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

 描述（由申请人提供）：Corti器官的细胞组织复杂而精确。它的机械感受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。破坏这种细胞 网络导致器官功能障碍，从而导致听力损失。多种发育途径在耳蜗和毛细胞的发育过程中起着重要作用。Wnt通路已被证明控制毛细胞分化和模式化，然而，分泌的Wnt的来源和身份以及分泌如何被调节仍然不完全清楚。简言之，Wnt配体产生细胞分泌被Wnt应答细胞识别的Wnt蛋白，其表达膜结合的Wnt受体，导致细胞应答。在Wnt分泌之前，Wnt蛋白被脂质修饰并分别由蛋白Porcupine（Porcn）和Wntless（Wls）转运。在这里，我们建议消融Porcn和WLS来表征它们在毛细胞发育过程中的作用。在初步的实验中，我们已经观察到不同的时空表达的Porcn和Wls，并删除这两个基因的Pax 2-Cre表达结构域导致不同的表型在胚胎耳蜗。具体来说，Porcn缺陷型耳蜗短小，毛细胞排列紊乱.另一方面，Wls缺陷内耳显示没有可辨别的耳蜗。在三个具体的目标，我们首先提出消融这两个基因使用组织特异性诱导Cre驱动程序，以确定它们是否需要毛细胞分化和图案。在Aim中 3，我们将表征单个Wnt配体的时空表达模式以鉴定在Porcn和Wls表达细胞中表达的成员。在这些目标的完成，我们将获得一个更好的了解Wnt通路在胚胎耳蜗发育过程中的作用，并可能开辟新的探索性途径来治疗听力损失。

 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.