IN VIVO IMAGING OF CIS REGULATORY EVENTS

荧光指示剂染料的开发、天然荧光蛋白质的分离以及活体显微镜技术的改进，为研究活的、完整的胚胎中的细胞和分子事件提供了前所未有的机会。与此同时，在海胆和海鞘胚胎发育过程中的基因调控研究也取得了巨大的进展，最终提出了用于发育模式的基因调控网络。因为它们的大小、形状和 透明，海胆胚胎和海鞘胚胎是用光学显微镜研究的理想系统。拟议的研究计划将结合成像科学和发育生物学的这些最新进展，以达到三个相关目标：第一，更好地定义完整胚胎中基因活动的时空方面；第二，改进被认为参与模式基因表达的因素的成像和扰动的工具；第三，表征关键时期转录因子的动员和活性。 发育和基因调控事件。双光子激光扫描显微镜(TPLSM)由于具有高灵敏度、低光毒性和对组织的深度穿透性，将作为这些实验的主要成像工具。第一个实验目标将通过将TPLSM与编码绿色荧光蛋白(GFP)及其基因的报告基因相结合来实现 在完整胚胎中产生基因激活的定量分析的颜色变体。平行实验将使用荧光原位杂交技术来检测固定胚胎中的基因激活。第二个目标将涉及TPLSM与GFP融合和特定抗体的结合，以更好地定义转录因子的区域和时间重新分布，以及笼子内的吗啉，以允许光激活的基因调控网络的扰动。第三个目标将涉及转录因子与绿色荧光蛋白突变体的融合，以允许先进的荧光技术随后进行转录因子动员和对接。 这个项目的长期目标是使用分子和光学技术的组合来定义驱动模式基因调控的细胞事件，以及反过来由模式基因调控驱动的发育事件。这将提供测试本计划项目其他组成部分中提出的遗传调控网络所需的工具。

Recent advancements in the development of fluorescent indicator dyes, the isolation of proteins that naturally fluoresce, and the refinement of techniques for in vivo microscopy offer unprecedented opportunities to study the cellular and molecular events within living, intact embryos. In parallel, there has been dramatic progress in the study of gene regulation during the embryonic development of the sea urchin and the ascidian, culminating in the proposal of Gene Regulatory Networks for developmental patterning. Because of their size, shape and transparency, the sea urchin embryo and ascidian embryos are ideal systems for study with light microscopy. The proposed research plan will combine these recent advances in imaging science and developmental biology to attack three related goals: first, to better define the spatio-temporal aspects of gene activity within intact embryos; second, to refine tools for the imaging and perturbation of factors thought to be involved in the patterned gene expression; third, to characterize the mobilization and activity of transcription factors during key developmental and gene regulatory events. Two-photon laser-scanning microscopy (TPLSM) will be used as the major imaging tool in these experiments, because of its high sensitivity, low photo-toxicity and deep penetration into tissues. The first experimental goal will be achieved by combining TPLSM with reporter genes that encode green fluorescent protein (GFP) and its color variants to yield quantitative assays of gene activation in intact embryos. Parallel experiments will employ fluorescent in situ hybridization technologies to assay gene activation in fixed embryos. The second goal will involve TPLSM combined with GFP fusions and specific antibodies to better define the regional and temporal redistribution of transcription factors, and caged morpholinos to permit photoactivatable perturbation of the gene regulatory network. The third goal will involve fusions of transcription factors with green fluorescent protein mutants to permit transcription factor mobilization and docking to be followed by advanced fluorescence techniques. The long-term goal of this project is use a combination of molecular and optical techniques to define the cellular events that drive patterned gene regulation and the developmental events that are in turn driven by patterned gene regulation. This will provide the tools needed to test the genetic regulatory networks proposed in the other components of this Program Project.