Genetic Regulation of Eye Development in Zebrafish

描述（由申请人提供）：我们研究的长期目标是阐明脊椎动物眼部形态发生的机制基础。在眼形态发生过程中，视原基经历了一系列复杂的形态发生运动，最终形成了包含未来视网膜和RPE的双层视杯。每个视神经原基的神经外胚层必须沿着其近端-远端轴融合，这样视网膜和RPE在眼形态发生的早期阶段将被限制在视神经杯的范围内。融合发生在视神经杯上一个叫做脉络膜裂的特殊区域。促进脉络膜裂隙闭合的分子机制和细胞过程尚未在任何脊椎动物中得到很好的表征。脉络膜裂隙闭合缺陷导致脉络膜视网膜结肠瘤，使人眼衰弱的先天性畸形。绒毛膜视网膜结肠瘤的分子基础尚不清楚，研究其病因的动物模型也很少。本实验将利用斑马鱼，Danio rerio，来阐明眼形态发生和脉络膜裂隙闭合的分子和细胞机制。这些研究的结果将是重要的，因为它们将开始揭示导致人类绒毛膜视网膜结肠瘤的分子和形态发生缺陷。（具体目标1）我们将确定斑马鱼突变井喷中结肠瘤表型的分子、细胞和形态发生基础。我们克隆了该突变体中的断裂位点，并在patche1基因中发现了一个突变，该基因是Hedgehog通路的负调控因子。在这里，我们将测试井喷中的结肠瘤表型是由视柄过度增殖引起的假设，我们将确定这种表型的分子机制。人类PATCHED1基因突变可导致基底细胞痣综合征或Gorlin综合征（BCNS），这是一种眼部缺陷的疾病，包括胶质源性视网膜前膜的形成、视网膜发育不良和结肠瘤。这些缺陷的分子和细胞基础尚不清楚，我们将利用成人blowout/patched1和leprechaun/patched2突变体来验证BCNS患者的眼部缺陷源于构成性刺猬依赖性靶基因表达导致的视网膜内Muller胶质细胞不受控制的增殖的假设。（Specific Aim 3）在Hedgehog通路的下游，vax1和vax2在介导腹侧视杯形成和脉络膜裂隙闭合的基因调控网络中占据关键节点。利用微阵列方法，我们已经确定了20多个基因，其在腹视杯和脉络膜裂中的表达依赖于vax1和vax2。在本研究中，我们将重点关注一个特定的vax1/vax2靶点：整合素a5，这是一种纤维连接蛋白结合整合素，我们假设它是脉络膜裂隙细胞与裂隙内富含纤维连接蛋白的细胞外基质相互作用以刺激其闭合所必需的。我们将利用组织学、分子和体内成像技术来验证这一假设。公共卫生相关性：这项拨款提出的研究将阐明脉络膜裂隙闭合的分子、细胞和形态发生基础。在人类中，脉络膜裂隙闭合缺陷导致脉络膜视网膜结肠瘤：先天性眼部缺陷，眼部大片区域缺乏视网膜和视网膜色素上皮，因为这些结构在眼部形态形成过程中未能包含在杯中。确定脉络膜裂隙闭合所需的因素将对我们对结肠形成的理解和可能治疗的发展产生直接影响。

DESCRIPTION (provided by applicant): The long-term goal of our research is to elucidate the mechanistic basis of ocular morphogenesis in vertebrates. During ocular morphogenesis, the optic primordia undergo a complex series of morphogenetic movements that ultimately result in a bilayered optic cup containing the prospective retina and RPE. The neuroectodermal layers of each optic primordium must fuse along its proximo-distal axis such that the retina and RPE will be confined within the limits of the optic cup during the early phases of ocular morphogenesis. Fusion occurs along a distinct region of the optic cup called the choroid fissure. The molecular mechanisms and the cellular processes that facilitate choroid fissure closure have not been well characterized in any vertebrate organism. Defects in choroid fissure closure result in chorioretinal colobomas, debilitating congenital malformations of the human eye. The molecular basis of the defects underlying chorioretinal colobomas is largely unknown and there are few animal models for studying their etiology. Experiments in this proposal will utilize the zebrafish, Danio rerio, to elucidate the molecular and cellular mechanisms that underlie ocular morphogenesis and choroid fissure closure. The results of these studies will be significant, as they will begin to shed light on the molecular and morphogenetic defects that lead to chorioretinal colobomas in humans. (Specific Aim 1) We will determine the molecular, cellular and morphogenetic bases for coloboma phenotypes in the zebrafish mutant blowout. We have cloned the disrupted locus in this mutant and identified a mutation in the patched1 gene, a negative regulator of the Hedgehog pathway. Here we will test the hypothesis that coloboma phenotypes in blowout result from overproliferation of the optic stalk and we will determine the molecular mechanisms underlying this phenotype. (Specific Aim 2) Mutations in human PATCHED1 lead to Basal Cell Naevus Syndrome or Gorlin Syndrome (BCNS), a disorder that presents with ocular defects that include the formation of a glia-derived epiretinal membrane, retinal dysplasia and colobomas. The molecular and cellular basis of these defects is not known and we will utilize adult blowout/patched1 and leprechaun/patched2 mutants to test the hypothesis that the ocular defects in BCNS patients stem from unchecked proliferation of Muller glia within the retina that results from constitutive Hedgehog-dependent target gene expression. (Specific Aim 3) Downstream of the Hedgehog pathway, vax1 and vax2 occupy a key node in the gene regulatory network mediating ventral optic cup formation and choroid fissure closure. Utilizing a microarray approach we have identified over twenty genes whose expression in the ventral optic cup and choroid fissure is dependent on vax1 and vax2. In this Aim, we will focus on one specific vax1/vax2 target: integrin a5, a fibronectin-binding integrin that we hypothesize is required for choroid fissure cells to interact with a fibronectin-rich extracellular matrix within the fissure to stimulate its closure. We will utilize histological, molecular and in vivo imaging techniques to test this hypothesis. PUBLIC HEALTH RELEVANCE: The research proposed in this grant will shed light on the molecular, cellular and morphogenetic underpinnings governing choroid fissure closure. In humans, defects in choroid fissure closure result in chorioretinal colobomas: congenital ocular defects where large regions of the eye lack both retina and retinal pigmented epithelium because these structures failed to be contained within the eyecup during ocular morphogenesis. Identification of factors required for choroid fissure closure will have a direct impact on our understanding of coloboma formation and on the development of possible treatments.