Figure 4

Figure 4

Visualization and analysis of innate immune cell migration to the head of 3 pdf larvae infected through different routes. The transgenic zebrafish lines (A) Tg(lyz:DsRed2), (B) Tg(mpx:GFP) and (C) Tg(mpeg:mCherry) were used to analyse the migration of myeloid precursors with lysozyme activity, neutrophils and macrophages, respectively, to the cephalic region. Larvae were infected through the 4 infection routes analysed in this study, and the cells were counted at 1, 2 and 3 dpi. Fluorescent immune cells were counted using ImageJ, and the graphs represent the difference in fold change of the number of cells located in the head from infected larvae compared to their corresponding uninfected control larvae (control FC = 1 in the dotted line). Representative images of the three transgenic lines at 2 dpi were included. (D) Whole-mount immunofluorescence of Tg(mpx:GFP) transgenic larvae infected via the brain with NNV at 1 dpi; neutrophils are displayed in green, NNV are displayed in red, and nuclei are displayed in blue. No colocalization between NNV particles and neutrophils was observed. (E) Expression analysis of marker genes of the two major innate immune cells (mpx – neutrophils, marco – macrophages) in NNV-infected and uninfected larvae at different times postinfection. Each sample (5 biological replicates, pools of 4 larvae each) was normalized to the 18S gene. The normalized expression values were standardized against their respective controls (Control FC = 1, dotted lines). For (A–C, E), the graphs represent the mean ± SEM of the biological replicates. Statistically significant differences are displayed as follows: ***, 0.0001 > p value > 0.001; **, 0.001 > p value > 0.01; *, 0.01 > p value > 0.05.