MYDGF regulates neutrophil response to tissue damage, but not infection. (A) Ribbon and surface representation of the human MYDGF NMR solution structure (PDB accession no. 6O6W). Residues identical in zebrafish MYDGF are shown in red. (B) Schematic of zebrafish mydgf gene, with exon 5 gRNA sequences highlighted for CRISPR-Cas9 mutagenesis. gRNA sequence is shown in gray and protospacer adjacent motif is shown in magenta; scale bar = 100 bp. (C) Exon 5 DNA sequence of WT (top) and mydgf^−/− (bottom) zebrafish mutants showing a 12-bp deletion. (D) Representative Western blot for zebrafish MYDGF and β-tubulin from pooled 3 dpf zebrafish larvae. Data are representative of three independent replicates. Size marker (mol wt [MW]) in left lane. (E) InstantBlue stain of 12% SDS-PAGE of zebrafish MYDGF in CCM by HEK293 cells at 72 h after transfection by empty or zebrafish MYDGF-expressing pCS2 constructs. (F and G) Representative images (F) and quantification (G) of mCherry-labeled neutrophils at the otic vesicle of WT or mydgf^−/− larvae, following microinjection with CCM ± zebrafish MYDGF protein at 2 h after injection; three independent replicates with n = 29 +/+ control, 32 +/+ zebrafish MYDGF, 54 −/− control and 52 −/− zebrafish MYDGF; scale bar = 50 µm. (H and I) Otic vesicle of 3 dpf WT or mydgf^−/− zebrafish larvae were microinjected with P. aeruginosa (Pa; 5000 CFU), followed by fixation at 2 h after injection. Neutrophils are visualized by Sudan Black staining. In H and I, representative brightfield images (H) and quantification (I) of the number of neutrophils at the otic vesicle are shown; three independent replicates with n = 43 +/+ and 57 −/−; scale bar = 100 µm. In G and I, data are expressed as mean with 95% CI; each symbol represents one larva, and different colors represent independent replicates. ****, P < 0.0001. P values were calculated by ANOVA with Tukey’s multiple comparisons. Ctrl, control; hpi, h postinjection; zMYDGF, zebrafish MYDGF.