Fig. 1.

M2^H37A channel variant is toxic to zebrafish embryos and ablates neurons more rapidly than NTR. (A) Wild-type zygotes were injected with the designated amount of M2^H37A mRNA and cultured in the absence or presence of 100 μg/ml rimantadine. Representative bright-field micrographs of 24 hpf embryos from two independent experiments are shown. Scale bar: 500 μm. (B) Phenotypic distributions of embryos injected with the designated amount of M2^H37A mRNA. The embryos were scored at 24 hpf, and the number of embryos (n) per condition from two or three independent experiments is indicated. Statistical analyses: χ²=389, d.f.=14, P<0.00001. (C) Comparison of Tg(tuba1a:Gal4VP16;myl7:GFP) and Tg(tuba1a:Gal4VP16; UAS:M2^H37A;myl7:gfp,mCherry) embryos cultured in the absence or presence of 100 µg/ml rimantadine. Neuronal expression of M2^H37A induces loss of the midbrain-hindbrain boundary (arrowheads) at 24 hpf and increasingly severe CNS deficits as development continues. Treatment with rimantadine starting at 10 hpf rescues these neuronal defects in Tg(tuba1a:Gal4VP16;UAS:M2^H37A;myl7:gfp,mCherry) embryos. Representative bright-field and epifluorescence micrographs (overlays) from three independent experiments are shown. (D) Comparison of Tg(tuba1a:Gal4VP16;UAS:NTR-mCherry;myl7:GFP) embryos cultured with 5 mM metronidazole or an equivalent amount of DMSO, starting at 10 hpf. Metronidazole-treated embryos first exhibit CNS defects at 48 hpf. Representative bright-field and epifluorescence micrographs (overlays) from two independent experiments are shown. All embryos are shown in lateral view, anterior left. Scale bars: 250 µm. Statistics for the observed phenotypes are in Table S1.