Analysis of Wilms tumour gene 1 (wt1) regulation and function in zebrafish

肿瘤抑制基因WT1/wt11编码锌指转录因子，其失活可导致Wilms肿瘤（一种小儿肾癌）以及泌尿生殖系统发育异常。尽管经过多年的研究，主要是在人和老鼠身上进行的，但关于各自基因产物的精确生理和分子功能的知识仍然很少。我们想以硬骨鱼斑马鱼为系统研究wt1的调控及其生理作用。这项建议的具体目标是双重的。首先，我们想知道wt1在斑马鱼中的表达是如何调控的。这将通过产生绿色荧光蛋白转基因鱼和随后的生化实验来分析。第二个目标是阐明wt1在发展中的作用。我们想用morpholinos进行过表达和敲低实验。最后，我们想要确定介导wt1活性的分子。为此，我们将采用敲低wt1功能和比较正常细胞和敲低wt1细胞中基因表达的方法。考虑到物种发育过程的保护，我们期望通过这里概述的实验获得的结果将有助于理解高等脊椎动物的基因调控、发育以及最后但并非最不重要的癌症生物学。

The tumour suppressor gene WT1/wt11 encodes a zinc finger transcription factor whose inactivation leads to Wilms tumour, a pediatric kidney cancer as well as to developmental anomalies concerning the urogenital system. Despite many years of research, mostly in man and mouse, knowledge about the precise physiologic and molecular function of the respective gene-product is scarce. We want to use the teleost fish Danio rerio (zebrafish) as a system to study the regulation and the physiologic role of wt1. The specific goals of this proposal are twofold. First, we want to know how wt1 expression is regulated in zebrafish. This will be analysed by the generation of GFP transgenic fish and subsequent biochemical experiments. The second goal is to elucidate the function of wt1 in development. We want to conduct overexpression and knockdown experiments using morpholinos. Finally, we want to identify molecules that mediate the activity of wt1. For this, knockdown of wt1 function and comparison of gene expression in normal and wt1-knockdown cells will be employed. Given the conservation of developmental processes across species we expect that the results which will be obtained through the experiments outlined here will contribute to the understanding of gene regulation, development and last but not least cancer biology also in higher vertebrates.