Neutrophil-specific miR-99 overexpression hinders neutrophil recruitment and motility. (A) Schematic of the Tol2-lyzC:miR-99/Dendra2 plasmid, injected into wild-type AB zebrafish embryos to generate the transgenic line Tg(lyzC:miR-99/Dendra2) (miR-99) and Tg(lyzC:Dendra2).(B) The relative expression level of miR-99, miR-223, and let-7e (normalized to U6 expression) in vector and miR-99 lines determined by RT-qPCR, mean ± s.d. (N = 3 biological replicates with ten larvae at each time point in each group). (C, D) Representative images and quantification of total neutrophils in vector and miR-99 lines. Scale bar: 500 μm. (E, F) Representative images and quantification of neutrophil recruitment to tail transection site in vector or miR-99 larvae at 1-hour post-injury. Neutrophils in the boxed region were quantified. Scale bar: 100 μm. (G, H) Representative images and quantification of neutrophil recruitment to localized ear infection in vector or miR-99 larvae at 1-hour post-infection. The infected ear is indicated with the circle. Scale bar: 100 μm (I, J) Tracks and quantification of neutrophil motility in vector or miR-99 larvae. Scale bar: 50 μm. All experiments were performed with F2 larvae at three dpf. One representative result of three independent experiments was shown (n=20). P-value was calculated with unpaired Student’s t-test.
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