Laser-induced gene expression in specific cells of transgenic zebrafish

Over the past few years, a number of studies have described the generation of transgenic lines of zebrafish in which expression of reporters was drive n by a variety of promoters. These lines opened up the real possibility tha t transgenics could be used to complement the genetic analysis of zebrafish development. Transgenic lines in which the expression of genes can be regu lated both in space and time would be especially useful. Therefore, we have cloned the zebrafish promoter for the inducible hsp70 gene and made stable transgenic lines of zebrafish that express the reporter green fluorescent protein gene under the control of a hsp70 promoter, At normal temperatures, green fluorescent protein is not detectable in transgenic embryos with the exception of the lens, but is robustly expressed throughout the embryo fol lowing an increase in ambient temperature, Furthermore, we have taken advan tage of the accessibility and optical clarity of the embryos to express gre en fluorescent protein in individual cells by focussing a sublethal laser m icrobeam onto them. The targeted cells appear to develop normally: cells mi grate normally, neurons project axons that follow normal pathways, and prog enitor cells divide and give rise to normal progeny cells. By generating ot her transgenic lines in which the hsp70 promoter regulates genes of interes t, it should be possible to examine the in vivo activity of the gene produc ts by laser-inducing specific cells to express them in zebrafish embryos. A s a first test, we laser-induced single muscle cells to make zebrafish Sema 3A1, a semaphorin that is repulsive for specific growth cones, in a hsp70-s ema3A1 transgenic line of zebrafish and found that extension by the motor a xons was retarded by the induced muscle.