IN VIVO IMAGING OF CIS REGULATORY EVENTS

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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.