Figure 5

Figure 5

Dominant-negative Rorα suppresses neutrophil motility and chemotaxis. (A) Schematic of the construct for neutrophil-specific expression of vector control or rorα dominant-negative (DN). (B, C) Representative images and quantification of total neutrophil numbers in vector or rorα DN zebrafish line. Scale bar: 500 μm. (D, E) Representative images and velocity of random neutrophil migration in vector or rorα DN zebrafish line. Scale bar, 100 µm. Three embryos each from three different founders were imaged, and quantification of neutrophils in one representative video is shown, Kruskal–Wallis test. (F, G) Representative images and quantification of neutrophils recruited to the infected ear in vector or rorα DN zebrafish line. Scale bar, 100 µm. (H, I) Representative images and quantification of neutrophil recruitment to the tailfin transection sites in vector or rorα DN zebrafish line. Scale bar, 200 µm. The result is presented as mean, Mann–Whitney test. (J) Simultaneous imaging of utrophin-GFP distribution in neutrophils expressing either mCherry alone or with Rora DN. Data are representative of more than three separate time-lapse videos. Scale Bar, 50 µm. (B, C, F–I) Representative results for three individual trials are shown. (K) Schematics of the plasmid constructs for neutrophil-specific Cas9 expression (upper construct) and for ubiquitous expression of sgRNAs with a neutrophil-specific expression of GFP (lower construct). (L, M) Representative images (L) and quantification (M) of neutrophil motility at head region of 3dpf larvae from Tg(LyzC: Cas9, Cry: GRFP, U6a/c: rora guides, LyzC: GFP) fish. Student’s t-test. Scale bar, 100 µm. (N) Deep sequencing of the rora loci targeted by guide RNAs in (K). The sequences on the top are wild-type sequences, and the five most frequent types of mutations are shown. Point mutations, deletions, and insertions are all observed.