Transgenic Studies of Vertebrate Retinal Development

描述（由申请人提供）：为了更详细地检查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，在视杆细胞特异性启动子下表达绿色荧光蛋白（GFP）的转基因斑马鱼将以两种方式使用：A）正向遗传筛选以鉴定影响视杆细胞发育的隐性突变。纯合雄性转基因斑马鱼将通过暴露于乙基亚硝基脲进行诱变。将突变的斑马鱼与纯合转基因雌性交配以产生转基因纯合的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，表明视杆细胞数量和/或视网膜分化的改变。B）化学遗传筛选以鉴定破坏参与正常视网膜发育和视杆细胞分化的途径的分子。将转基因胚胎以3个胚胎/96孔板的孔的密度排列，并暴露于来自已知生物活性物质文库的小分子。小分子文库由2489种化合物组成，并被组装以包含具有已知生物活性和先前鉴定的蛋白质靶标的分子。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（hpf），视杯开始形成; 24 hpf，成视网膜细胞增殖但在细胞分化之前; 48 hpf，视杆细胞分化开始。将通过标准光学显微镜检查胚胎以筛选一般形态学缺陷，通过微分干涉对比（DIC）显微镜检查胚胎以筛选视网膜分层缺陷，以及通过荧光显微镜检查胚胎以筛选视杆细胞分化缺陷。在识别出特定突变或小分子后，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。这些研究的目标是确定在常规的遗传筛查中可能错过的视网膜发育途径。

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.