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

项目总结/摘要 Li-Fraumeni综合征患者易患肿瘤生殖系突变导致的癌症 抑制基因，p53。p53突变的遗传以常染色体显性模式发生，并导致 发生多种恶性肿瘤的终生风险增加，早期发病的可能性很大。虽然 先前使用小鼠敲除的研究已经证明p53在肾形成中起作用， 发育性肾脏异常与Li-Fraumeni患者中观察到的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 一般人口。因此，本提案的目标是利用非洲爪蟾胚胎来评估p53的基本功能。 在肾脏发育中的作用，并产生动物模型，以确定是否在Li中观察到p53突变， Fraumeni患者导致肾脏异常。这些实验将检验p53蛋白在细胞内表达的假设。 Li-Fraumeni患者的功能障碍导致肾脏发育异常。假设将被检验 通过以下目标：目标1。评价非洲爪蟾胚胎中p53破坏导致的肾缺陷。 先前的研究表明，p53基因缺失的小鼠胚胎具有发育性肾脏缺陷，包括双链体， 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p53水平在爪蟾胚胎的肾脏中降低时存在，这是一个有用的解剖模型。 导致发育缺陷的机制。为今后分析 p53活性的分子机制，p53调节肾单位分化的假设将在 非洲爪蟾的肾这将通过靶向CRISPR敲除p53的新策略来实现。 非洲爪蟾肾，前肾发育特征的定量，以及 肾脏发育目标二。确定在Li-Fraumeni患者中观察到的p53突变是否会导致肾脏疾病。 异常初步的MRI数据表明，Li-Fraumeni患者的泌尿生殖系统疾病的发生率增加， 异常假设导致Li-Fraumeni综合征的p53突变会导致 将测试非洲爪蟾肾单位的发育，从而产生Li-Fraumeni肾的动物模型 为今后的研究做准备。考虑到这些患者的p53突变预计会通过 功能获得机制，这将通过靶向表达p53改变来实现。 非洲爪蟾肾，研究将是困难的，在其他模型的时间限制。总的来说，实验 本申请中提出的建议将有助于我们了解p53通常如何影响肾单位形成 以及Li-Fraumeni患者p53突变如何破坏这一过程。

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.