Wnt5a and Planar Cell Polarity in Kidney Development and Cyst formation

描述（由申请人提供）：囊肿是上皮器官（如肾脏）的“构建模块”，囊肿发生的异常调节导致常染色体显性多囊肾病（ADPKD）等疾病。影响50万美国人的ADPKD是最常见的潜在致命遗传疾病。初级纤毛是一种从顶面突出的细胞器，与ADPKD发病机制密切相关，被认为是一种感觉天线，将有关血流动力学的信息传递给细胞。当肾小管细胞的初级纤毛被破坏时，细胞通过尚不清楚的机制将其误解为去分化和增殖的信号，导致形成破坏肾脏的肾囊肿。 本文的目的是了解高度保守的八蛋白外囊复合物在肾脏发育中的作用，特别是外囊如何与Wnt蛋白相互作用。外囊是分泌途径的重要组成部分，穿梭含有膜蛋白的囊泡从trans-Golgi网络到靶向亚细胞区域，包括初级纤毛。Sec 10又是外囊的关键成分。Lipschutz实验室（导师的实验室）通过对肾小管细胞的体外研究表明，Sec 10对于初级纤毛和囊肿的正确形成至关重要。此外，在人ADPKD细胞中显示，外囊在细胞内错误定位，从而建立了外囊与ADPKD细胞之间的联系。 位置和ADPKD。Wnt基因编码一个大的、高度保守的分泌性糖蛋白家族，在控制组织模式、细胞命运和从果蝇到人类的增殖中发挥重要作用。脊椎动物Wnt根据其下游信号通路分为两个功能组。经典Wnt通过核2-catenin/TCF-LEF信号传导，而非经典Wnt通过包括Ca 2 +/PKC和RhoA/JNK的交替信号传导级联起作用。经典的Wnt通路是成人肾脏稳态所必需的，并且该信号传导的废除导致囊性肾病。Lipschutz实验室表明Rho GTP酶调节外囊，初步研究表明在肾脏发育期间Wnt和外囊mRNA共表达，以及体外Sec 10敲低后的差异Wnt表达。我们提出的假设是，Wnt基因与外囊一起调节肾脏发育，特别是纤毛发生和囊肿发生。因此，我们将使用胚胎小鼠肾脏检查发育过程中的时空外囊和Wnt表达模式（目的1）。然后，我们将使用体外肾上皮细胞模型来表征外囊和Wnt相互作用的分子途径（目的2）。最后，我们将利用肾脏特异性Sec 10过表达、Sec 10敲除和Pkd 2 WS 25/WS 183小鼠（代表ADPKD的杰出动物模型）研究外囊在体内调节肾脏发育和Wnt信号传导中的作用（目的3）。这些实验的成功完成将导致ADPKD治疗干预的新候选靶点的鉴定，ADPKD是一种没有批准的治疗方法的疾病。

DESCRIPTION (provided by applicant): Cysts are "building blocks" for epithelial organs, such as the kidney, and abnormal regulation of cystogenesis results in disorders such as autosomal dominant polycystic kidney disease (ADPKD). ADPKD, affecting 500,000 Americans, is the most common potentially lethal genetic disease. The primary cilium, an organelle projecting from the apical surface, has been strongly implicated in ADPKD pathogenesis and is thought to act as a sensory antenna that transmits information regarding flow dynamics to the cell. When primary cilia of renal tubule cells are disrupted, the cells, through a still unclear mechanism, misinterpret this as a signal to dedifferentiate and proliferate, resulting in the formation of lare cysts that destroy the kidney. The goal here is to understand the role of the highly conserved eight-protein exocyst complex in kidney development, especially how the exocyst interacts with Wnt proteins. The exocyst is a critical component of the secretory pathway, shuttling vesicles containing membrane proteins from the trans-Golgi network to targeted subcellular locales, including primary cilia. Sec10, in turn, is a critical component of the exocyst. The Lipschutz Lab (the mentor's lab) has shown through in vitro studies of renal tubule cells that Sec10 is critical for proper formation of both primary cilia and cysts. In addition, it was shown in human ADPKD cells that the exocyst is mislocalized intracellularly, thereby establishing a link between exocyst location and ADPKD. Wnt genes encode a large, highly conserved family of secreted glycoproteins that play essential roles in controlling tissue patterning, cell fate, and proliferaton from drosophila to humans. Vertebrate Wnts have been divided into two functional groups by reference to their downstream signaling pathways. The canonical Wnts signal through nuclear 2-catenin/TCF-LEF while the noncanonical Wnts function through alternate signaling cascades that include Ca2+/PKC and RhoA/JNK. The canonical Wnt pathway is necessary for adult kidney homeostasis, and abrogation of this signaling leads to cystic kidney disease. The Lipschutz Lab showed that Rho GTPases regulate the exocyst, and Preliminary Studies show Wnt and exocyst mRNA co-expression during kidney development, as well as differential Wnt expression following Sec10 knockdown in vitro. The hypothesis we propose testing is that Wnt genes work with the exocyst to regulate kidney development, especially ciliogenesis and cystogenesis. Accordingly, we will use embryonic mouse kidneys to examine spatial-temporal exocyst and Wnt expression patterns during development (Aim 1). We will then use in vitro renal epithelial cell models to characterize the molecular pathways by which the exocyst and Wnts interact (Aim 2). Finally, we will study the role of the exocyst in regulating kidney development and Wnt signaling in vivo utilizing kidney- specific Sec10-overexpressing, Sec10 knockout, and Pkd2WS25/WS183 mice, representing an outstanding animal model of ADPKD (Aim 3). Successful completion of these experiments will result in the identification of novel candidate targets for therapeutic intervention in ADPKD, a disease for which no approved treatments exist.