Figure 4

Zebrafish expressing mutant FUS show abnormal neuromuscular junctions and orphaned pre-synaptic endings. (A) Confocal images of long pec stage zebrafish trunk, lateral view, anterior to the right, imaged after microinjection with eGFP, eGFP-FUS^WT, eGFP-FUS^R514G or eGFP-FUS^ΔNLS. Merged images show the GFP-FUS (green) and SV2 (cyan) and BTX staining (red) which is indicated by a white arrow for each condition. Analysis was carried out on the greyscale images. Scale bar = 100 μm. (B) Quantitative analysis of the synaptic density of BTX, SV2 and synaptic colocalization. There was a significant reduction in the number of NMJs as shown by a reduction in both BTX (top) and SV2 (middle) staining in axons expressing eGFP-FUS^R514G (p < 0.05) and eGFP-FUS^ΔNLS (p < 0.05) compared to eGFP-FUS^WT. The colocalisation between the SV2 and the BTX (bottom) was also reduced in axons expressing eGFP-FUS^R514G (p < 0.05) and eGFP-FUS^ΔNLS (p < 0.0001) compared to WT. Statistical analysis was performed using a One-Way ANOVA with a post-hoc Tukey’s multiple comparisons test; error bars are ± SEM. N = six different independent injections for each plasmid. *p < 0.05, **p < 0.01, ***p < 0.001 ****p < 0.0001.