IN VIVO IMAGING OF CIS REGULATORY EVENTS

荧光指示剂染料的开发，天然荧光蛋白的分离，以及体内显微镜技术的改进，为研究活的完整胚胎内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进展，最终提出了基因调控网络的发展模式。由于它们的大小，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。拟议的研究计划将联合收割机结合成像科学和发育生物学的最新进展，以实现三个相关目标：第一，更好地定义完整胚胎内基因活动的时空方面;第二，改进被认为参与模式化基因表达的因素的成像和扰动工具;第三，表征关键过程中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）将被用作这些实验中的主要成像工具，因为它具有高灵敏度，低光毒性和深入组织的穿透性。第一个实验目标将通过将TPLSM与编码绿色荧光蛋白（GFP）及其编码的报告基因组合来实现。 颜色变体以产生完整胚胎中基因激活的定量测定。平行实验将采用荧光原位杂交技术来测定固定胚胎中的基因激活。第二个目标将涉及TPLSM与GFP融合和特异性抗体的组合，以更好地定义转录因子的区域和时间再分布，以及笼状吗啉代，以允许基因调控网络的光活化扰动。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，以允许转录因子的动员和对接，随后是先进的荧光技术。 该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化基因调控的细胞事件以及反过来由模式化基因调控驱动的发育事件。这将提供所需的工具来测试本计划项目的其他组成部分中提出的遗传调控网络。

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.