ROLE OF WT1 IN GENITOURINARY DEVELOPMENT AND TUMORIGENESIS

Denys-Drash （DDS）是一种罕见的人类疾病，其严重的泌尿生殖系统异常导致肾病，性腺条纹，生殖器模糊，男性假雌雄同体和Wilms肿瘤。这种情况主要影响XY型个体。DDS患者在生殖器异常的程度上表现出很大的差异，一些人同时具有穆勒氏和沃尔夫氏衍生结构，而另一些人则两者都没有。WT1基因锌指区错义突变是DDS发病过程中的一个关键事件。最突出的突变热点之一是外显子0的氨基酸394 (R394W)，发生在40%的DDS患者中。WT1的一些肾脏和性腺特异性靶点已经被确定，并且可能是DDS表型发展的关键组成部分。为了确定R394W突变是否会引起小鼠的DDS表型，我们将使用PCR位点定向mutage4nesis和cre-lox基因靶向策略生成一个密码子394上含有一个arg到trp替换的小鼠模型。这种突变对泌尿生殖系统（GU）发育的影响将通过检查大体形态表型来评估。此外，将通过原位杂交和免疫组织化学来确定该突变对各种肾脏和性腺发育特异性分子标记表达的影响，从而研究该突变在DDS发展中的作用。如果突变导致不育、胚胎死亡或两者兼而有之，将产生DDS嵌合动物，以确定突变细胞在泌尿生殖器官发育中的作用。此外，本研究将有助于确定Wt1点突变是否会导致小鼠Wilms肿瘤。对肿瘤发展的易感性，也将在与杂合p53突变小鼠杂交后进行分析。建立小鼠模型来分析DDS将非常有助于确定WT1点突变如何导致DDS患者的一系列泌尿生殖系统影响。

Denys-Drash (DDS) is a rare human condition in which severe urogenital abnormalities result nephropathy, streak gonads, ambiguous genitalia, male pseudohermaphroditism and Wilms' tumor. The condition affects mostly XY individuals. DDS patients show large variability in the extent of the genital abnormalities some have both Mullerian- and Wolffian-derived structures, and others have neither. Missense mutation in the zinc-finger region of the WT1 gene is a critical event in the elaboration of DDS. One of the most prominent mutational hot spots is amino acid 394 in exon 0 (R394W), which occurs in 40% of the DDS patients. A number of kidney- and gonadal-specific targets of WT1 have identified, and are likely to be critical components in the development of the DDS phenotypes. To determine whether the R394W mutation will cause the DDS phenotypes in mice as seen in human, a mouse model containing an arg to trp substitution at codon 394 will be generated using PCR site-directed mutage4nesis and the cre-lox gene-targeting strategies. The effects of this mutation on genitourinary (GU) development will be assessed by examination of gross morphological phenotypes. In addition, the role of this mutation in the development of DDS will be studied by in situ hybridization and immunohistochemistry by determining the effects on the expression of various kidney- and gonadal-development specific molecular markers. If the mutation results in infertility, embryonic lethality or both, DDS chimeric animals will be generated to determine the contribution of the mutant cells in the development of the urogenital organs. Moreover, this study will help to determine if this point mutation in Wt1 causes Wilms' tumor in mice. Susceptibility to tumor development, also will be analyzed after cross- breeding with heterozygous p53 mutant mice. The establishment of a mouse model to analyze DDS will be extremely useful in determining, how a point mutation in WT1 causes a constellation of urogenital effects in patients with DDS.