Generation of novel models of kidney defects using the zebrafish

斑马鱼是脊椎动物发育（包括肾器官发生）的一个强大的遗传模型。先前的基因筛选分离出斑马鱼肾脏突变体，其中肾单位肾脏的功能单位显示囊肿形成。许多实验室的工作已经揭示了囊性突变体在纤毛发生基因中存在缺陷，因此与多囊肾病的研究有关。近年来，人们已经认识到，斑马鱼肾单位被分割成近端和远端上皮细胞的功能离散域，类似于哺乳动物，并且肾单位祖细胞在这些物种中表达相似的基因。由于哺乳动物肾脏发育的复杂性，从肾干细胞向肾单位细胞的特化和分化的许多过程很难研究，今天仍然知之甚少。我们推测肾发生的遗传途径在脊椎动物中是保守的。斑马鱼胚胎的特征使诱变基因组的遗传筛选和小分子的遗传途径的询问成为可能，我们已经用小分子来开始描绘肾发生的遗传配方。然而，对肾单位细胞类型形成进行系统的遗传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。我们建议进行一个前向筛选的遗传性突变，破坏斑马鱼肾单位的形成，并使用化学文库，以确定小分子，可以调节斑马鱼肾祖细胞图案。在用ENU诱变的斑马鱼进行的试验性单倍体筛选中，我们分离出一组独特的肾发生突变体。这些发现确立了我们的筛查企业可以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选，然后进行互补和基因表达分析，以记录肾脏缺陷。在一个平行的方法，我们将采用化学遗传学筛选库的生物活性分子与已知的目标，并确定化合物，改变肾。我们还将确定肾单位基因和已知的肾祖细胞信号，视黄酸和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子集。该项目的总体目标是建立一个遗传工具包，可以促进和激励斑马鱼在肾脏学研究中的蓬勃发展。斑马鱼信息网络（ZFIN）将免费提供一个带有突变体描述、图像和化学表型的完整注释界面，斑马鱼国际资源中心（ZIRC）将协调肾脏突变体的分布。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GUDMAP，并促进使用斑马鱼来制定和测试先进的假设，这些假设可以在解剖肾发生的遗传途径方面取得重大进展。

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.