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

Journal of Cell Science 121, 3196-3206 (2008)
Published by The Company of Biologists 2008
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Research Article

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

Ana Cvejic1,2, Chris Hall3, Magdalena Bak-Maier1, Maria Vega Flores3, Phil Crosier3, Michael J. Redd4 and Paul Martin1,*

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

* Author for correspondence (e-mail: paul.martin{at}bristol.ac.uk)

Accepted 8 July 2008

Wiskott-Aldrich syndrome protein (WASp) is haematopoietically restricted, and is the causative protein underlying a severe human disorder that can lead to death due to immunodeficiency and haemorrhaging. Much is known about the biochemistry of WASp and the migratory capacity of WASp-defective cells in vitro, but in vivo studies of immune-cell behaviour are more challenging. Using the translucency of zebrafish larvae, we live-imaged the effects of morpholino knockdown of WASp1 (also known as Was) on leukocyte migration in response to a wound. In embryos at 22 hours post-fertilisation, primitive macrophages were impaired in their migration towards laser wounds. Once a circulatory system had developed, at 3 days post-fertilisation, we observed significantly reduced recruitment of neutrophils and macrophages to ventral fin wounds. Cell-tracking studies indicated that fewer leukocytes leave the vessels adjacent to a wound and those that do exhibit impaired navigational capacity. Their cell morphology appears unaltered but their choice of leading-edge pseudopodia is more frequently incorrect, leading to impaired chemotaxis. We also identified two zebrafish mutants in WASp1 by TILLING, one of which was in the WIP-binding domain that is the hotspot for human lesions, and mutants exhibited the same deficiencies in wound inflammation and thrombus formation as WASp1 morphants.

Key words: WASP, Wound, Inflammation, Macrophage, Neutrophil, TILLING


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