Gal80 expression inhibits Gal4 activity.

(A–H) Embryos resulting from a cross between Tg[hsp70:Gal4] and Tg[UAS:Kaede] fish were either non-injected (NI, A–D) or injected with 100 pg of mRNA encoding full-length Gal80 (E–H). Representative specimens are depicted at 24 hpf (A–B, N = 18 GFP⁺/72 fish; E–F, N = 0 GFP⁺/75 fish) and 5 dpf (C–D, N = 18 GFP⁺/77 fish; G–H, N = 19 GFP⁺/82 fish). In H, asterisks indicate the fish displaying GFP. (I–N) Embryos resulting from a cross of Tg[UAS:Kaede] fish were injected with a DNA encoding full-length Gal4 under the control of the HuC promoter either alone (NI, I–K, N = 32) or with 100 pg of mRNA encoding full-length Gal80 (L–N, N = 37). Representative specimens are depicted at 48 hpf, 3 and 7 dpf.

Gal80 RNA temporarily inhibits Gal4 activity in a dose-dependant manner.

(A) Embryos resulting from a cross of Tg[UAS:mem-TdTomato] fish were injected with a DNA encoding full-length Gal4 under the control of the β-actin promoter alone (a–c) or with 50 pg (d–f, N = 21), 100 pg (g–i, N = 23) or 200 pg (j–l, N = 20) of mRNA encoding full-length Gal80. Representative specimens are depicted at 3, 4 and 6 dpf. (Scale bars: 300 μm). (B) Red fluorescence quantification of tdTomato expressing cells. (a–c) Maximal projection of muscle fibers from a embryo resulting from a cross of Tg[UAS:mem-TdTomato] fish injected with the β-actin:Gal4 construct alone. The white arrowheads indicate the quantified cell. (d–f) Maximal projection of muscle fibers from a embryo resulting from a cross of Tg[UAS:mem-TdTomato] fish injected with the β-actin:Gal4 construct and 200 pg of mRNA encoding full-length Gal80. Z-stacks have been captured at the same settings as in a–c. (g–i) Same as d-f with over-exposure. (Scale bars: 20 μm) (h) Quantitative values of fluorescence per μm². Each value corresponds to the average of the mean values obtained for three different regions of each cell. (C) Anti-tdTomato western blotting of 3dpf fish resulting from a cross of Tg[UAS:mem-TdTomato] fish injected with the β-actin:Gal4 construct alone or with 50, 100 or 200 pg of mRNA encoding full-length Gal80.

Temporal control of Gal80 expression.

(A) Schematic representation of the construct hsp70:Gal80-myc. (B) Anti-myc western blotting of 48 hpf fish resulting from a cross of Tg[hsp70:Gal80-myc] fish after 0, 30 and 60 minutes of heat-shock. (C) Embryos resulting from a cross between Tg[hsp70:Gal80-Myc] and Tg[UAS:Kaede] fish were not submitted (a–c) or submitted to a one-hour heat-shock (d–f). Representative specimens are depicted for anti-Gal80 in situ hybridization at 30 hpf (a, d) or for Kaede expression at 2 dpf (b–c, d–e). (Scale bars: 150 µm). (D) Embryos resulting from a cross between Tg[hsp70:Gal80-Myc] and Tg[UAS:Kaede] fish were injected with a DNA encoding full-length Gal4 under the control of the β-actin promoter. At 24 hpf, the embryos were subjected to a 30-minute heat-shock at 39 degrees and immediately photo-converted. The following day, immunostaining anti-myc (c, g) was performed on fish expressing Gal80 (a–d) or not expressing Gal80 (e–h). The figure depicts muscle fibers for the expression of Kaede^red (a, e) and Kaede^green (b, f). (Scale bars: 20 μm).

Gal4 morpholino temporarily inhibits Gal4 expression in Gal4-VP16 lines.

(A) Embryos resulting from a cross between Tg[hsp70:Gal4] and Tg[UAS:mem-TdTomato] fish were either non-injected (NI, Aa-b, N = 27) or injected with 3 ng of Gal4 MO (Ac-d, N = 37). Representative specimens are depicted at 2 dpf. (B) Embryos resulting from a cross of Tg[ET(hsp:Gal4VP16^s1006t);UAS:Kaede] double transgenic animals were either non-injected (NI, Ba-d, N = 47) or injected with 3 ng of Gal4 MO (Be-h, N = 52). Representative specimens are depicted at 24 hpf (Ba, e), 4 dpf (Bb, f) and 6 dpf (Bc-d, Bg-h). (C) Embryos resulting from a cross of Tg[hspGFF53A;UAS:EGFP] double transgenic animals were either non-injected (NI, Ca-b, N = 64) or injected with 3 ng of Gal4 MO (Cc-d, N = 58). Representative specimens are depicted at 48 hpf (Ca, c) and 4 dpf (Cb, d). (D). Embryos resulting from a cross of Tg[hspGFF53A;UAS:EGFP] double transgenic animals were either non-injected (NI, Da), injected with 5 ng of Gal4 MO (Db) or injected with 5 ng of a control MO (Dc). Representative specimens are depicted at 48 hpf. (Scale bars: 600 μm for A; 300 μm for B and C, 150 μm for D).

Gal4 morpholino temporarily inhibits Gal4 expression in a dose-dependant manner.

Embryos resulting from a cross of Tg[hspGFF53A;UAS:EGFP] double transgenic animals were either non-injected (A–E) or injected with 1 ng (F–J), 3 ng (K–O) or 5 ng (P–T) of Gal4 MO (N = 94). Representative specimens are depicted at 2, 4 and 6 dpf. White arrows indicate green fluorescence at the level of the posterior afferent lateralis ganglion. (Scale bars: 150 μm).

Gal4 morpholino allows bypassing of early developmental stages.

(A–F) Embryos resulting from an incross between Tg[ET(hsp:Gal4VP16^s1001t);UAS:Kaede] double trangenics (A,D) or from Tg[ET(hsp:Gal4VP16^s1001t);UAS:Kaede] with Tg[UAS:N^ICD-Myc] animals (B–C, E–F) were either non-injected (NI, A–B, D–E) or injected with 3 ng of Gal4 MO (C,F). Representative specimens are depicted at 3 dpf. The lower panel shows a magnification of the different specimens after DiASP treatment (D–F). (G–J) Embryos resulting from a cross between Tg[ET(hsp:Gal4VP16^s1006t)] and Tg[UAS:N^ICD-Myc] animals were either non-injected (G and magnification in H) or injected with 3 ng of Gal4 MO (I and magnification in J). Representative specimens are depicted at 6 dpf. (K–P) Embryos resulting from a cross between Tg[hspGFF53A;UAS:EGFP] double transgenic animals and either wt (K, N) or Tg[UAS:N^ICD-Myc] (L–M, O–P) animals were either non-injected (K–L, N–O) or injected with 3 ng of Gal4 MO (M, P). Representative specimens are depicted at 7 dpf. (K–M) Maximal projections of posterior lateralis afferent ganglion (dashed circles) and central projection (white arrowheads). (N–P) Maximal projections of posterior lateralis afferent nerve. (Scale bars: 150 μm for A–F, H and J; 300 μm for G and I; 100 μm for K–P).