Role of p53 in Kidney Development: Modeling Renal Anomalies of Li-Fraumeni Patients

项目摘要/摘要 Li-Fraumeni综合征的患者容易患上癌症，这是由于肿瘤的种系突变引起的 抑制者，P53。P53突变的遗传以常染色体显性模式发生，并导致 罹患多种恶性肿瘤的终生风险增加，并极有可能早发。虽然 先前使用小鼠基因敲除的研究表明，P53在肾脏形成中发挥作用， 发育中的肾脏异常与Li-Fraumeni患者中看到的P53突变无关。 初步的MRI数据表明，这些患者的泌尿生殖系统异常的发生率高于 普通人口。因此，这项建议的目标是利用非洲爪哇胚胎来评估p53的S基础 在肾脏发育中的作用，并建立动物模型，以确定在LI中是否存在P53突变。 Fraumeni患者会导致肾脏异常。拟议的实验将检验这样一种假设，即P53 Li-Fraumeni患者的功能障碍会导致肾脏发育异常。这一假设将得到检验。 通过以下目的：目的1.评估非洲爪哇胚胎中P53基因突变所致的肾脏缺陷。 以前的研究表明，p53基因缺失的小鼠胚胎有发育性肾脏缺陷，包括双肾畸形。 输尿管和肾发育不全。初步数据表明，类似的肾脏发育缺陷也是 当非洲爪哇胚胎肾脏中的P53水平降低时，一个有用的模型可以用来解剖 导致发育缺陷的机制。为将来对 P53活性的分子机制，P53调节肾单位分化的假说将在 非洲爪哇的肾脏。这将通过以CRISPR基因敲除P53为目标的新策略来实现 非洲爪哇前肾发育特征的量化及其标志物的表达分析 肾脏发育。目的2.确定在Li-Fraumeni患者中发现的p53突变是否会导致肾脏 反常现象。初步核磁共振数据显示，Li-Fraumeni患者的泌尿生殖系统发病率增加 反常现象。假设导致Li-Fraumeni综合征的P53突变导致 将对非洲爪哇肾脏发育进行测试，从而产生Li-Fraumeni肾脏的动物模型 未来研究的异常现象。鉴于这些患者的p53突变预计会通过 功能获得机制，这将通过靶向p53改变的表达来实现 非洲爪哇肾脏，在其他模型中进行的研究将是困难和耗时的。总的来说，这些实验 将有助于我们理解P53是如何影响肾单位形成的。 以及Li-Fraumeni患者p53突变可能如何扰乱这一过程。

PROJECT SUMMARY/ ABSTRACT Patients with Li-Fraumeni syndrome are predisposed to cancer resulting from germline mutations in the tumor suppressor, p53. Inheritance of mutations in p53 occurs in an autosomal dominant pattern and results in an increased lifetime risk for developing multiple malignancies with a significant likelihood of early onset. Although prior studies using mouse knockouts have demonstrated that p53 plays a role in kidney formation, developmental kidney anomalies have not been associated with p53 mutations seen in Li-Fraumeni patients. Preliminary MRI data indicate that these patients have a higher incidence of urogenital anomalies than the general population. Thus, the goal of this proposal is to utilize Xenopus embryos to assess p53's fundamental role in kidney development and to generate animal models to determine whether p53 mutations seen in Li- Fraumeni patients result in renal anomalies. The proposed experiments will test the hypothesis that the p53 dysfunction in Li-Fraumeni patients results in kidney developmental anomalies. The hypothesis will be tested through the following aims: Aim 1. Evaluate kidney defects resulting from p53 disruption in Xenopus embryos. Previous studies showed that p53-null mouse embryos have developmental kidney defects, including duplex ureter and renal hypoplasia. Preliminary data indicate that similar nephric developmental defects are also present when p53 levels are reduced in the kidneys of Xenopus embryos, a useful model for dissecting the mechanisms that contribute to developmental defects. To set the foundation for future analysis of the molecular mechanism of p53 activity, the hypothesis that p53 regulates nephron differentiation will be tested in the Xenopus kidney. This will be achieved through novel strategies targeting CRISPR knockout of p53 to the Xenopus kidney, quantitation of pronephric developmental features, and analysis of expression of markers of kidney development. Aim 2. Determine whether p53 mutations seen in Li-Fraumeni patients result in renal anomalies. Preliminary MRI data indicate that Li-Fraumeni patients have an increased incidence of urogenital anomalies. The hypothesis that the p53 mutations that cause Li-Fraumeni syndrome result in disruption of Xenopus nephron development will be tested, thereby generating animal models of Li-Fraumeni kidney anomalies for future research. Given that the p53 mutations from these patients are anticipated to act through gain-of-function mechanisms, this will be carried out by targeting expression of the p53 alterations to the Xenopus kidney, studies that would be difficult and time-prohibitive in other models. Overall, the experiments proposed in this application will facilitate our understanding of how p53 affects nephron formation generally and how Li-Fraumeni patient mutations in p53 may disrupt this process.