Cilia Assembly and Transport in the Vertebrate Retina

项目描述（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。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。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。在具体目标1，我们将测试的假设，动力蛋白/动力肌动蛋白复合物调节顶端运输的基础机构纤毛形成之前，通过检查斑马鱼的动力蛋白和p150和p50亚基的动力肌动蛋白突变体。在具体目标2中，我们提供了初步证据，表明成年斑马鱼视网膜内的基底体显示出高度极化的排列。我们将直接测试的假设，PCP途径调节这种模式，是至关重要的感光细胞的生存。在具体目标3中，我们将检查在Joubert综合征基因arl 13 b中携带无效突变的斑马鱼的视网膜表型。提出的实验还将测试GTdR结构域和纤毛靶向序列RdR Px对Arl 13 b功能的需求。我们还将测试arl 13 b与Bardet-Biedl综合征（BBS）基因和PCP途径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。这些研究的结果将揭示纤毛形成之前基底体放置所需的新机制，并确定影响遗传性失明的新遗传相互作用。

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. 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. 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. In Specific Aim 1, we will test the hypothesis that the dynein/dynactin complex regulates the apical transport of basal bodies preceding cilia formation by examining zebrafish mutants in dynein and the p150 and p50 subunits of dynactin. In Specific Aim 2, we provide preliminary evidence that basal bodies show a highly polarized arrangement within the adult zebrafish retina. We will directly test the hypothesis that the PCP pathway regulates this patterning and is essential for photoreceptor survival. In Specific Aim 3, we will examine zebrafish carrying null mutations in the Joubert Syndrome gene arl13b for retinal phenotypes. Proposed experiments will also test the requirement of the GTPase domain and a ciliary-targeting sequence RVxPx for Arl13b function. We will also test arl13b for functional interactions with Bardet-Biedl Syndrome (BBS) genes, and components of the PCP pathway. These interactions will identify potential second-site modifiers that enhance expression of photoreceptor phenotypes. The results of these studies will reveal novel mechanisms required for basal body placement prior to cilia formation and to identify novel genetic interactions that influence hereditary blindness.