Generation of novel models of kidney defects using the zebrafish

描述(由申请人提供)：使用。斑马鱼是脊椎动物发育的强大遗传模型，包括肾脏器官发生。以前的基因筛查分离出斑马鱼肾脏的突变体，在这些突变体中，肾脏的功能单位-肾单位-显示出包囊的形成。许多实验室的研究表明，囊性突变体在纤毛发生基因上存在缺陷，因此与多囊肾病的研究有关。近年来，人们认识到斑马鱼的肾单位被划分为功能离散的近端和远端上皮细胞区域，类似于哺乳动物，并且肾单位祖细胞在这些物种中表达类似的基因。由于哺乳动物肾脏发育的复杂性，从肾干细胞分化为肾单位的过程中有许多过程很难研究，至今仍知之甚少。我们假设肾脏发生的遗传途径在脊椎动物之间是保守的。斑马鱼胚胎的特征使突变基因组的遗传筛选和小分子遗传途径的询问成为可能，我们已经开始利用这些小分子来描述肾脏发生的遗传配方。然而，对肾单位细胞类型的形成进行系统的遗传学分析将提供大量有意义的新见解，最终适用于先天性肾缺陷的预防和治疗。我们建议对扰乱斑马鱼肾单位形成的可遗传突变进行正向筛选，并使用化学文库来识别可以调节斑马鱼肾脏祖细胞模式的小分子。在ENU诱变斑马鱼的中试单倍体筛选中，我们分离到了一组独特的肾脏发生突变。这些发现确立了我们的筛查企业能够成功发现影响肾单位细胞类型形成的新突变的先例。我们将进行大规模的筛查，然后进行互补和基因表达分析，以记录肾脏缺陷。在一种平行的方法中，我们将使用化学遗传学来筛选具有已知靶点的生物活性分子库，并识别改变肾脏发生的化合物。我们还将确定肾单位基因和已知的肾脏前体信号维甲酸和Notch之间的上位关系。最后，我们将克隆并进一步研究肾脏突变的一个子集。该项目的总体目标是建立一个遗传工具包，以促进和促进斑马鱼在肾脏病研究中的迅速使用。斑马鱼信息网络(ZFIN)将免费提供带有突变描述、图像和化学表型的完全注释的界面，并将通过斑马鱼国际资源中心(Zirc)协调肾脏突变的分布。这些研究将产生一种有价值的商品，将补充现有的肾脏研究工具，如哺乳动物的GUDMAP，并促进使用斑马鱼来制定和测试先进的假说，这些假说可以在解剖肾脏发生的遗传途径方面取得重大进展。

DESCRIPTION (provided by applicant): Generation of novel models of kidney defects using the. The zebrafish is a powerful genetic model of vertebrate development, including kidney organogenesis. Previous genetic screens isolated zebrafish kidney mutants in which nephrons-the functional units of the kidney-evince cyst formation. Work by many labs has revealed that cystic mutants harbor defects in ciliogenesis genes, and thus are relevant to study polycystic kidney disease. In recent years, it has become appreciated that zebrafish nephrons are segmented into functionally discrete domains of proximal and distal epithelial cells, similar to mammals, and that nephron progenitors express similar genes across these species. Many processes in the specification and differentiation of nephron cells from renal stem cells are difficult to study, and today remain poorly understood, due to the complex nature of mammalian kidney development. We hypothesize that the genetic pathways of nephrogenesis will be conserved between vertebrates. The features of the zebrafish embryo enable genetic screens of mutagenized genomes and interrogation of genetic pathways with small molecules, which we have used to begin delineating the genetic recipe of nephrogenesis. However, implementing a systematic genetic analysis of nephron cell type formation would proffer a wealth of meaningful new insights ultimately applicable to the prevention and treatment of congenital kidney defects. We propose to conduct a forward screen for heritable mutations that disrupt zebrafish nephron formation, and to use chemical libraries to identify small molecules that can modulate zebrafish renal progenitor patterning. In a pilot haploid screen with ENU-mutagenized zebrafish, we isolated a cohort of unique nephrogenesis mutants. These findings establish the precedence that our screen enterprise can successfully uncover novel mutations affecting nephron cell type formation. We will conduct a large-scale screen, then complementation and gene expression analyses to chronicle the kidney defects. In a parallel approach, we will employ chemical genetics to screen libraries of bioactive molecules with known targets and identify compounds that alter nephrogenesis. We will also determine the epistatic relationship between nephron genes and the known renal progenitor signals, retinoic acid and Notch. Finally, we will clone and further study a subset of the kidney mutations. The overall goal of this project is to establish a genetic toolkit that can facilitate and energize the burgeoning use of zebrafish in nephrology research. A fully annotated interface with mutant descriptions, images, and chemical phenotypes will be made freely available on the Zebrafish Information Network (ZFIN), and kidney mutant distribution will be coordinated through the Zebrafish International Resource Center (ZIRC). These studies will produce a valuable commodity that will complement existing kidney research tools like the mammalian GUDMAP, and foster the use of zebrafish to formulate and test advanced hypotheses that can forge significant inroads into dissecting the genetic pathways of nephrogenesis.