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. Because of its size, shape and transparency, the sea urchin embryos is an ideal system for study with light microscopy. The proposed research plan will combine these recent advances in imaging science and reporter gene activity within intact embryos; second, to characterize the post-translational modification of transcription factors thought to be involved in the patterned gene expression; third, to characterize the occupancy of cis-regulatory domains by 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 an deep penetration into tissues. The first experimental goal will be achieved by combining TPLSM with reported genes that encode gene fluorescent protein (GFP) or beta-lactamase (and a fluorescent reporter dye), resulting in quantitative assays of gene activation in intact embryos. The second goal will involved TPLSM combined with phospho-peptide specific antibodies to better define the post-translational modification thought to be critical for the regional and temporal control of transcription factor activity. The third goal will involve fusions of transcription factors with green fluorescent protein mutants to permit resonance energy transfer between and within transcription factors to be used as assays of transcription factor binding. The long-term goal of this project is use a combination of molecular and optical techniques to define of the cellular events that drive patterned gene regulation and the developmental events that are driven by patterned gene regulation.
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