilia genes will be targeted with antisense morpholino oligos and morphant embryos subjected to a battery of assays including live imaging, confocal immunofluorescence of cilia and basal body markers, and cilia sensory signaling. In Aim 3 we will positionally clone the schmalhans gene, a novel gene associated with cilia motility and a candidate gene for human primary ciliary dyskinesia. The overall goal of these studies is to 1) identify novel ciliogenic genes 2) generate new tools to study cilia formation and 3) categorize the large number of cilia associated proteins in terms of their function(s).