Wnt5a and Planar Cell Polarity in Kidney Development and Cyst formation

描述（由申请人提供）：先天性肾脏和尿路异常（CAKUT）占产前发现的异常的20-30%，估计在高达50%的儿童终末期肾脏疾病病例中起致病作用。ckut影响了大约60万美国人，包括一大批由肾脏异常发育过程引起的疾病。平面细胞极性（PCP）是细胞和细胞结构在上皮表面平面内沿轴的协调方向。当新形成的小管通过定向细胞迁移和细胞分裂而逐渐延长时，PCP在肾脏发育过程中起着核心作用。Wnt家族蛋白已被证明在许多发育系统中控制细胞极性和定向细胞运动。Wnt信号通路整合受体、细胞器和细胞骨架蛋白组织以赋予细胞极性和定向运动的机制尚不完全清楚。目的是了解Wnt5a整合下游细胞器和细胞骨架蛋白以及PCP蛋白的机制，从而在肾脏发育过程中赋予细胞极性和定向细胞运动。Wnt5a是一种非规范的Wnt，它与受体frizzed结合，通过disheded （Dsh）起作用，并激活Wnt/Ca2+和PCP途径。我之前的研究表明，Wnt5a缺失小鼠在前列腺流明化和平面细胞极性方面存在缺陷，而出生时死亡的Wnt5a缺失小鼠缺乏肾脏。Wnt5a似乎在发育中的小鼠肾脏的间质/间质中表达。因此，我们提出的测试假设是Wnt5a通过作用于PCP通路调节肾脏发育。因此，在Aim 1中，我将使用体外细胞培养来研究Wnt5a调节PCP的详细机制。在Aim 2中，我将利用反义Wnt5a morpholinos在斑马鱼中确定Wnt5a信号传导在指导肾原肾发育中的体内作用。使用斑马鱼将使我能够快速确认和扩展Aim 1的体外机制结果。最后，在Aim 3中，我将利用Cre/loxP二元小鼠系统产生肾特异性Wnt5a敲除小鼠，确定Wnt5a在体内控制PCP和调节后肾发育中的作用。我们可以获得Wnt5a固定小鼠，我计划在发育中的小鼠肾脏中做一个Wnt5a的全表达谱，以证实Wnt5a在发育中的肾脏间质/间质中的表达。间充质肾Cre驱动因子，如FoxD1，是市售的，我可以通过将Wnt5a固定小鼠与FoxD1- creert2小鼠交配，以肾脏特异性和时间性的方式敲除Wnt5a。我将在Wnt5a敲除模型中评估定向细胞分裂和其他PCP参数，然后研究包括PCP蛋白在内的下游元件。这些实验的成功完成将导致对CAKUT治疗干预的新候选靶点的鉴定，目前尚无批准的治疗方法。

DESCRIPTION (provided by applicant): Congenital anomalies of the kidney and urinary tract (CAKUT) constitutes 20-30% of anomalies identified in the prenatal period, and is estimated to play a causative role in up to 50% of cases of end-stage kidney disease in children. CAKUT, affecting approximately 600,000 Americans, encompases a large group of disorders that result from abnormal renal developmental processes. Planar cell polarity (PCP) is the coordinated orientation of cells and cellular structures along an axis within the plane of an epithelial surfac. PCP plays a central role during renal development when newly formed tubules undergo progressive lengthening by directed cell migration and cell division. Wnt family proteins have been shown to control cell polarity and directional cell movement in many developmental systems. The mechanisms by which Wnt signaling pathways integrate the organization of receptors, organelles, and cytoskeletal proteins to confer cell polarity and directional cell movement are not completely understood. The goal here is to understand the mechanism by which Wnt5a integrates downstream organelles and cytoskeletal proteins, as well as PCP proteins, to confer cell polarity and directional cell movement during kidney development. Wnt5a is a noncanonical Wnt that binds to its receptor Frizzled, acts through disheveled (Dsh), and activates both the Wnt/Ca2+ and PCP pathways. I previously showed that Wnt5a nul mice develop defects in prostate gland lumenization and planar cell polarity, and Wnt5a null mice, which die at birth, lack kidneys. Wnt5a appears to be expressed in the mesenchyme/stroma of the developing mouse kidney. The hypothesis, therefore, that we propose testing is that Wnt5a regulates kidney development via actions on the PCP pathway. Accordingly, in Aim 1, I will use in vitro cel culture to investigate the detailed mechanism by which Wnt5a regulates PCP. In Aim 2, I will determine the in vivo role of Wnt5a signaling for guiding pronephric kidney development, using antisense Wnt5a morpholinos in zebrafish. Use of zebrafish will allow me to rapidly confirm and expand on the mechanistic in vitro results from Aim 1. Finally, in Aim 3, I will determine the role of Wnt5a in controlling PCP and regulating metanephric kidney development in vivo by utilizing the Cre/loxP binary mouse system to generate kidney-specific Wnt5a knockout mice. Wnt5a floxed mice are available to us and I plan to do a whole expression profile of Wnt5a in the developing mouse kidney to confirm reports of Wnt5a expression in developing kidney mesenchyme/stroma. Mesenchymal kidney Cre drivers, such as FoxD1, are commercially available, and I can knockout Wnt5a in both a kidney-specific and temporal manner by mating Wnt5a floxed mice with FoxD1-CreERT2 mice. I will evaluate oriented cell division and other PCP parameters in the Wnt5a knockout model and then investigate downstream elements including PCP proteins. Successful completion of these experiments will result in the identification of novel candidate targets for therapeutic intervention in CAKUT, a disorder for which no approved treatments currently exist.