Transgenic Studies of Vertebrate Retinal Development

描述(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，将在斑马鱼中使用新的筛选方法。具体地说，在杆状光感受器特异性启动子下表达绿色荧光蛋白(GFP)的转基因斑马鱼将用于两种方式：a)正向遗传筛选，以确定影响杆状发育的隐性突变。纯合子雄性转基因斑马鱼将通过接触乙基亚硝脲进行诱变。突变的斑马鱼将被培育成纯合子的转基因雌性斑马鱼，以产生转基因纯合子的F2代家庭。为了识别隐性突变，F3代FISH将通过光学和荧光显微镜检查GFP表达的变化，表明杆状感光细胞数量和/或视网膜分化的变化。B)化学遗传筛选，以确定扰乱参与视网膜正常发育和视杆分化的途径的分子。转基因胚胎将以96孔板的3个胚胎/孔的密度排列，并暴露于已知生物活性物质库中的小分子。小分子文库由2489个化合物组成，它们被组装成包含具有已知生物活性和先前确定的蛋白质靶标的分子。胚胎将在三个对眼睛发育具有重要意义的时间点暴露于小分子：受精后9小时，视杯形成开始；24小时，视网膜母细胞增殖，但细胞分化之前；48小时，视杆感光细胞分化开始。胚胎将用标准光学显微镜来筛选一般的形态缺陷，用差分干涉对比(DIC)显微镜来筛选视网膜板层中的缺陷，用荧光显微镜来筛选杆状感光细胞分化中的缺陷。在鉴定出特定的突变或小分子后，必要时将通过组织学、免疫组织化学、原位杂交和镶嵌分析来表征表型。这些研究的目标将是确定常规正向遗传筛查可能遗漏的视网膜发育途径。

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.