Cilia Assembly and Transport in the Vertebrate Retina

描述（由申请人提供）：本项目的长期目标是了解脊椎动物感光细胞中纤毛形成和维持的分子基础以及纤毛基因突变如何导致视网膜变性。在脊椎动物中，光感受器外节的组装和维持始于连接纤毛的形成。连接纤毛包含一个微管为基础的轴丝是锚定的顶端内节的基体。纤毛的形成始于内节顶端表面的基体对接。纤毛轴丝的延伸和维持需要鞭毛内转运（IFT）的双向运动过程，以携带来自感光细胞内节和外节的蛋白质。遗传突变破坏基体和/或纤毛的组装、结构或功能，导致一系列被称为纤毛病的疾病。这些多综合征疾病通常表现为视网膜变性、肾脏疾病、智力低下和多指（趾）畸形。在当前的应用中，我们将利用斑马鱼的功能丧失策略来研究控制基体定位的机制，并确定cep 290和arl 13 b中的突变如何导致Joubert综合征和其他睫状体病，导致视网膜变性。在具体目标1中，我们将检查携带cep 290和arl 13 b无效突变的斑马鱼的视网膜表型。我们还将测试cep 290和arl 13 b与Bardet-Biedl综合征（BBS）基因，IFT基因和平面细胞极性（PCP）途径的组分的功能相互作用。这些相互作用将确定潜在的第二位点修饰剂，增强感光细胞表型的表达。在具体目标2中，我们将测试的假设，dynactin复合物调节细胞质动力蛋白马达在基体对接和纤毛通过检查斑马鱼突变体的p150和p50亚基的dynactin。在具体目标3中，我们将直接测试PCP通路在光感受器中起作用以控制基底体的极化定位以及PCP信号传导中的缺陷可导致光感受器变性的假设。我们的初步证据表明，成年斑马鱼视网膜内的基底体确实显示出高度极化的排列。我们将确定这种模式在发育过程中是否存在。然后，我们将在光感受器中表达显性阴性形式的核心PCP蛋白Disheveled（Dvl），并确定这是否会导致视网膜变性。这些研究的结果将使我们能够研究在纤毛组装和维持过程中发挥关键作用的基因，并将确定遗传性失明的新基因候选者，以便开发预防视力丧失的疗法。 公共卫生相关性：在脊椎动物视网膜中，感光细胞的存活取决于连接纤毛和外节的适当形成和维持。纤毛是一种复杂的细胞器，具有重要的临床意义，因为纤毛组装或功能障碍可导致视网膜变性、肾脏疾病、智力低下、内脏逆位、多指（趾）畸形和其他病症。了解控制纤毛的形成、定位和结构完整性的机制将有助于发展纤毛疾病的治疗方法。

DESCRIPTION (provided by applicant): The long-term goal of this project is to understand the molecular basis of cilia formation and maintenance in vertebrate photoreceptor cells and how mutations in cilia genes cause retinal degeneration. In vertebrates, the assembly and maintenance of photoreceptor outer segments begins with the formation of a connecting cilium. The connecting cilium contains a microtubule-based axoneme that is anchored to the apical inner segment by a basal body. Cilia formation begins with the docking of basal bodies at the apical surface of the inner segment. Extension and maintenance of the ciliary axoneme requires the bi-directional motility process of Intraflagellar Transport (IFT) to carry proteins from the photoreceptor inner segment and outer segment. Genetic mutations disrupting the assembly, structure, or function of basal bodies and/or cilia result in a spectrum of diseases known as ciliopathies. These multisyndromic disorders often present with retinal degeneration, kidney disease, mental retardation, and polydactyly. In the current application, we will utilize loss-of-function strategies in zebrafish to investigate the mechanisms controlling basal body localization and determine how mutations in cep290 and arl13b, which are causative for Joubert Syndrome and other ciliopathies, lead to retinal degeneration. In Specific Aim 1, we will examine zebrafish carrying null mutations in cep290 and arl13b for retinal phenotypes. We will also test cep290 and arl13b for functional interactions with Bardet-Biedl Syndrome (BBS) genes, IFT genes, and components of the Planar Cell Polarity (PCP) pathway. These interactions will identify potential second-site modifiers that enhance expression of photoreceptor phenotypes. In Specific Aim 2, we will test the hypothesis that the dynactin complex regulates cytoplasmic dynein motors during basal body docking and ciliogenesis by examining zebrafish mutants in the p150 and p50 subunits of dynactin. In Specific Aim 3, we will directly test the hypothesis that the PCP pathway functions in photoreceptors to control the polarized positioning of basal bodies and that defects in PCP signaling can contribute to photoreceptor degeneration. Our preliminary evidence indicates that basal bodies indeed show a highly polarized arrangement within the adult zebrafish retina. We will determine if this patterning exists during development. We will then express dominant-negative forms the core PCP protein Disheveled (Dvl) in photoreceptors and determine if this leads to retinal degeneration. The results of these studies will allow us to study genes that play critical roles during both cilia assembly and maintenance and will identify novel gene candidates for hereditary blindness so that therapies can be developed to prevent vision loss. PUBLIC HEALTH RELEVANCE: In the vertebrate retina, photoreceptor survival depends on the proper formation and maintenance of the connecting cilium and outer segment. The cilium is a complex organelle that is of great clinical importance because dysfunction in cilia assembly or function can lead to retinal degeneration, kidney disorders, mental retardation, situs inversus, polydactyly, and other conditions. An understanding of the mechanisms that control the formation, positioning, and structural integrity of cilia will lead to the development of treatments for ciliary diseases.