TERMINAL DIFFERENTIATION OF THE RENAL EPITHELIUM

描述（由申请人提供）：终末分化是一个关键的发育过程，其中细胞周期退出与生理功能的表达相协调。了解控制未成熟和增殖的上皮细胞转化为成熟分化细胞类型的机制对于我们理解肾脏发育以及损伤后修复和再生的机制非常重要。肿瘤抑制蛋白p53由于其在细胞周期调控和转录调控中的双重作用而在终末分化调控因子中占据独特的地位。在这个提议中要测试的总体假设是，p53在发育中的哺乳动物肾脏中的终末上皮细胞分化的转录控制中起关键作用。第一个目标是检验p53刺激肾上皮生化分化的假设;将使用凝胶迁移和瞬时转染试验检查p53与肾功能基因和其他终末分化基因的启动子的体外结合和激活。染色质免疫沉淀试验将测试在体内终末分化过程中，与靶启动子结合的p53是否被选择性激活。免疫组织化学研究将检查内源性p53和靶向终末分化基因的细胞共定位。第二个目标将研究p53在肾脏形态和功能分化中的作用。将评估在各种遗传背景下繁殖的发育中的p53缺失小鼠的终末分化标志物的异常时空表达、不受控制的上皮细胞增殖、肾小管发育不全和肾功能受损的体征。将使用发育阶段特异性标志物和免疫组织化学/原位杂交技术鉴定导致肾发育不全的形态发生事件。为了进一步证实p53在肾细胞分化中的作用，我们将确定p53无效的肾表型是否可以通过在肾特异性启动子的控制下转基因过表达显性阴性突变体p53来重现。此外，通过诱导肾缺血/再灌注损伤对p53缺失小鼠进行应激，并检查肾上皮进行修复和再生的能力。第三个目标将阐明p53在终末分化命运的特化中的作用。将测试在所选终末分化基因的启动子中具有野生型或突变型p53结合位点的报告转基因小鼠体内转基因表达的适当时空特异性。这些结果将促进我们对上皮细胞分化的转录程序的理解，并阐明p53在调节肾脏上皮细胞分化中的新功能。

DESCRIPTION (provided by applicant): Terminal differentiation is a crucial developmental process in which cell cycle exit is coordinated with the expression of physiological functions. Understanding mechanisms that control the transformation of immature and proliferating epithelial cells into mature differentiating cell types is important for our understanding of renal development as well as mechanisms of repair and regeneration after injury. The tumor suppressor protein, p53, occupies a unique position among regulators of terminal differentiation due to its dual roles in cell cycle control and transcriptional regulation. The overall hypothesis to be tested in this proposal is that p53 plays a key role in the transcriptional control of terminal epithelial cell differentiation in the developing mammalian kidney. The First Aim will test the hypothesis that p53 stimulates the biochemical differentiation of the renal epithelium; p53 will be examined for in vitro binding to and activation of promoters of renal function genes and other terminal differentiation genes using gel shift and transient transfection assays. Chromatin immunoprecipitation assays will test if p53 binding to target promoters is selectively activated during the process of terminal differentiation in vivo. Immunohistochemical studies will examine the cellular co-localization of endogenous p53 and target terminal differentiation genes. The Second Aim will examine the role of p53 in the morphological and functional differentiation of the kidney. Developing p53 null mice bred on various genetic backgrounds will be assessed for signs of aberrant spatiotemporal expression of terminal differentiation markers, uncontrolled epithelial cell proliferation, tubular dysgenesis, and impaired renal function. The morphogenetic events leading to the renal dysgenesis will be identified using developmental stage-specific markers and immunohistochemistry/in situ hybridization techniques. To further confirm the role of p53 in renal cell differentiation, we will determine whether the p53 null renal phenotype can be recapitulated by transgenic over expression of dominant negative mutant p53 under the control of kidney-specific promoters. In addition, p53 null mice will be stressed by induction of renal ischemia/reperfusion injury and the capacity of the renal epithelium to undergo repair and regeneration will be examined. The Third Aim will elucidate the role of p53 in specification of terminal differentiation fate. Reporter transgenic mice harboring wild-type or mutant p53-binding sites in the promoter of a selected terminal differentiation gene will be tested for appropriate spatio-temporal specification of transgene expression in vivo. The results should advance our understanding of the transcriptional program of epithelial cell differentiation and elucidate a novel function for p53 in regulating epithelial cell differentiation in the kidney.