Disruption of Wnt secretion via conditional deletion of Porcupine and Wntless in the developing mouse cochlea

 描述(申请人提供)：科尔蒂器官的细胞结构复杂而精确。它的机械感受性毛细胞排列成三排外毛细胞和一排内毛细胞由支持细胞交错排列。对这种细胞的破坏 网络会导致器官功能障碍，从而导致听力损失。多种发育途径在耳蜗毛细胞发育过程中发挥着重要作用。Wnt途径已被证明控制毛细胞的分化和模式，然而，分泌的Wnts的来源和身份以及分泌是如何调控的仍不完全清楚。简而言之，产生WNT配体的细胞分泌一种WNT蛋白，该蛋白可被WNT反应细胞识别，该蛋白表达膜结合的WNT受体，导致细胞反应。在Wnt分泌之前，Wnt蛋白被脂质修饰，并分别被豪猪蛋白(PORCN)和无Wnless蛋白(WLS)转运。在这里，我们建议去除PORCN和WLS来表征它们在毛细胞发育中的作用。在初步实验中，我们观察到了PORCN和WLS的不同时空表达，这两个基因在Pax2-Cre表达区域的缺失导致了胚胎耳蜗中不同的表型。具体地说，PORCN缺失的耳蜗很短，含有杂乱的毛细胞。另一方面，WLS缺陷的内耳没有显示出可辨认的耳蜗线。在三个特定的目标上，我们首先建议使用组织特异性的可诱导Cre驱动程序来去除这两个基因，以确定它们是否是毛细胞分化和模式形成所必需的。在AIM 3，我们将表征单个Wnt配体的时空表达模式，以确定在PORCN和WLS表达细胞中表达的成员。随着这些目标的完成，我们将更好地了解Wnt通路在胚胎耳蜗发育中的作用，并有可能开辟治疗听力损失的新的探索性途径。

 DESCRIPTION (provided by applicant): Cellular organization of the organ of Corti is complex yet precise. Its mechanoreceptive hair cells are arranged such that three rows of outer hair cells and one row of inner hair cells are interdigitated by supporting cells. Disruption of this cellular network causes dysfunction of the organ and thus hearing loss. Multiple developmental pathways have been shown to play important roles during cochlear and hair cell development. The Wnt pathway has been shown to govern hair cell differentiation and patterning, however, the source and identity of secreted Wnts and how secretion are regulated remain incompletely understood. Briefly, a Wnt ligand-producing cell secretes a Wnt protein that is recognized by a Wnt-responsive cell, which expresses membrane bound Wnt receptors, leading to a cellular response. Prior to Wnt secretion, Wnt proteins are lipid modified and transported by the proteins Porcupine (Porcn) and Wntless (Wls), respectively. Here, we propose to ablate Porcn and Wls to characterize their roles during hair cell development. In preliminary experiments, we have observed distinct spatiotemporal expression of Porcn and Wls, and that deletion of these two genes in the Pax2-Cre expression domain leads to distinct phenotypes in the embryonic cochlea. Specifically, Porcn- deficient cochleae were short and contained disorganized hair cells. On the other hand, Wls-deficient inner ear displayed no discernible cochlea. Over three specific aims, we first propose to ablate these two genes using tissue-specific inducible Cre drivers to determine whether they are required for hair cell differentiation and patterning. In Aim 3, we will characterize the spatio-temporal expression pattern of individual Wnt ligands to identify members expressed in Porcn and Wls-expressing cells. At the completion of these aims, we will have gained a better understanding of the roles of Wnt pathway during embryonic cochlea development and potentially open new exploratory avenues to treat hearing loss.