ZFIN ID: ZDB-FIG-131210-21
Jones et al., 2013 - Modelling human Wiskott-Aldrich syndrome protein mutants in zebrafish larvae using live in vivo imaging. Journal of Cell Science   126(Pt 18):4077-84 Full text @ J. Cell Sci.
ADDITIONAL FIGURES
EXPRESSION / LABELING:
Gene:
Antibody:
Fish:
Condition:
Anatomical Terms:
Stage: Protruding-mouth
PHENOTYPE:
Fish:
Condition:
Observed In:
Stage: Protruding-mouth

Fig. 1

The Zebrafish WASp mutant has a defect in leukocyte wound recruitment. (A) Schematic of WASp protein domains with the site of the STOP codon in washu3280 mutant indicated (red). (B) Schematic of 3dpf zebrafish larva showing haematopoietic cell location (green) and wound (red arrow). (C) Time course of Sudan-Black-positive neutrophil recruitment in WT versus mutant larvae (i). Two-way ANOVA, Bonferroni post-test at each time-point 0.5hour (NS), 1hour (***), 1.5hours (***), 2hours (***), 2.5hours (**), 3hours (***), 4hours (**), 5hours (***), 6hours (NS). Representative images of Sudan-Black-stained neutrophils recruited to the 90minute wound site (ii) in WT (a) and mutant (b). (D) Time-course of macrophage recruitment in WT versus mutant mpx:GFP+ larvae (i). Two-way ANOVA, Bonferroni post-test at each time point 1hour (NS), 2hours (*), 3hours (**), 5hours (***), 7hours (***), 9hours (***), 24hours (NS). Representative images of 3hour wounds immunostained for L-Plastin to reveal all leukocytes (red) and mpx:GFP+ neutrophils (green) at the wound site (ii) in WT (a) and mutant (b). (E) Representative tracking analysis of mpx:GFP+ neutrophils during the first 90minutes post wounding of a WT larva (i); each neutrophil track is in a different colour and the wound is indicated by dotted lines. Graphic representation of the velocity of migrating cells (µm/second) (ii), pause duration (seconds) with number of pauses >1minute during wound migration (in brackets) (iii) and cell meandering index (iv), taken from tracks of WT (n = 20) and mutant (n = 20) mpx:GFP+ neutrophils. (F–H) Sample still images from confocal movies to illustrate protrusion/retraction analysis (i); neutrophils are green and new protrusive areas are indicated in magenta (F,G); and the retracting uropod in blue (H). WT cells exhibit greater persistence of polarity towards the wound (red asterisk). (F) (ii) Comparison of protrusive area (front versus back ratio analysis) in paused cells of WT vs mutant larvae. (G) (ii) As in F, but for migrating WT versus mutant cells, showing reduced protrusion in mutant cells. (H) (ii) Retraction analysis applied to migrating WT versus mutant cells, revealing mutant has defect in rate of uropod retraction. Error bars: s.e.m. (C,D); s.d. (E–H); *P<0.05; **P<0.01; ***P<0.001 by one-way ANOVA (C,D) and Student′s t-test (E–H). NS, not significant. Scale bars: 10μm (C,D); 20μm (E); 5μm (F–H).

Gene Expression Details
Gene Antibody Fish Conditions Stage Anatomy Assay
GFP i114Tg physical alteration: anatomical structure Protruding-mouth neutrophil IHC
wasbhu3280/hu3280; i114Tg physical alteration: anatomical structure Protruding-mouth neutrophil IHC
Antibody Labeling Details
Antibody Assay Fish Conditions Stage Anatomy
Ab2-lcp1 IHC i114Tg physical alteration: anatomical structure Protruding-mouth leukocyte
IHC wasbhu3280/hu3280; i114Tg physical alteration: anatomical structure Protruding-mouth leukocyte
Phenotype Details
Fish Conditions Stage Phenotype
wasbhu3280/hu3280 physical alteration: anatomical structure Protruding-mouth neutrophil chemotaxis decreased process quality, abnormal
Protruding-mouth neutrophil decreased amount, abnormal
wasbhu3280/hu3280; i114Tg physical alteration: anatomical structure Protruding-mouth macrophage chemotaxis decreased process quality, abnormal
Protruding-mouth macrophage decreased amount, abnormal
Protruding-mouth neutrophil chemotaxis decreased process quality, abnormal
Protruding-mouth neutrophil chemotaxis duration, abnormal
Protruding-mouth neutrophil decreased amount, abnormal
Protruding-mouth neutrophil decreased velocity, abnormal
Protruding-mouth neutrophil pseudopodium assembly decreased rate, abnormal
Protruding-mouth neutrophil uropod retraction decreased rate, abnormal
Acknowledgments:
ZFIN wishes to thank the journal Journal of Cell Science for permission to reproduce figures from this article. Please note that this material may be protected by copyright. Full text @ J. Cell Sci.