Genetic analysis of zebrafish kidney development

描述（由申请人提供）：纤毛已成为肾脏囊性疾病、视网膜变性、脑积水和左右不对称缺陷病理学中的关键细胞器。现在，大量的蛋白质已被确定为“纤毛蛋白质组”的一部分，我们提出的核心假设，本申请的一个显着数量的人类疾病的基因将被链接到新的基因，目前代表纤毛蛋白质组，但尚未确定。我们已经利用斑马鱼来鉴定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中的活胚胎中进行研究，2）感觉和运动纤毛都可以进行研究，3）与纤毛缺陷相关的表型已经得到很好的表征，以及4）基因功能的正向和反向遗传方法已经很好地建立。在目标1中，我们计划解决缺乏有用的表型分析工具的纤毛蛋白质组的高通量分析，通过产生新的表位标记的纤毛蛋白质表达转基因系的斑马鱼图像纤毛，基体和IFT蛋白质在活胚胎中的定位。我们还将制造报告感觉纤毛活性的转基因（钙指示剂转基因）。蛋白质组学和基因组学方法已经鉴定了数百种与纤毛相关的蛋白质，并且每一种都是候选的人类疾病基因。在目标2中，我们将进行纤毛蛋白质组基因，是高度保守的，但完全未知的系统分析。斑马鱼同系物的新纤毛基因将有针对性的反义吗啉代寡核苷酸和morphant胚胎进行一系列的测定，包括活成像，共聚焦免疫荧光的纤毛和基体标记，和纤毛感觉信号。在目标3中，我们将定位克隆schmalhans基因，一个新的基因与纤毛运动和人类原发性纤毛运动障碍的候选基因。这些研究的总体目标是1）鉴定新的纤毛发生基因2）产生研究纤毛形成的新工具和3）根据其功能对大量纤毛相关蛋白进行分类。

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).