RNA foci formation in transgenic zebrafish. a, b Cy3-labeled in situ probe detected dot-like structures in spinal cord in GA80-GFPa/b and ggggcc80-GFP zebrafish. b Foci were only detected in GA80-GFP and ggggcc-GFP fish whereas no foci were detected in wild-type and GA2-GFPa/b fish at 28 hpf. GA80-GFPa zebrafish were treated with RNaseA or DNase. Scale bar 10 μm. c Pericardial edema phenotype observed in ggggcc80-GFP zebrafish at 4 dpf. Phenotypic features are classified as wild-type, mild edema, and severe edema. d The average percentages of phenotypic fish of the three different phenotypic classes at 4 dpf are indicated in the bargraph (at least three independent clutches were analyzed with n ≥ 14)

Poly-GA protein elicits a toxic phenotype. a In vivo image of GA-GFP polypeptides at 2 and 4 dpf. Genotypes as indicated. No GFP sibling (GA80-GFP) refers to a sibling from a cross between a Gal4 driver and a UAS GA80-GFP responder fish that is GFP negative, and hence is either negative for the driver or the responder construct, or both constructs. GFP fluorescent images shown are merged with DIC pictures. Lowest panel is a magnification of the middle panel at 4 dpf. Lateral views of the trunk musculature. Scale bar 20 μm. b A strong pericardial edema phenotype was observed in GA80-GFPa/b zebrafish at 4 dpf. The average percentages of phenotypic fish of the three different classes are indicated in the bargraph. c GA80-GFPa/b zebrafish had mostly no circulation at 2 dpf. Red blood cells accumulate due to circulation defects (arrow). d The average percentages of fish with or without circulation are indicated in the bargraph (at least three independent clutches were analyzed with n ≥14)

Antisense morpholino rescued the toxic edema phenotype. a Representation of process of intervention for each morpholino during the generation of GA80-GFP protein in vivo. b GAL4 targeting AMO efficiently blocked Gal4 translation at 2 dpf shown by immunoblotting (upper panel). Quantification of the pericardial edema phenotype observed in GA80-GFP with injection of ctrl AMO or GAL4 targeting AMO at 4 dpf are shown as a bar graph (lower panel) (p < 0,001, 3 independent experiments with 3 clutches n ≥ 6 are shown, unpaired t test). c ATG targeting morpholino efficiently inhibited the ATG dependent translation of poly-GA at 2 dpf (upper panel). Quantification of the pericardial edema phenotype observed in GA80-GFP upon injection of ctrl AMO or ATG targeting AMO at 4 dpf shown as a bar graph (lower panel) (p < 0,005, 3 independent experiments with 3 clutches n ≥ 19 are shown, unpaired t test). d Semi-quantitative RT-PCR analyses of injected embryos at 2 dpf. e RNA foci formation was not affected upon injection with ctrl AMO or ATG AMO at 2 dpf. Scale bar 10 μm

RNA foci formation overview. Embryos of the indicated genotypes stained with a Cy3-labeled probe to visualize RNA foci formation by in situ hybridization. Between 13–33 cells per field of view showed RNA foci in the GA80-GFP larvae. All images were taken without DAPI fluorescence. Scale bar 10 μm.

Spinal motor neuron axonal outgrowth is not affected. (A) Spinal motor neuron axon of GA80-GFP fish (Gal4 driver + UAS:ATG-80xGGGGCC-GFP responder) and the GFP negative siblings (Gal4 driver or UAS:ATG-80xGGGGCC-GFP alone, or wild-type) at 28 hpf. (B) Length of outgrowing spinal motor neuron axons measured from the exit point of the spinal cord to the tip of the growth cone in the 5 somites anterior of the end of the yolk expansion at 28 hpf (indicated by the numbers 1–5). Embryos are sorted by the genotypes wild-type, driver, responder, and driver + responder. Statistical analyses was performed in indicated genotypes. Scale bar 20 μm. Mean ± SD.

Overall neuronal outgrowth is not affected. Overall neuronal outgrowth was analyzed in embryos stained with an antibody against acetylated tubulin at 2 dpf. Siblings of GA80-GFPa zebrafish expressing GFP (A) or not expressing GFP (B). Scale bar 100 μm.

Muscle patterning is not affected. (A) Quantification of GFP inclusions in GA80-GFPa and GA80-GFPb larvae subdivided into mild and strong edema phenotypes at 4 dpf. (n = 4 per subgroup, mean ± SD). Amount of inclusions from GA80-GFP line a and b with mild and strong phenotypes were not significantly different (paired t-test). Inclusions were exclusively detected in the musculature in both lines. (B) The overall structure of the muscle was analyzed by α-actinin staining at 2 dpf in a GFP negative GA80-GFP embryo and (B) GFP positive sibling. Scale bar 20 μm.

Vascular patterning is not affected. The vasculature was analyzed by incrossing with Tg(kdrl:HsHRAS-mCherry)^s896 into the GA80-GFP expressing lines. mCherry expressed from the Tg(kdrl:HsHRAS-mCherry)^s896 transgene is shown in Ga80-GFP-a transgenic zebrafish not expressing GFP (A) and siblings expressing GFP (B) at 2.5 dpf. Scale bar 20 μm. (PDF 4003 kb)