Figure 4—figure supplement 3.

(a) csf3r morpholino-injected animals have less neutrophils (arrows) compared with controls as assessed by RNA in situ hybridization for a neutrophil marker myeloid-specific peroxidase mpx. Right bar graph, quantification of the area of mpx expression in the body posterior to the head using ImageJ show an average of a 27% reduction. Statistical significance was determined by a two-tailed t-test with Welch’s correction. (b) Reduction of neutrophils leads to a decrease in macrophage infiltration as assessed by RNA in situ hybridization as an alternative approach in addition to live transgenic imaging as shown in Figure 4. As a control, we also verified the level of neutrophil reduction in csf3r morpholino-injected animals by mpx in situ. Right bar graphs show frequency of liver infiltration by each myeloid cell type. N is shown in parenthesis and represents number of independent animals analyzed. (c) Left, scatter plot representing the effects of csf3r morpholino-mediated knockdown on macrophages and neutrophils showed no significant change to overall macrophage numbers, but a significant reduction in neutrophils in csf3r morpholino-injected animals. In vivo confocal imaging of double transgenic zebrafish carrying the macrophage reporter (mpeg1:GFP) and the neutrophil reporter (lyz:mCherry) was conducted. Right, representative fluorescent maximum projection, multi-tiled images of the transgenic zebrafish body. Dotted region shows area of cell number quantification. Statistical significance was determined by a one-tailed t-test. Each symbol represents an individual larva. Scale bar represents 500 µm. ns, not significant.