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First published online 9 September 2008
doi: 10.1242/jcs.032235

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Analysis of WASp function during the wound inflammatory response – live-imaging studies in zebrafish larvae

¹ Departments of Biochemistry and Physiology, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, UK
² Department of Haematology, University of Cambridge, Cambridge CB2 2PT, UK
³ Department of Molecular Medicine and Pathology, School of Medical Sciences, The University of Auckland, Auckland, New Zealand
⁴ Huntsman Cancer Institute, University of Utah, Salt Lake City, UT 84112, USA

View larger version (56K): [in this window] [in a new window]   Fig. 1. WASp bioinformatics and in situ hybridisation studies. (A) Schematic diagram illustrating the predicted domains in WASp: WASp homology region 1 (EVH1, green), basic (B, blue), minimal high-affinity Cdc42-binding site (C, orange), poly-proline (pro-rich, pink) and VCA (yellow) domains are shown. (B) Alignment of the predicted amino acid sequences of zebrafish (zf prefix) WASp1 and WASp2 with those of human (h prefix) and mouse (m prefix). Multiple alignments were made using the CLUSTALW program as part of the sequence-analysis tools available at the EBI (European Bioinformatics Institute). The underlined sequences represent: green, EVH1 domain; blue, basic domain; orange, minimal high-affinity Cdc42-binding domain site; red, proline-rich domain; yellow, VCA domain. (C) Unrooted dendrogram of the WASp family, including the new zebrafish homologues (circled in blue) of human WASp (circled in red). This phylogenetic tree was generated using the GenomeNet Computation Service program. (D-G, I-K) Whole-mount in situ hybridisation expression patterns for WASp1 and WASp2 in 3-dpf zebrafish larvae. WASp1 (D,E,G,I, lateral view; F, dorsal view) and WASp2 (J,K, lateral view) staining indicates expression in haematopoietic cells, leukocytes and thrombocytes (zebrafish platelets). (G) A high-magnification view of an intersomitic vessel indicates WASp-expressing cells both within the vessel and surrounding the adjacent neuromast (lateral-line precursor); staining in the latter corresponds to cells that are also positive for the pan-leukocyte marker L-plastin (H). Scale bars: D, 100 µm; E,J, 250 µm; F, 125 µm; G,H, 20 µm; I, 85 µm; K, 75 µm.

View larger version (91K): [in this window] [in a new window]   Fig. 2. The requirement for WASp1 during the primitive macrophage response to a laser wound. (A,B) Whole-mount views of 22-hpf Tg(fli1a:EGFP) transgenic fish with the typical distribution of primitive macrophages (green cells) over the yolk (enclosed by the white broken line in the inset) at 0 minutes (left) and 60 minutes (right) post-wounding of control (A) and WASp1-morphant (B) embryos (wound centre indicated by an asterisk; see supplementary material Movies 1 and 2). (C) A graphic illustration of the number (n, y-axis indicates number of cells) of macrophages recruited to control versus morphant wounds and the mean speed (v, y-axis indicates mean speed in µm/minute) of these cells. (D,E) Illustrate two typical cell morphologies each of control (D) versus morphant (E) macrophages en route to the wound. Scale bars: A, 20 µm (inset, 50 µm); D,E, 4 µm.

View larger version (96K): [in this window] [in a new window]   Fig. 3. Ultrastructural studies of the fin-wound response. (A,B) Whole-mount 3-dpf larvae prior to wounding that were histochemically stained with Sudan Black to reveal neutrophils (A) or immunostained with L-plastin antibody to reveal macrophages and neutrophils (B), indicating the absence of any leukocytes in the undamaged fin. (C) Scanning electron micrograph (SEM) of a 3-dpf zebrafish larvae fixed immediately post-wounding, indicating the site of lesion (arrow). (D,E) High-magnification views of the wound region at 0 minutes (D) and 10 minutes (E) post-wounding, indicating the rapidity of repair in control larvae. (F) Higher-magnification view of the boxed area in E shows complete epithelial sealing by 10 minutes. (G) Transverse resin section through a Methylene-Blue stained wounded larval tail region; red arrow indicates the route taken by leukocytes from the ventral vein to the site of wound damage in the ventral fin. (H) Transmission electron micrograph (TEM) transverse reconstruction of the entire fin, from the ventral vein (asterisk) to the damaged-fin tip (black arrow). The white arrow indicates fin epithelium. (I-M) False-coloured, high-magnification TEM images corresponding to various steps in the leukocyte migration route from the blood vessel towards the wound. (I) A neutrophil (purple) with characteristic electron-dense cigar-shaped granules is captured as it adheres to the vessel wall (green). (J) Once outside the vessel, the neutrophil changes its morphology, becoming more elongated and polarised as it follows chemotactic cues towards the wound (in the direction of the arrow). (K) Low-magnification TEM illustrating a number of neutrophils and a single macrophage (blue) at the wound site. (L) Illustrates a macrophage that has recently engulfed matrix debris. Areas circled with red indicate desmosomes at junctions between macrophages and adjacent cells at the wound site. The high-magnification inset shows engulfed collagen. (M) Illustrates a macrophage that has engulfed a cell corpse (arrow). Scale bars: A-C, 500 µm; D,E, 20 µm; F, 5 µm. G, 15 µm; H, 5 µm; I,J, 2.5 µm; K, 4 µm; L,M, 2 µm.

View larger version (75K): [in this window] [in a new window]   Fig. 4. The consequences of WASp1- and WASp2-morpholino injections on leukocyte recruitment to a fin wound in 3-dpf larvae. (A-C) Typical 90-minute wounds in control- (A), WASp1-morpholino-injected (B) and WASp2-morpholino-injected (C) larvae after staining with Sudan Black to reveal neutrophil influx to the fin wound. (D-F) Similar wounds, but harvested at 3 hours and immunostained with antibody against L-plastin to reveal the influx of macrophages and neutrophils. (G) Images from a typical fluorescent time-lapse movie of control- and WASp1-morpholino-injected (MO) Tg(lyz:EGFP) fish after fin wounding; the white dotted ovals indicate wound location. See also supplementary material Movies 3 and 4. (H) qPCR analysis of egfp, lysC, wasp1 and wasp2 expression within EGFP-marked myelomonocytic cells of 3-dpf Tg(lyz:EGFP) larvae and Tg(lyz:EGFP) whole kidney marrow, relative to that within EGFP-negative cells. Error bars represent standard deviations. (I) FACScan analysis of EGFP-labelled compartments within wild-type, WASp1- and WASp2-depleted 3-dpf larvae. The y-axis indicates the percentage of green cells from total cells analysed, and reveals no significant difference between control and WASp-morphant fish. Averages and standard deviations calculated from two separate experiments for each sample are shown. Scale bars: A-F, 25 µm; G, 15 µm.

View larger version (60K): [in this window] [in a new window]   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.

View larger version (63K): [in this window] [in a new window]   Fig. 6. Identification of WASp1 mutants by TILLING. (A) Schematic to illustrate the sites of the two mutations in the wasp1 gene identified from our TILLING screen. (B,C) Graphic illustrations of the number of neutrophils drawn to 90-minute fin wounds that were made to 3-dpf larvae with the genotypes +/+ (wild type), +/– (heterozygote) and –/– (mutant) derived from +/– x +/– crosses of our exon-2 (B) or exon-10 (C)-mutant lines. Amino acids remaining post stop codon are highlighted in red. (D) Illustrates fluorescent immunostaining of typical wild-type, heterozygote and WASp1-mutant wounds at 3 hours post-wounding to indicate the differential extent of leukocyte recruitment; the broken white line indicates the ventral margin of the vein and the asterisk marks the centre of the wound. (E) Blood smears from 1-month-old WASp1-mutant (exon 10) fish versus control sibs to illustrate morphologies of erythrocytes (e), thrombocytes (t), neutrophils (n) and lymphocytes (l). Scale bars: D, 15 µm; E, 4 µm.

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