Transgenic Studies of Vertebrate Retinal Development

描述（由申请人提供）：为了更详细地研究脊椎动物视网膜的发育过程，我们将在斑马鱼中使用新的筛选方法。具体来说，在杆状光感受器特异性启动子下表达绿色荧光蛋白（GFP）的转基因斑马鱼将被用于两种方式：a)通过正向遗传筛选来识别影响杆状体发育的隐性突变。纯合子雄性转基因斑马鱼暴露在乙基亚硝基脲中会发生突变。诱变的斑马鱼将被培育成纯合子转基因雌性，产生转基因纯合子的F2代家族。为了鉴定隐性突变，F3代鱼将通过光学和荧光显微镜检查GFP表达的变化，表明杆状光感受器数量和/或视网膜分化的改变。B)化学遗传筛选，以识别干扰视网膜正常发育和视杆分化通路的分子。转基因胚胎将在96孔板上以3个胚胎/孔的密度排列，并暴露于已知生物活性物质文库中的小分子中。小分子文库由2489个化合物组成，并被组装成含有已知生物活性和先前确定的蛋白质靶点的分子。胚胎将在眼睛发育的三个重要时间点接触小分子：受精后9小时，视杯形成开始；24 hpf，成视网膜细胞增殖时间，但在细胞分化之前；48hpf，杆状光感受器分化开始。胚胎将通过标准光学显微镜检查，以筛查一般形态学缺陷，鉴别干涉对比（DIC）显微镜检查，以筛查视网膜层压缺陷，荧光显微镜检查，以筛查杆状光感受器分化缺陷。在确定特定突变或小分子后，表型将通过组织学，免疫组织化学，原位杂交和马赛克分析来表征，必要时。这些研究的目标将是确定视网膜发育的途径，可能在常规的正向遗传筛查中被遗漏。

DESCRIPTION (provided by applicant): To examine in more detail the developmental processes in the vertebrate retina, new screening methods will be used in zebrafish. Specifically, transgenic zebrafish expressing the green fluorescent protein (GFP) under a rod photoreceptor specific promoter will be used in two ways: A) A forward genetic screen to identify recessive mutations affecting rod development. Homozygous male transgenic zebrafish will be mutagenized by exposure to ethylnitrosourea. Mutagenized zebrafish will be bred to homozygous transgenic females to produce F2 generation families that are homozygous for the transgene. To identify recessive mutations, F3 generation fish will be examined by light and fluorescence microscopy for changes in GFP expression, indicating an alteration in rod photoreceptor number and/or retinal differentiation. B) Chemical genetic screens to identify molecles that disrupt pathways involved in proper retinal development and rod differentiation. Transgenic embryos will be arrayed at a density of 3 embryos/well of a 96-well plate and exposed to small molecules from libraries of known bioactive substances. The small molecule libraries consist of 2489 compounds and were assembled to contain molecules with known biological activity and with previously identified protein targets. Embryos will be exposed to small molecules at three timepoints significant in eye development: 9 hours post fertilization (hpf), when optic cup formation begins; 24 hpf, a time of retinoblast proliferation but prior to cellular differentiation; 48 hpf, the onset of rod photoreceptor differentiation. Embryos will be examined by standard light microscopy to screen for general morphological defects, differential interference contrast (DIC) microscopy to screen for defects in retinal lamination, and fluorescence microscopy to screen for defects in rod photoreceptor differentiation. Upon identification of specific mutations or small molecules, the phenotypes will be characterized by histology, immunohistochemistry, in situ hybridization, and mosaic analysis, when necessary. The goal of these studies will be to identify pathways in retinal development that could be missed in routine forward genetic screens.