This laboratory has focused its attention on retinoid signaling in the developing mouse embryo. This occurs through ligand-inducible transcription factors named retinoic acid receptors(RARs) or retinoid-X receptors(RXRs). These receptors can heterodimerize and bind to response elements associated with the transcriptional promoters of genes. We have used molecular, cellular and animal techniques to identify regions of active retinoic acid receptor activity in embryos, sources of retinoic acid in embryos, and the molecular structure of some of the genes encoding these receptors. In the mouse, we are currently using procedures that should allow us to identify genes that are regulated via these receptors.

In addition to this work, we have been involved in using magnetic resonance microscopy to image and render in three dimensions, developing mouse embryos. This has resulted in a CD ROM 'Digital Atlas of Mouse Embryology' (B.R. Smith, G.A. Johnson and E. Linney).

We recognized certain limitations in working with complex signaling in developing mouse embryos and have recently begun to work with an alternative vertebrate system, the zebrafish. This system allows us to obtain quite easily hundreds of developing embryos and to use simple injections to introduce proteins, mRNAs and DNAs into the embryos.

Because the zebrafish embryo does not markedly grow in overall size during embryonic development, it can be 'optically sectioned' completely throughout embryonic development using fluorescent markers and the scanning confocal laser microscope.

For the above reasons and more, we have begun to use the zebrafish system in our studies of retinoid signaling during embryonic development and are adapting methods and techniques which should allow us to 'see' gene expression in developing zebrafish embryos using fluorescent reporter molecules. Because of its small embryonic size, one can examine the pattern of a gene throughout development using the technique of whole mount in situ hybridization. We are adapting this technique to provide us with a fluorescent signal so that we can capture, in three dimensions, the localization of specific mRNAs and then use computer reconstruction and rendering techniques to construct the location of the mRNA in three dimensions.

With the above technologies, we are now addressing several different questions:

1) Are there localized regions of retinoic acid receptor activity in the developing zebrafish embryo?

2) Can we interfere and re-direct this signaling through introducing of in vitro synthesized mRNAs that would inhibit receptor activity?

3) Can we identify adaptor molecules which allow the receptor to communicate with the transciption complex using the yeast two-hybrid selection system?

4) Can we develop the zebrafish embryo as a biosensor for environmental estrogens using the above technology in combination with the creation of transgenic indicator zebrafish for estrogen receptor activity?