RETINOID SIGNALING DURING EMBRYOGENESIS

Duke University, Durham NC

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Abstract

The proposal is focused upon using the zebrafish embryo as a model system for examining the nature of retinoid signaling during embryogenesis. This laboratory's past experience in working with mouse embryos has provided us with a picture of retinoid signaling during embryogenesis but the mouse system is much more difficult to examine in real-time because of the absence of in vitro culture systems that would allow for following events directly and continuously throughout development. In this proposal we plan to use fluorescent reporter techniques (the expression of green fluorescent protein and mutants thereof as a reporter gene and fusion proteins and the use of fluorescent whole mount in situ hybridization), transgenic approaches, microinjection of in vitro synthesized fusion mRNAs, confocal laser scanning microscopy, and three-dimensional computer reconstruction of digitized fluorescent data to develop a picture of retinoid signal during development of the embryo. We plan to computer archive the appearance of the zebrafish retinoic acid receptors and retinoid-X receptors during embryonic development; to develop transgenic zebrafish which will allow us to determine where retinoic acid receptor activity occurs; to introduce the expression of fusion proteins which will interfere with this expression. Our experience in the mouse embryo suggests that one does not observe retinoid signaling just because receptors and retinoids co-exist at specific locations in the embryos. We have isolated a putative zebrafish homologue to the human SMRT co-repressor and plan to examine and isolate a zebrafish homologue to the N-Cor co-repressor. These co-repressor will be characterized, their 3-dimensional expression patterns will be determined and computer archived using fluorescent whole mount in situ hybridization, and their possible role in selecting or limiting retinoid signaling in the embryo will be examined.