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 tran sgene expression ill living zebrafish embryos. In comparison to conventiona l expression vectors, Gal4-VP16-amplified expression results in a significa nt higher number of cells which express a transgene at detectable levels. T he Gal4-VP16-activator and the Gal4-VP16-dependent transgene can be placed on a single expression vector. Using tissue-specific regulatory elements, w e show that expression of a Gal4-VP16-dependent transgene can be reliably r estricted to muscle, notochordal, or neuronal tissues. Furthermore, Gal4-VP 16 can drive the expression of two or more transgenes from the same constru ct resulting in simultaneous coexpression of both genes in virtually all ex pressing cells. The reported expression system works effectively not only i n zebrafish embryos but also in Xenopus embryos, chicken, mouse, and human cultured cells and is thus applicable to a broad variety of vertebrates. Th e high frequency of transgene expression together with the linked coexpress ion of more than one transgene opens the possibility of easily monitoring t he behavior of individual transgene-expressing cells in real time by labeli ng 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 consfquences. (C) 2001 Academic Press.