Figure 7
Figure 7. Depletion of SDF1a and photoactivation of Rac are sufficient to restore WHIM-GFP neutrophil–directed migration in vivo. (A) Time-lapse imaging (from supplemental Video 10) of GFP fluorescence showing WHIM-GFP neutrophils responding to a wound (*) in the ventral tailfin in a 3-dpf SDF1a morphant larvae. White arrows indicate WHIM-GFP neutrophils. (B-C,E-F) Sudan Black staining of neutrophil response to wounding (* or line) in the ventral tailfin (B-C) or to tail transection (E-F) of 3-dpf WHIM-GFP transgenic larvae injected with control (B,E) or SDF1a (C,F) MO. (D) Quantification of WHIM-GFP neutrophil response in wounded 3-dpf morphant larvae fixed 2 hours after wound as in panels B and C; *P < .01. (G) Quantification of WHIM-GFP neutrophil response to tail transection in 3-dpf morphant larvae fixed 2 hours after transection as in panels E and F; *P < .05. (H-J) Laser stimulation with a 458-nm light induces directed migration of WHIM-GFP neutrophils also expressing mCherry-PA-Rac from the CHT. Repeated photoactivation was used to direct a single WHIM-GFP/mCherry-PA-Rac neutrophil away from the cell aggregate in the CHT into the tailfin. (H) Z-stack images from the indicated time points in supplemental Video 11 were summed into a single 2-dimensional image and then consolidated into a semi–1-dimensional line. Stars indicate time points and position of laser stimulations. (I) Two examples of the WHIM-GFP/mCherry-PA-Rac neutrophil protruding after stimulation from supplemental Video 11. Black circles indicate position of laser stimulation; white circles are included as reference points. (J) Composite differential interference contrast image of the posterior CHT, blood vessels, and tailfin from supplemental Videos 11 and 12 overlaid with the track (black line) of the directed WHIM-GFP/mCherry-PA-Rac neutrophil migration away from and return to the CHT. The starting and stopping points of photoactivation are indicated. Note that after termination of photoactivation the neutrophil immediately returns to the neutrophil aggregate in the CHT. Similar observations were made in 3 different experiments with 3 different larvae. CHT indicates caudal hematopoietic tissue; BV, blood vessel. Arrows indicate direction of migration. Bars = 200 μm (B-D,F); 100 μm (A); 40 μm (J); and 20 μm (I).

Depletion of SDF1a and photoactivation of Rac are sufficient to restore WHIM-GFP neutrophil–directed migration in vivo. (A) Time-lapse imaging (from supplemental Video 10) of GFP fluorescence showing WHIM-GFP neutrophils responding to a wound (*) in the ventral tailfin in a 3-dpf SDF1a morphant larvae. White arrows indicate WHIM-GFP neutrophils. (B-C,E-F) Sudan Black staining of neutrophil response to wounding (* or line) in the ventral tailfin (B-C) or to tail transection (E-F) of 3-dpf WHIM-GFP transgenic larvae injected with control (B,E) or SDF1a (C,F) MO. (D) Quantification of WHIM-GFP neutrophil response in wounded 3-dpf morphant larvae fixed 2 hours after wound as in panels B and C; *P < .01. (G) Quantification of WHIM-GFP neutrophil response to tail transection in 3-dpf morphant larvae fixed 2 hours after transection as in panels E and F; *P < .05. (H-J) Laser stimulation with a 458-nm light induces directed migration of WHIM-GFP neutrophils also expressing mCherry-PA-Rac from the CHT. Repeated photoactivation was used to direct a single WHIM-GFP/mCherry-PA-Rac neutrophil away from the cell aggregate in the CHT into the tailfin. (H) Z-stack images from the indicated time points in supplemental Video 11 were summed into a single 2-dimensional image and then consolidated into a semi–1-dimensional line. Stars indicate time points and position of laser stimulations. (I) Two examples of the WHIM-GFP/mCherry-PA-Rac neutrophil protruding after stimulation from supplemental Video 11. Black circles indicate position of laser stimulation; white circles are included as reference points. (J) Composite differential interference contrast image of the posterior CHT, blood vessels, and tailfin from supplemental Videos 11 and 12 overlaid with the track (black line) of the directed WHIM-GFP/mCherry-PA-Rac neutrophil migration away from and return to the CHT. The starting and stopping points of photoactivation are indicated. Note that after termination of photoactivation the neutrophil immediately returns to the neutrophil aggregate in the CHT. Similar observations were made in 3 different experiments with 3 different larvae. CHT indicates caudal hematopoietic tissue; BV, blood vessel. Arrows indicate direction of migration. Bars = 200 μm (B-D,F); 100 μm (A); 40 μm (J); and 20 μm (I).

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