Expression of GST-GFP in zebrafish embryos. (A) An uninjected embryo (lower left) and an embryo injected at the single cell stage with GST-GFP cRNA (upper right). (B) Only the GST-GFP injected embryo fluoresces and the uninjected embryo gives little background fluorescence. (F) A confocal micrograph of another embryo 4 hr after being injected with GST-GFP cRNA at the one-cell stage, demonstrating the homogeneity GST-GFP expression. (C, D, and E) Micrographs of the same embryo taken at 4 (C and D) and 30 (E) hr after injection with GST-GFP cRNA into one cell at the two-cell stage. (G and H) Confocal fluorescence micrographs of the same embryo at 3.5 and at 30 hr after injection of GST-GFP cRNA into one cell at the eight-cell stage. Fluorescence can easily be detected only 3-4 hr after injection and at 30 hr the fluorescence is extremely intense, allowing the ready identification of embryonic tissues such as the eye (e), brain (b), and tail musculature (m). Using confocal microscopy at 30 hr single cells comprising embryonic organs could easily be identified (r, retina; l, lens; d, diencephalon). (H) Persistent fluorescence at 4 days after injection at the one-cell stage with GFP-GST cRNA. Magnifications: A-E and I, 200x: F-G, 250x.

Overlapping distribution of green and red fluorescence in embryos co-injected with GST-GFP cRNA and Texas red dextran. A mixture of GST-GFP cRNA (1 μg/ml) and Texas red dextran (2000 K; 2mg/ml) was injected into a single blastomere of embryos at the eight-cell stage. The distribution patterns of GST-GFP protein and Texas red dextran was compared using dual fluorescence microscopy. (A and B) The overlapping distribution of green and red fluorescence, respectively, in a group of cells cascading over the surface of the yolk, possibly endothelial cells forming the venous return to the embryonic heart. (D and E) The overlapping distribution of green and red fluorescence, respectively, in individual cells of the notochord. (C and F) Computer-generated subtractions of the green images from the red images (B minus A and E minus F, respectively) clearly demonstrating the high degree of overlap in the distribution GST-GFP and the Texas red dextran. Cells contained within the white boxes A, B, D, and E were enlarged in G, H, I, and J, respectively, to allow inspection of overlap between green and red fluorescence on a cell by cell basis. Embryos injected with GST-GFP cRNA or Texas red dextran alone showed no detectable GFP fluorescence using the Texas red filter set or Texas red fluorescence using the fluorescein filter set (data not shown). Original magnification: A-C, 100x; D-F, 200x.

Short half-life of GST-GFP cRNA in zebrafish embryos. GST-GFP cRNA was synthesized in the presence of ³⁵S-UTP and injected into one-cell zebrafish embryos. Total RNA was isolated from embryos at 0, 4, and 24 hr after injection (15 embryos at each time point), ethanol-precipitated, resuspended, and analyzed on a 5% polyacrylamide gel. After overnight exposure of the gel to a phosphorimager screen, a single radiolabeled band was evident at 0 hr but by 24 hr was barely detectable. The estimated half-life of the injected GST-GFP cRNA was less than 2 hr based on quantitation of the bands by phosphorimager analysis.