FISH and Chips: Applying Microarray Technology and in Situ Hybridization to Understanding Light-Adaptation in Zebrafish

生物研究的一项重大突破是DNA微阵列的发展，它可以将数千个不同的基因片段“打印”到一个小的载玻片或芯片上。这些芯片在特定的生理条件下表现出不同的基因表达。到目前为止，还不可能评估暴露于不同照明方案或经历不同昼夜节律阶段的鱼视网膜中基因表达的变化。该提案将使用微阵列技术来鉴定在适应光和黑暗的斑马鱼视网膜中表达的基因差异。脊椎动物视网膜中的视紫红质、参与调节昼夜节律的各种基因、褪黑素及其生物合成的酶、多巴胺和GABA以及ph都发生昼夜节律或光驱动振荡。早期nsf资助的研究表明，乙酰胆碱刺激从蓝鳃鱼眼睛分离的视网膜色素上皮（RPE）中的光适应性色素颗粒分散，并作用于g蛋白偶联的毒碱受体。因此，合成毒蕈碱受体信号通路上游和下游效应器所需的基因受到了特别的关注。在这项研究中，提出了两种方法：(1)比较中午和午夜从适应12/12光/暗循环的鱼类中分离的视网膜的mRNA水平；(2)进行原位杂交研究以定位感兴趣基因的表达。视网膜中基因表达的昼夜调节的微阵列分析将为研究参与细胞信号传导、神经递质生物合成和神经递质受体的基因表达之间的相关性奠定基础。结果将用于将表达阶段与受体对配体的亲和力联系起来，并更好地理解节律性表达的生理含义。原位杂交研究将允许将表达的基因置于细胞环境中。微阵列研究增加了相关人员的研究技能。这项研究将提供意外收获的数据，这些数据可能会揭示与信号传导途径无关的基因，但这些基因调节着色素颗粒的运动。因此，学生和其他研究人员将能够使用这些数据来开展自己的研究事业。此外，本提案将支持至少一名硕士生和至少一名博士生的论文研究。

A major breakthrough in biological research is the development of DNA microarrays in which thousands of different gene segments can be "printed" onto a small slide, or chip. Such chips show differential gene expression under specific physiological conditions. To this point, it has not been possible to assess changes in gene expression in the retina of fish exposed to different lighting regimens or experiencing different circadian phases. This proposal will use microarray technology to identify differences in genes expressed in light- versus dark-adapted zebrafish retinas. Circadian or light-driven oscillations occur in the vertebrate retina for rhodopsin, for various genes involved in regulation of circadian rhythms, for melatonin and the enzymes involved in its biosynthesis, for dopamine and GABA, and for pH. Earlier NSF-funded studies show that acetylcholine stimulates light-adaptive pigment granule dispersion in retinal pigment epithelium (RPE) isolated from the eye of bluegill, and that it acts upon G-protein-coupled muscarinic receptors to do so. Therefore, genes required for synthesis of both upstream and downstream effectors of the muscarinic receptor signaling pathway are of special interest. For this study, two approaches are proposed: (1) comparing mRNA levels between retinas isolated at midday and midnight from fish acclimated to a 12/12 light/dark cycle, and (2) performing in situ hybridization studies to localize expression of genes of interest. Microarray analysis of diurnal regulation of gene expression in the retina will lay the foundation for examining correlations between expression of genes involved in cell signaling, neurotransmitter biosynthesis, and neurotransmitter receptors. Results will be used to relate the phase of expression to the affinity of receptors for ligand, and to better understand the physiological implications of the rhythmic expression. In situ hybridization studies will permit the expressed genes to be placed in the cellular context. The microarray study add to the research skill set for those involved. The research will provide a windfall of data that will likely reveal genes not related to the signaling pathways, but that regulate pigment granule movement. Therefore, students and other researchers will be able to use those data to launch their own research careers. In addition, this proposal will support the thesis research of at least one master's student and at least one Ph.D. student.