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形态发生的机制基础。在眼的形态发生过程中，视原基经历一系列复杂的形态发生运动，最终形成包含预期视网膜和RPE的双层视杯。每个视原基的神经外胚层层必须沿着其近-远轴融合，使得在眼形态发生的早期阶段，视网膜和RPE将被限制在视杯的范围内。融合发生在沿着视杯的一个独特区域，称为脉络膜裂。促进脉络膜裂闭合的分子机制和细胞过程在任何脊椎动物生物体中都没有得到很好的表征。脉络膜裂闭合缺陷导致脉络膜视网膜缺损，使人眼的先天性畸形衰弱。脉络膜视网膜缺损的分子基础在很大程度上是未知的，很少有动物模型来研究其病因。在这个建议中的实验将利用斑马鱼，Danio rerio，阐明分子和细胞机制，眼睛形态发生和脉络膜裂关闭。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脉络膜视网膜缺损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缺损表型的分子，细胞和形态发生基础。我们已经克隆了这个突变体中被破坏的位点，并确定了patched 1基因中的突变，patched 1基因是刺猬途径的负调节因子。在这里，我们将测试这一假设，即缺损表型井喷导致过度增殖的视柄，我们将确定这种表型的分子机制。（特定目的2）人PATCHED 1中的突变导致基底细胞痣综合征或Gorlin综合征（BCNS），这是一种表现为眼部缺陷的病症，包括胶质源性视网膜前膜的形成、视网膜发育不良和缺损。这些缺陷的分子和细胞基础尚不清楚，我们将利用成人井喷/patched 1和小妖精/patched 2突变体来测试这一假设，即BCNS患者的眼部缺陷源于视网膜内Muller神经胶质细胞的不受抑制的增殖，这是由组成性刺猬依赖性靶基因表达引起的。（具体目的3）在Hedgehog通路的下游，vax 1和vax 2在介导腹侧视杯形成和脉络膜裂闭合的基因调控网络中占据关键节点。利用微阵列的方法，我们已经确定了超过20个基因，其在腹侧视杯和脉络膜裂的表达依赖于vax 1和vax 2。在这个目标中，我们将专注于一个特定的vax 1/vax 2目标：整合素a5，我们假设是必要的脉络膜裂细胞与富含纤维连接蛋白的细胞外基质的裂缝，以刺激其关闭的纤维连接蛋白结合的整合素。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：这项研究将揭示脉络膜裂隙闭合的分子、细胞和形态发生基础。在人类中，脉络膜裂闭合缺陷导致脉络膜视网膜缺损：先天性眼部缺陷，其中眼睛的大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发生期间未能包含在眼杯内。确定脉络膜裂闭合所需的因素将直接影响我们对缺损形成的理解和可能治疗方法的发展。

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.