Wilms tumor 1 (Wt1) mutation reveals mechanisms of cell lineage crosstalk in the developing kidney

项目摘要/摘要 NIH导师职业发展奖描述了一项为期三年的计划，提供培训和 为候选人发展成为研究基因和基因的独立研究人员所必需的指导 肾发育的分子调控，特别关注肾母细胞瘤1(Wt1)的突变 影响发育中肾脏的细胞谱系串扰。为了实现这一点，候选人开发了一种 详细的职业发展计划，融合了德克萨斯大学卓越的导师和科学的环境 西南大学在生物信息学和体外技术方面进行了重点培训，以成功过渡到 独立。WT1是第一个在肾母细胞瘤中发现的基因，对正常肾脏有多方面的调节作用 在发育过程中，它在肾单位祖细胞(NPC)中起着关键作用。 差异化。特异性靶向鼻咽癌的Wt1功能丧失突变肾脏的进一步检查 谱系(Six2cre；Wt1c/c)表现出对肾源性间质的额外影响，间质扩张 祖细胞、间质模式的中断以及几种间质标志物的表达增加 先前显示在肾母细胞瘤中上调，导致假设来自NPC的信号 和/或早期肾单位结构非自主地调节间质发育。对此的测试 假说将询问鼻咽癌到基质的串扰如何指导基质祖细胞的维持和/或 正常肾脏发育中的分化，这将通过两个拟议的目标来实现。在目标1中， 利用基因工程小鼠模型的实验旨在确定信号是如何从 肾单位血统来源的细胞调节肾间质。要做到这一点，间质增殖、细胞凋亡和 将在针对不同肾单位血统的突变小鼠模型中检查模式(即： 自我更新的神经干细胞与早期肾单位结构)对间质的影响 Wt1c/c突变肾脏。目的2将研究鼻咽癌到基质的串扰机制(S)，使用 识别候选受体/配体相互作用的生物信息学和功能验证的体外测试 通过确定干扰特定的信号通路是否会导致对照肾脏的基质缺陷， 同样，如果恢复信号通路，可以挽救突变肾脏正常的基质分化 外植体。鉴于肾脏发育研究的进步对 对肾母细胞瘤的认识，反之亦然，对肾母细胞瘤生物学的研究促进了 发展性肾病，这项提案中概述的目标有可能进一步揭示 相互信号相互作用调节正常肾脏祖细胞的增殖和/或分化 并为未来研究祖细胞串扰的中断提供了基础 肾单位和基质之间的关系可能会改变胚胎的微环境，并易患上 肾母细胞瘤的发生发展。

PROJECT SUMMARY/ABSTRACT This NIH Mentored Career Development Award describes a three-year proposal providing the training and mentorship necessary for the candidate to develop into an independent investigator studying the genetic and molecular regulation of kidney development, with a specific focus on how mutations in Wilms tumor 1 (Wt1) affect cell lineage crosstalk in the developing kidney. To accomplish this, the candidate has developed a detailed career development plan integrating the outstanding mentorship and scientific environment at UT Southwestern with focused training in bioinformatics and in-vitro techniques to successfully transition to independence. Wt1, the first gene identified in Wilms tumor, regulates multiple aspects of normal renal development and has been previously shown to play a critical role in nephron progenitor cell (NPC) differentiation. Further examination of mutant kidneys with Wt1 loss-of-function specifically targeting the NPC lineage (Six2cre;Wt1c/c) shows additional effects on the nephrogenic stroma, with an expansion of stromal progenitor cells, disruptions in stromal patterning, and an increase in expression of several stromal markers previously shown to be upregulated in Wilms tumors, leading to the hypothesis that signals from the NPCs and/or early nephron structures non-autonomously regulate stromal development. The testing of this hypothesis will interrogate how NPC-to-stromal crosstalk directs stromal progenitor maintenance and/or differentiation in normal kidney development, which will be accomplished through two proposed aims. In aim 1, experiments utilizing genetically engineered mouse models are directed at determining how signals from nephron lineage derived cells regulate the nephrogenic stroma. To do this, stromal proliferation, apoptosis, and patterning will be examined in mutant mouse models targeting different populations of the nephron lineage (ie: self-renewing NPCs vs. early nephron structures) in comparison to the effects on the stroma identified in Six2cre;Wt1c/c mutant kidneys. Aim 2 will investigate the mechanism(s) of NPC-to-stromal crosstalk, using bioinformatics to identify candidate receptor/ligand interactions and in-vitro assays to functionally validate these pathways by determining if disrupting specific signaling pathways results in stromal defects in control kidneys, and likewise, if restored signaling pathways can rescue normal stromal differentiation in mutant kidney explants. Given that advances in renal development research have significantly contributed to the understanding of Wilms tumor, and vice versa, with the study of Wilms tumor biology leading to advances in developmental nephrology, the aims outlined in this proposal have the potential to further uncover how intricate reciprocal signaling interactions regulate progenitor cell proliferation and/or differentiation in normal kidney development and provide the foundation for future work examining how disruptions in progenitor cell crosstalk between the nephron and stromal lineages may alter the embryonic microenvironment and predispose to the development of Wilms tumor.