Wnt5a and Planar Cell Polarity in Kidney Development and Cyst formation

描述（由申请人提供）：先天性肾脏和泌尿道异常（CAKUT）占产前发现的异常的20-30%，估计在高达50%的儿童终末期肾病病例中起致病作用。CAKUT影响大约600，000名美国人，包括由异常肾脏发育过程引起的一大组疾病。平面细胞极性（PCP）是上皮表面平面内细胞和细胞结构沿着轴的协调取向。PCP在肾脏发育过程中起着重要作用，此时新形成的小管通过定向细胞迁移和细胞分裂进行进行性延长。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的组织以赋予细胞极性和定向细胞运动的机制尚未完全理解。这里的目标是了解Wnt 5a整合下游细胞器和细胞骨架蛋白以及PCP蛋白的机制，以在肾脏发育过程中赋予细胞极性和定向细胞运动。Wnt 5a是一种非经典的Wnt，与其受体Frizzled结合，通过Dsh起作用，并激活Wnt/Ca 2+和PCP途径。我以前的研究表明，Wnt 5a裸小鼠在前列腺管腔化和平面细胞极性方面存在缺陷，而出生时死亡的Wnt 5a裸小鼠缺乏肾脏。Wnt 5a似乎在发育中的小鼠肾脏的间充质/基质中表达。因此，我们提出的假设是，Wnt 5a通过对PCP途径的作用来调节肾脏发育。因此，在目的1中，我将使用体外细胞培养来研究Wnt 5a调控PCP的详细机制。在目标2中，我将确定Wnt 5a信号转导在体内的作用，指导原肾发育，使用反义Wnt 5a吗啉代斑马鱼。使用斑马鱼将使我能够快速确认和扩展Aim 1的体外结果。最后，在目标3中，我将确定Wnt 5a在控制PCP和调节后肾发育中的作用，通过利用Cre/loxP二元小鼠系统产生肾特异性Wnt 5a敲除小鼠。我们可以获得Wnt 5a floxed小鼠，我计划在发育中的小鼠肾脏中进行Wnt 5a的完整表达谱，以确认Wnt 5a在发育中的肾脏间充质/基质中表达的报告。间充质肾Cre驱动因子，如FoxD 1，是市售的，我可以敲除Wnt 5a在肾脏特异性和时间的方式通过交配Wnt 5a floxed小鼠与FoxD 1-CreERT 2小鼠。我将在Wnt 5a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在CAKUT，目前没有批准的治疗方法存在的疾病的治疗干预的新的候选目标的识别。

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.