GAVPO-mediated toxicity in UAS:NTR-mCherry and UAS:M2H37A zebrafish lines. (A) Tg(UAS:NTR-mCherry) zygotes were injected with the designated amounts of GAVPO mRNA, and their embryonic shields were irradiated at 6 hpf using an epifluorescence microscope equipped with a 470/40 nm filter. An iris diaphragm was used to restrict illumination to a 100 µm diameter region. The 10 hpf embryos were dechorionated and treated with DMSO or 5 mM metronidazole, and imaged at 32 hpf. Representative bright-field and epifluorescence micrographs from two independent experiments are shown. Shield-irradiation of the GAVPO-expressing embryos induces NTR-mCherry expression in the notochord, head mesoderm and hatching gland (arrowheads). (B) The designated amounts of GAVPO mRNA were injected into Tg(UAS:UAS:M2H37A; myl7:mCherry) zygotes, and the embryos were either cultured in the dark or globally exposed to blue LED light (3 mW/cm2) from 6 to 8 hpf and then cultured in the absence or presence of 100 μg/ml rimantadine. Developmental phenotypes were scored at 24 hpf, using the indicated number of embryos (n) per condition from two to four independent experiments. Statistical analyses: χ2=301, d.f.=30, P<0.00001. (C) RT-PCR analyses of M2H37A expression in Tg(UAS:UAS:M2H37A;myl7:mCherry) embryos injected with GAVPOmRNA and treated as described above. The expression of the translation elongation factor eef1a1l1 was used as a normalization control. The amplification reaction was performed using a standard PCR with 40 cycles for M2 and 35 cycles for eef1a1l1 amplification. (D) Tg(UAS:M2H37A;myl7:mCherry) zygotes were injected with the designated amounts of GAVPO mRNA, cultured in the absence or presence of 100 µg/ml rimantadine, shield-irradiated at 6 hpf as described in A, and imaged at 30 hpf. Representative bright-field and differential interference contrast (DIC) micrographs from three independent experiments are shown. Shield irradiation of the GAVPO-expressing embryos disrupts development of the notochord (inset), head and hatching gland. All embryos are shown in lateral view, anterior left. Scale bars: 250 µm in A,D; 50 µm in D (insets). Statistics for the observed phenotypes are in Table S3.

Light-inducible, neuron-specific gene expression using the GAVPO system. (A) Tg(elavl3:GAVPO;UAS:NTR-mCherry)embryos were irradiated for varying durations, using a blue LED lamp and starting at 48 hpf. The embryos were then imaged 8 h later, and representative bright-field and epifluorescence micrographs (with short or long exposure times) are shown. NTR-mCherry was expressed in neural tissues, at levels that increased with the length of irradiation. Dashed outlines indicate the region used to determine the average pixel intensity (API) for each micrograph. (B,C) The heads of 48 hpf Tg(elavl3:GAVPO;UAS:NTR-mCherry) embryos were irradiated using an epifluorescence microscope equipped with a 470/40 nm filter and either a 20× (B) or 63× (C) objective. The embryos were imaged 8 h later, and representative bright-field and epifluorescence micrographs are shown. NTR-mCherry expression was observed in anterior neural tissues, at levels that increased with the duration and intensity of irradiation. Dashed outlines indicate the region used to determine the average pixel intensity (API) for each micrograph. (D-F) Tg(elavl3:GAVPO;UAS:NTR-mCherry) embryos were illuminated either globally using a blue LED lamp (D) or within a 100 µm diameter region in the head using an epifluorescence microscope equipped with a 470/40 nm filter, 20× objective and iris diaphragm (E,F). Representative bright-field and confocal fluorescence micrographs from two independent experiments are shown, and total sample sizes are indicated. LED illumination induced NTR-mCherry expression throughout the head, whereas spot-irradiated embryos exhibited mCherry fluorescence in only a localized anterior region. Embryo orientations: A-D,F, lateral view and anterior left; E, dorsal view, anterior up. Scale bars: 200 µm in A; 300 µm in B,C; 50 µm in D-F. Statistics for the observed phenotypes are in Table S4.

Light-inducible neural ablation using the GAVPO/M2H37Asystem. (A) Heterozygous Tg(elavl3:GAVPO;UAS:M2H37A;myl7:mCherry) embryos were injected with an elavl3:mCherry-CAAX construct to label neurons in a mosaic manner. The injected embryos were globally irradiated with a blue LED lamp at 48 hpf for 8 h, fixed 12 h later and then imaged using a confocal microscope. Representative micrographs from a hindbrain slice just above the otic vesicle are shown. Irradiated embryos have fewer intact neurons (arrowheads) than those cultured in the dark or treated with 100 µg/ml rimantadine during blue-light illumination. Neurons were counted in ImageJ in a blinded manner, and the average number of neurons per slice±s.d. for each experimental condition is shown. (B) Representative micrographs from a maximal intensity projection of the spinal cord just above the cloaca, with axonal tract widths in selected regions indicated by the dashed lines. The average axonal tract width±s.d. for each experimental condition are shown. Values for individual fish are shown. The box extends from the 25th to 75th percentiles. The whiskers extend to the minimum and maximum values. The horizontal line indicates the average value. Embryo orientations: A,B, lateral view, anterior left. Scale bars: 25 µm.