Tracing transgene expression in living zebrafish embryos.

Ectopic expression by injection of plasmid DNA is rarely used in zebrafish embryos due to a low frequency of cells expressing a transgene of interest at detectable levels. Furthermore, the mosaic nature of ectopic expression by plasmid injection requires the direct detection of transgene-expressing cells. We have used the transcriptional activator Gal4-VP16 to amplify transgene expression in living zebrafish embryos. In comparison to conventional expression vectors, Gal4-VP16-amplified expression results in a significant higher number of cells which express a transgene at detectable levels. The Gal4-VP16-activator and the Gal4-VP16-dependent transgene can be placed on a single expression vector. Using tissue-specific regulatory elements, we show that expression of a Gal4-VP16-dependent transgene can be reliably restricted to muscle, notochordal, or neuronal tissues. Furthermore, Gal4-VP16 can drive the expression of two or more transgenes from the same construct resulting in simultaneous coexpression of both genes in virtually all expressing cells. The reported expression system works effectively not only in zebrafish embryos but also in Xenopus embryos, chicken, mouse, and human cultured cells and is thus applicable to a broad variety of vertebrates. The high frequency of transgene expression together with the linked coexpression of more than one transgene opens the possibility of easily monitoring the behavior of individual transgene-expressing cells in real time by labeling them with the fluorescent reporter GFP. The combinatorial nature of the expression system greatly facilitates changing the tissue-specificity, the transgene expressed, or the cell compartment-specific GFP reporter, making it simpler to address a gene's function in different tissues as well as its cell biological consequences. Copyright 2001 Academic Press.