Fig. 5. Live-imaging of the wound inflammatory response in WASp-morphant fish and comparison with mutants. (A,B) Cell tracking, from DIC movies, of the inflammatory response at a wound in the ventral tail fin of 3-dpf zebrafish larvae treated with control morpholino (A) or WASp1 morpholino (B). Cell tracks were generated by marking the centre of the leukocyte every 45 seconds (three frames) from the DIC movie as cells migrated from the vessels to the wound (asterisk). (C,D) Several parameters were analysed: the number of cells that left the vessel up to 3 hours post-wounding (D), the number of cells that successfully navigated to the wound (D), the speed of leukocytes (D, y-axis indicates mean speed in µm/minute), and the persistence of their directionality, as indicated by their chemotactic index (CI) (C). (D) Error bars are the standard error of the mean (s.e.m.). (E) Three typical cell tracks, with cell outlines traced at 1- to 2-minute intervals to illustrate cell morphologies during a meandering (orange), a fairly direct (green) or a return (red) journey to and from the wound in a control larvae. (F) False-coloured cell profiles equivalent to those in E, indicating that the morphology of migratory cells (i-iii) does not significantly differ in WASp1-morphant larvae compared with controls, but the pseudopodia choice is frequently `wrong' and takes the cell away from the wound (wound direction indicated by white arrowhead). When a cell stops, it extends pseudopodia in all directions (see supplementary material Movie 5) and can become elongated (iv). (G) Graphic illustration of the proportion of `correct' pseudopodia choices made by control versus morphant leukocytes in response to wound signals. (H) DIC micrographs to illustrate the extent of thrombus formation after laser-wounding the aorta at 60, 120 and 180 seconds post-lesion in wild-type, WASp1-morphant and WASp1-mutant fish. Arrow indicates direction of vessel flow. Scale bars: A,B, 30 µm; E, 20 µm; F,H, 10 µm.