smh mutant neutrophils display directed migration defects. (A) Schematic outlining the wound generated with the multiphoton laser. The dashed line box indicates the imaging area. (B,C) Representative confocal projection images of wounded WT (n=6) and smh (n=6) embryos with the mpx:GFP transgene at 24 hpf. (D) Quantification of migration efficiency scores calculated from point position data generated in Imaris. Each data point represents an individual cell from a minimum of three separate experiments, per genotype. (E) Quantification of the maximum track speed of migrating cells. (F,G) Maximum confocal projections of wounded WT and smh embryos bearing the mpx:GFP transgene with individual cells projected as 3D surfaces and color coded according to sphericity index, as calculated by Imaris. (H) Quantification of the mean cell sphericity index. Box and whisker plots represent individual cells from all independent experiments at each time point imaged, in order to capture changes in cell sphericity over the course of the time-lapse sequence. In E and H, the box and whisker plots represent the median value (center line), with the box including all values from the two median quartiles and the whiskers representing minimum and maximum values (with no outliers excluded). (I,J) Representative single z-slices from high-resolution live confocal imaging of individual mpx:GFP+ cells from WT (n=10) and smh (n=12) embryos. In I, the arrow indicates a uropod and the arrowhead indicates lamellipodia. In J, the arrow indicates cytoplasmic extension. (K) Mean circularity index for individually imaged cells as calculated in Imaris. **P<0.005, *P<0.05 (two-tailed unpaired Student's t-test). Scale bars: 100 μm (B,C,F,G), 10 μm (I,J).
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