Fig. 7

a Schematic showing brain region and categories of neutrophils analyzed in 5–6 dpf larvae from nlrc3l heterozygous intercross with (+GH) and without (no GH) the macrophage rescue transgene (mpeg1-nlrc3l). Lingering (blue arrow) defines infiltrated neutrophils that remain in the brain over the entire 15-min period of imaging, while circulating (red dotted circle) defines neutrophils observed only at a single timepoint (imaged every 30-s) that flow through the brain. GH, GFP + heart. b Representative 3D volumetric images of neutrophils (lyz:GFP+) in the brain at baseline and after macrophage rescue. See associated Supplementary Movies 9, 10, 12, and 13 showing the same zebrafish larvae as shown here. c Scatter bar plots show quantifications of brain lingering and circulating neutrophils, and total neutrophil numbers in the head periphery. Macrophage rescue reversed neutrophil infiltration in the nlrc3l larval brain, but not an overall increase in neutrophil numbers. Error bars show standard error of mean. Significance was determined by two-tailed t-tests. d Still images extracted from time-lapse confocal imaging showing circulating and infiltrating neutrophils in the brain at embryonic stage of 2 dpf and larval stage of 6 dpf (see associated Supplementary Movies 6 and 7). Individual neutrophils lingering in the parenchyma are labeled by arrows in different colors over three timepoints. Dotted circles mark circulating neutrophils passing through. tele telenchephalon, dien diencephalon, cp chorid plexus, max proj maximum projection of a z-stack. e Genetic ablation of neutrophils using combined morpholinos against csf3r and spi1b and single morpholino against csf3r (top diagram). Uninjected and control morpholino injected groups show the expected reduced daytime locomotion in nlrc3l mutants. By contrast, daytime locomotion was significantly improved in nlrc3l mutants after neutrophil ablation using both morpholino strategies. No effect on locomotion was found in wild-type and heterozygous siblings after morpholino-mediated neutrophil ablation compared with baseline controls. Scatter plots show mean ± standard deviation from data of individual animals. Two-tailed t-tests were used to determine statistical significance. n number of animals analyzed shown below each bar graph. See also associated Supplementary Movies 8–13.