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Files in this Data Supplement:
Fig. S1. Distribution of neutrophils and macrophages in 3-dpf zebrafish larvae. (A,D,E,H) Sudan-Black staining for neutrophils. Lateral view of the head and yolk sac in control (A) and WASp1-morphant (E) larvae. High-magnification view of the ventral vein region in a wt larvae (D) and WASp1 morphant (H). (B,C,F,G) Whole-mount immunostaining for L-plastin to reveal macrophages. Overview of a control larvae (B) and WASp1 morphant (F) showing an approximately equivalent number and distribution of macrophages. High-magnification views of the ventral vein region in control (C) and morphant larvae (G). Scale bars: A, E=85 µm; B, F=250 µm; C, D, G, H=30 µm.
Fig. S2. (A-C) Illustrate the effects of either control, WASp1- or WASp2-specific morpholinos on expression of either WASp1-EGFP or WASp2-EGFP reporter constructs injected together at the one-cell stage. Scale bars: A-C′=150 µm.
Fig. S3. (A,B) Temporal characterization of the inflammatory response to fin wounding. (A) Recruitment of Sudan-Black-stained neutrophils (arrow) to the site of tissue damage (asterisk) over the time course from 30 minutes to 24 hours post-wounding. (B) Anti-L-plastin antibody staining of mixed leukocytes (arrow) over the same time course. (C) Graph to illustrate the time course of leukocyte recruitment to the fin wound. Error bars are s.e.m. (D,E) Graphs to indicate numbers of Sudan-Black-positive neutrophils and L-plastin leukocytes recruited to control versus, WASp1- and WASp2-morphant wounds at various time points. (F) To confirm our WASp1-morphant phenotype, as shown in Fig. 4, we repeated the study with a second, non-overlapping morpholino. (i,iii) Control wounds, at 3 hours and at 90 minutes, stained with L-plastin antibody and Sudan Black, respectively. (ii,iv) Equivalent wounds in larvae injected with this second WASp1 morpolino. (G) Graphic representation of the data using the second morpholino, showing a significant reduction in the number of leukocytes recruited to wounds, almost exactly mirroring our first WASp1-morphant results. Scale bars: A, B, F, G=25 µm.
Movie 1. Time-lapse movie of primitive macrophage chemotaxis towards a laser wound in a 22-hpf Tg(fli1a:EGFP)-expressing zebrafish embryos. A laser-induced wound at the centre of the yolk triggers a rapid recruitment of macrophages towards the site of epithelial damage. Up to 20 cells are drawn from a distance of 200 µm extending back from the wound margin. The movie was made at 15 seconds per frame over 1 hour (240 frames total), starting immediately after the wound was made. The first image is a DIC image of the yolk; all images thereafter are of EGFP fluorescence.
Movie 2. Time-lapse movie showing the effect of the WASp1 morpholino on primitive macrophage migration towards the wound. WASp1-morphant embryos appear to have normal numbers of primitive macrophages patrolling beneath the yolk epithelium prior to wounding, but exhibit a severely reduced wound response. Only those cells within 100 µm of the wound margin respond to the wound chemotactic signal. Images were recorded at 15 seconds intervals over a period of 1 hour (240 frames total), starting immediately after the wound was made. The first frame is a DIC image of the yolk; all images thereafter are EGFP fluorescence.
Movie 3. Time-lapse movie of a WASp1-morpholino-treated Tg(lyz:EGFP) transgenic larva. The wound inflammatory response in a WASp1-morpholino-treated Tg(lyz:EGFP) expressing larva demonstrates that very few morphant cells even commence migrating towards the wound. Three cells that begin migration stop for a period of time (upper left). Images were captured every 90 seconds over a period of 3 hours (120 frames total), starting 15 minutes after a wound was made.
Movie 4. Leukocytes within 3-dpf Tg(lyz:EGFP) transgenic larvae demonstrate a robust response to a mechanical wound. A small incision within the ventral fin triggers a strong infiltration of Tg(lyz:EGFP)-expressing neutrophils and macrophages towards the wound. Images were captured every 90 seconds over a period of 3 hours (120 frames total), starting 15 minutes after a wound was made.
Movie 5. WASp1 knockdown results in failure of some leukocytes to reach the wound site. After leaving the vessel, this leukocyte starts migrating towards the wound (bottom left). However, half way to the wound it starts to extend protrusions in all directions and appears unable to restart a polarised migration. The movie was captured over a period of 3 hours, frames were taken at 15 seconds intervals (720 frames total), starting 1 minute after a wound was made in the fin.
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