Analysis of WASp function during the wound inflammatory response--live-imaging studies in zebrafish larvae.
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.
Cvejic
Ana
A
Departments of Biochemistry and Physiology, School of Medical Sciences, University of Bristol, Bristol, UK.
Hall
Chris
C
Bak-Maier
Magdalena
M
Flores
Maria Vega
MV
Crosier
Phil
P
Redd
Michael J
MJ
Martin
Paul
P
D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified; D001777:Blood Coagulation / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002634:Chemotaxis, Leukocyte / Q000187:drug effects; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006410:Hematopoiesis / Q000187:drug effects; D007249:Inflammation / Q000378:metabolism*; D007814:Larva / Q000187:drug effects / Q000378:metabolism; D007962:Leukocytes / Q000166:cytology / Q000187:drug effects; D008264:Macrophages / Q000166:cytology / Q000187:drug effects; D008857:Microscopy, Interference; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009504:Neutrophils / Q000166:cytology / Q000187:drug effects; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D013623:Tail / Q000473:pathology / Q000648:ultrastructure; D013997:Time Factors; D051316:Wiskott-Aldrich Syndrome Protein / Q000737:chemistry / Q000378:metabolism*; D014947:Wounds and Injuries / Q000378:metabolism* / Q000473:pathology*; D015027:Zebrafish / Q000196:embryology / Q000378:metabolism*
D016376:Oligonucleotides, Antisense; D051316:Wiskott-Aldrich Syndrome Protein; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins