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Knockdown of semaphorin 3F by MO injection or by TALEN-induced mutation accelerates resolution of neutrophilic inflammation in zebrafish.(A–D) sema3fa and/or sema3fb MO (1 nL of 0.5 mM) was injected into 1-cell-stage zebrafish mpx:GFP embryos, with 1 nL of 0.5 mM control MO used as a negative control. Tail fin transection was performed at 2 dpf, and neutrophils were counted at 6 hpi and 24 hpi. (A) Neutrophil counts at the 6 hpi time point with (B) overlaid fluorescence and bright-field photomicrographs (recruitment). (C) Neutrophil counts at the 24 hpi time point with (D) overlaid fluorescence and bright-field photomicrographs (resolution). Scale bars: 60 μm (B and D). Data are mean ± SEM, with individual data points (n = 30) from 3 independent experiments. (E–G) sema3fa- or sema3fb-mutated F1 fish were incrossed and compared with mpx:GFP fish. Tail fin transection was performed at 2 dpf, and (E) neutrophils were counted at 6 hpi. Data are mean ± SEM, with individual data points (n = 30) from 4 independent experiments. (F) Whole-body total neutrophil numbers were counted at 3 dpf. Data are mean ± SEM, with individual data points (n = 60) from 4 independent experiments. (G) Tail fin transection was performed at 2 dpf and neutrophils were counted at 24 hpi. Data are mean ± SEM, with individual data points (n = 5–60) from 4 independent experiments. (H) sema3fa and or sema3fb MOs (1 nL of 0.5 mM) were injected into 1-cell-stage zebrafish mpx:kaede embryos, and tail fin transection was performed at 2 dpf. Neutrophils at 6 hpi were recruited to the wound and photoconverted, and red fluorescence neutrophils were tracked for 3.5 hours. Data are from 3 independent experiments (n = 9). Statistical analysis was by 1-way ANOVA and Bonferroni’s post hoc test. ***P < 0.001.
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