WHIM-GFP neutrophils fail to enter the blood stream and respond to wounding in the ventral tailfin. (A) Schematic of the tail of 3-dpf larvae and sample GFP frame from supplemental Video 3; red box is area of dorsal aorta where time-lapse imaging was performed. DA indicates dorsal aorta outlined by white lines, arrows indicate direction of blood flow; CHT, where GFP⁺ neutrophils not in circulation can be seen. White arrowheads indicate neutrophils in the circulation. (B) Quantification of neutrophils in the blood of MPO:GFP and WHIM-GFP 3-4 dpf transgenic larvae; *P < .01. Each dot represents a separate larva whose blood was analyzed by time-lapse imaging for 1 minute as in supplemental Video 3. (C) Time-lapse imaging of the wound response in WHIM-GFP transgenic larvae (from supplemental Video 4), GFP fluorescence overlaid with differential interference contrast image at indicated time points. (D-E) Sudan Black staining to show neutrophils at wounds in the ventral tailfin in 3-dpf control (D) or WHIM-GFP (E) 2 hours after wound. (F) Quantification of neutrophil recruitment to wounds in fixed larvae as in panels D and E; *P < .001; n = the number of individual larva wounded and counted; control = GFP⁻ siblings of WHIM-GFP larvae. (G) Time course of neutrophil wound recruitment in MPO:GFP and WHIM-GFP transgenic larvae. Error bars = SEM. **P < .001; *P < .01; n = 20-25 larvae at each time point. Bars = 200 μm (C-E); 20 μm (A).