Analysis Of Cell Fate Acquisition In the Retina

描述（由申请人提供）：脊椎动物视网膜由七种主要细胞类型组成，以惊人的精确度组织成层状模式。在胚胎发生过程中，所有的视网膜神经元都是由形态上均匀的神经上皮细胞群产生的。在成熟的视网膜中，各个神经元类别的特征在于不同的功能、位置和形态。我们想确定是什么遗传机制赋予视网膜神经元和神经胶质细胞这些独特的身份。 为了确定视网膜中细胞命运获取所涉及的遗传基因座，我们采取了反向遗传方法。我们的合作者和我们利用斑马鱼胚胎的子宫外发育和透明性来寻找在视网膜神经元发育的最早阶段以细胞类限制模式表达的基因。对7 000多个克隆进行的原位筛选显示，9个克隆显示出这些特征。这9个基因的结构和功能将进一步表征。结构表征将揭示分子基序，如蛋白质-蛋白质相互作用界面，DNA结合结构域和催化位点，为生物化学功能提供见解。功能表征将利用两种技术。首先，我们将使用最近引入的基因敲除技术来消除单个基因的功能。第二，我们将研究中的基因表达超过正常水平。这些实验操作产生的表型异常将揭示这些基因在视网膜发育中的作用。由于已知参与视网膜早期细胞命运获取的因素很少，这些研究可能为这一过程提供关键见解。 在这里描述的大规模原位杂交筛选中选择的细胞类限制性基因的表征将打开许多令人兴奋的研究机会。对它们在包括人类在内的高等脊椎动物中的功能的研究可以揭示与人类疾病如光感受器营养不良和青光眼的关系，并且可以提供用于治疗这些疾病的工具。

DESCRIPTION (provided by applicant): The vertebrate retina consists of seven major cell types organized with striking precision in a laminar pattern. During embryogenesis, all retinal neurons arise from a morphologically uniform population of neuroepithelial cells. In the mature retina, individual neuronal categories are characterized by distinct functions, positions, and morphologies. We would like to determine what genetic mechanisms confer these unique identities on the retinal neurons and glia. To identify the genetic loci involved in cell fate acquisition in the retina, we took a reverse genetic approach. Our collaborators and we took advantage of the extrauterine development and transparency of the zebrafish embryo to search for genes expressed in cell-class restricted patterns at the earliest stages of neuronal development in the retina. An in situ screen of over 7,000 clones revealed 9 that display these characteristics. The structure and function of these 9 genes will be characterized further. Structural characterization will reveal molecular motifs, such as protein-protein interaction interfaces, DNA binding domains, and catalytic sites providing insights into biochemical function. Functional characterization will take advantage of two techniques. First, we will use the recently introduced technique of gene knockdown to abolish function of individual genes. Second, we will express the genes under investigation in excess of the normal level. The phenotypic abnormalities produced by these experimental manipulations will reveal the roles these genes play in the development of the retina. As very few factors are known to be involved in early cell fate acquisition in the retina, these studies may provide key insights into this process. Characterization of cell-class restricted genes selected in a large-scale in situ hybridization screen described here will open a number of exciting research opportunities. Studies of their functions in higher vertebrates including humans may reveal relationships to human disorders such as photoreceptor dystrophies and glaucoma and may provide tools for the treatment of these disorders.