Köster et al., 2001 - Tracing transgene expression in living zebrafish embryos. Developmental Biology   233(2):329-346 Full text @ Dev. Biol.

Fig. 2 Gal4-VP16-mediated expression in comparison to conventional DNA-construct expression. Lateral views of the hindbrain (A, C, E) and trunk (B, D, F) of CMV-lyn-injected (A, B), CMV-GVP/Ulyn-coinjected, and EF-GVP/Ulyn-coinjected embryos at 3 dpf. Pictures represent pseudocolored composites of single optical sections using confocal microscopy. EGFP-expressing cells are displayed in green. Embryos where counterstained with Bodipyceramide TexasRed (Molecular Probes) displayed in red to visualize overall morphology. Note the precise membranelocalization of the lynEGFP-fluorescence in expressing cells (e.g., neuron in A or muscle cells in D). Abbreviations: nc, notochord; nt, neural tube; otv, otic vesicle; rh, rhombencephalon.

Fig. 3 Combined Gal4-VP16-activator/effector constructs result in strong EGFP expression in numerous cells of DNA-injected embryos. Single optical confocal microscopy section (lateral view) focusing on the nervous system of injected embryos at 37 hpf. Two embryos were embedded together in low-melting agarose to allow direct comparison of EGFP-expression levels and number of EGFP-expressing cells in a single optical section. Fluorescent (A pseudocolored in green) and transmitted light (B) images were taken at the same focal plane without moving the embryos. (A, B) EF-GVP-UG-injected embryos of the same batch. EGFP expression in EF-GVP-UG-injected embryos can vary strongly in intensity and frequency from a high number of embryonic GFP-expressing cells (A, upper embryo) to very few EGFP-expressing cells (A, lower embryo). Higher laser power and increased contrast, however, revealed a higher frequency of EGFP-expressing cells in the lower embryo than judged by the initial conditions (see A, inset).

Fig. 5 Gal4 is a weaker transcriptional activator in zebrafish embryos than Gal4-VP16. (A–D) Single optical confocal microscopy sections (lateral view) of injected embryos at 27 hpf. Two embryos were embedded together in low-melting agarose to allow direct comparison of GFP-expression levels and number of GFP-expressing cells in a single optical section. Fluorescent (A and C, pseudocolored in green) and transmitted light (B, D) images were taken at the same focal plane without moving the embryos. (A, B) While Gal4-mRNA/UG-coinjected embryos display a broad variety of EGFPexpression, including embryos with a high frequency of EGFP-expressing cells (upper embryo), EF-G-UG-injected embryos show no or very dim EGFP fluorescence in a few cells (lower embryos). In contrast to EF-GVP-UG-injected embryos (see Fig. 3A, lower embryo), increase in laser power and contrast did not reveal an increase in the frequency of EGFP-expressing cells in EF-G-UG-injected embryos (not shown). (C, D) EF-GVP/EF-G-UG-coinjected embryos display a broad variety of EGFP expression, including embryos with strongly expressing cells in a high frequency (upper embryos), while EF-G-UG-injected embryos show no or very dim EGFP fluorescence in a few cells (lower embryo).

Fig. 6 Gal4-VP16-activator/effector constructs can drive expression in different mesodermal tissues. (A–D) Lateral views of injected zebrafish embryos at 27 hpf; images were taken by using confocal microscopy. Pictures represent merged stacks of 10 individual optical sections covering 90 μm in depth. (A, B) Same twhh-GVP-UG-injected embryo imaged with fluorescent (A, EGFP fluorescence pseudocolored in green) and transmitted light (B) without moving the specimen. (A1, B1) Magnification of notochordal region of images in A and B, respectively, to demonstrate EGFP fluorescence in vacuolated cells (*) in the center of the notochord and notochordal sheath cells (arrowhead) surrounding the vacuolated cell type. (C, D) Same twhh-GVP-UG/EF-GVP-coinjected embryo imaged with fluorescent (C, EGFP fluorescence pseudocolored in green) and transmitted light (D) without moving the specimen. Note intense ubiquitous EGFP expression in comparison to notochord-specific twhh-GVP-UG-injected embryo shown in (A). This indicates that injected twhh-GVP-UG is distributed in cells over all germ layers but activates expression almost exclusively in the notochord. (E–H) Dorsal views of act-GVP-UG-injected embryos at 3 dpf; images were taken by using confocal microscopy and represent single optical sections. Pictures in (E, F) and (G, H) show the same embryo imaged with fluorescent (E and G, EGFP expression pseudocolored in green) and transmitted (F, H) light. Note almost exclusive EGFP expression in muscles of embryo shown in (E and F), while the embryo shown in (G and H) displays also expression in numerous cells of the hindbrain. Abbreviations: c, cerebellum; fp, floorplate; nt, neural tube; rh, rhombencephalon; sm, somitic muscles.

Fig. 7 Neural-specific expression in transient transgenic Gal4-VP16-activator/effector injected zebrafish embryos. Lateral views of -1696α1TIpEGFP- (A), tub-GVP-Uunc- (B, C), and tub-GVP-Uunc-inv-injected (D, E) embryos showing single optical sections which were taken at 2 (A) or 3 dpf (B–E) using confocal microscopy. Pictures represent pseudocolored composites with EGFP fluorescence displayed in green overlaid with either a transmitted light image (A, B, D) or Bodipyceramide-TexasRed counterstain to visualize overall morphology (C, E). (A) The lateral view of -1696α1TIpEGFP-injected embryo demonstrates that EGPF expression is almost exclusively confined to neurons (arrowheads). Only very few neurons show EGFP fluorescence at detectable levels. Diffusion of EGFP into the neuronal processes also visualizes axons, dendrites, and synaptic butons (see inset, magnification of EGFP-expressing neuron in trunk). (B, D) Overview of the head region of tub-GVP-Uunc- (B) and tub-GVP-Uunc-inv-injected (D) embryos, note the strong and high-frequent expression in the hindbrain and spinal chord. The tub-GVP-Uunc-inv-injected specimen (D) displays expression in the optic tectum demonstrating the linear arrangement of these neurons projecting into the tectal neuropil (*). Also, ventrally projecting axons leaving the spinal cord can be observed (e.g., arrowheads). (C, E) Higher magnification of the cerebellum (lateral views) of the injected embryos shown in (A) and (C). In the tub-GVP-Uunc-injected specimen (C), even tiny neurons (supposedly granule cells, arrowheads) and their processes can be visualized. In the tub-GVP-Uunc-inv-injected specimen (E), the predominant membrane localization of the unc76:EGFP-fusion protein can be seen highlighting the ventral projections (see arrowhead) of the labeled cerebellar neurons. Abbreviations: c, cerebellum; otec, optic tectum; rh, rhombencephalon.

Fig. 8 Gal4-VP16-mediates coexpression of two UAS effectors in a very high frequency. (A–L) Confocal microscopy pictures of cultured primary zebrafish cells transfected with different Gal4-VP16 activator/effector constructs. (A–C) EF-GVP/UH2B cotransfected cells, images of H2BEYFP fluorescence (A, pseudocolored in green) and Bodipyceramide-TexasRed counterstained cells (C, pseudocolored in red) were overlaid (B) to confirm nuclear localization of the histone2B-EYFP-fusion protein. (D–F) EF-GVP/Ulyn cotransfected cells, fluorescent (D), and transmitted (F) light images were overlaid (E) to confirm the membrane localization and nuclear exclusion of the lynEGFP-fusion protein. Comparison of EF-GVP/UH2B cotransfected (G, J), EF-GVP/Ulyn cotransfected (I, L), and EF-GVP-Ulyn-UH2B-transfected cells (H, K) shows characteristic localization, size, and shape of the different EGFP-targeted organelles (pseudocolored in green) to allow scoring of simultaneous coexpression of both EGFP variants in individual and EF-GVP-Ulyn-UH2B-transfected cells. Scoring results in percentages are shown in the diagram below.

Fig. 9 Gal4-VP16 is capable of mediating coexpression of two UAS-dependent transgenes in individual cells of living zebrafish embryos. (A–G) Single optical sections through EF-GVP-Ulyn-UH2B-injected embryo at 86 hpf taken by confocal microscopy (EGFP fluorescence pseudocolored in green). (A) Lateral view of EF-GVP-Ulyn-UH2B-injected embryo, the high frequency of EGPF-expressing cells allows one to observe the overall morphology and to distinguish different tissues like nervous system, body muscles, and yolk syncytial layer. (B–G) Individual optical sections of stacks of pictures taken from the skin (B–D) or somitic muscles (E–G) of the injected embryo. Arrowheads mark same individual cells through these stacks to demonstrate that cells indeed coexpress both EGFP variants while scoring single individual optical sections might often underestimate the degree of coexpression. Note syncytial nature of muscle cells containing several nuclei (E*). Abbreviations: ey, eye; ot, otic vesicle; otec, optic tectum; rh, rhombencephalon; sc, spinal cord; sm, somitic muscles; y, yolk.

Acknowledgments:
ZFIN wishes to thank the journal Developmental Biology for permission to reproduce figures from this article. Please note that this material may be protected by copyright.

Reprinted from Developmental Biology, 233(2), Köster, R.W. and Fraser, S.E., Tracing transgene expression in living zebrafish embryos, 329-346, Copyright (2001) with permission from Elsevier. Full text @ Dev. Biol.