This laboratory has developed technology which allows us to visualize signal transduction pathways in developing zebrafish embryos. This has been done by introducing fluorescent reporter genes (green fluorescent protein and yellow versions of the green fluorescent protein) coupled to transcriptional regulatory elements either through direct DNA injection into embryos or through pseudotyped retroviral vector infection. We have been exploring the roles of the retinoic acid response and the estrogen response using these technologies. At the same time we are working with transgenic embryos which express fluorescent reporter genes in every cell and in subsets of cells in the nervous system. Techniques have been developed to immobilize embryos in agar and perform time-lapse fluorescent microscopy throughout embryonic development so that Quick-time movies can be created of gene expression during embryonic development.

We are beginning to use these living reagents for two major purposes:

1. to genetically dissect signal transductions systems associated with embryonic development; and
2. to determine whether these transgenic lines can be used as biosensors for the presence of environmental toxicants which can affect embryonic development and growth.

Our genetic approaches with the retinoic acid response involve using either existing zebrafish mutants or imposed "morphant" phenocopying of mutations. "Morphants" are produced through the design and application of nuclease resistant anti-sense morpholinos. These are designed to selectively knockdown the expression of genes in zebrafish embryos so that the effects of reducing expression can be measured both morphologically and the effects upon the fluorescent pattern of our transgenic lines.

The combination of morpholino knockdown and fluorescent, transgenic examination of development after the inhibition of specific genes allows us to identify the morphological consequences of inactivating specific genes. To these technologies we are adding microarray analysis so that we can survey the effects of specific gene inactivation upon thousands of zebrafish genes. These combined technologies should aid us in dissecting pathways of normal embryonic development and genes affected by environmental toxicants.