FIGURE

Figure 5

ID

ZDB-FIG-200725-5

Publication

Poplimont et al., 2020 - Neutrophil Swarming in Damaged Tissue Is Orchestrated by Connexins and Cooperative Calcium Alarm Signals

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Figure 5

Neutrophil Cx43 Is Required for Intracluster Calcium Fluxes and Swarm Initiation

(A) Time-lapse sequence of two-photon confocal image projections showing neutrophils in Tg(lyz:GCamp6F) larvae in the presence of PI, without treatment (ctr), with 50 μM CBX, or with morpholinos against cx43/cx43.4 (cx43 MO). Scale bars represent 50 μm and 10 μm for zoomed-out and zoomed-in images, respectively. Time after LW is shown in minutes. Arrows indicate neutrophils in contact with the wound.

(B) Normalized GCamp6F levels in control (n = 8), CBX-treated larvae (n = 5), and cx43 MO-treated (n = 7) from 8, 2, and 3 experiments, respectively. One-way ANOVA with Dunnett’s post test is shown. GCamp6F intensity was normalized as in Figure 1D. Data are from Tg(lyz:GCamp6F) and Tg(lyz:GCamp6F)xTg(lyz:tRFP-5LO) larvae evenly distributed across the groups.

(C) Neutrophil radial speed over time post-wounding for neutrophils in control, cx43 MO-treated, and CBX-treated larvae. Data are pooled from Tg(lyz:GCamp6F), Tg(lyz:GCamp6F)xTg(lyz:tRFP-5LO), and Tg(mpx:GFP)ⁱ¹¹⁴ zebrafish larvae. n = 1,201–1,719 cell steps per bin from 12 control, n = 1,268–1,535 cell steps per bin from 8 CBX-treated, and n = 1,308–1,554 cell steps per bin from 11 cx43 MO-injected larvae from 8, 3, and 4 experiments, respectively. Kruskal-Wallis with Dunn’s post test results indicating significance found between ctr and MO and between ctr and CBX (gray) or only between ctr and CBX (black) are shown.

(D) Time-lapse sequence of two-photon confocal image projections showing neutrophils in Tg(lyz:GCamp6F)xTg(lyz:cx43DN-T2A-mCherry) zebrafish larvae, positive (cx43 DN) or negative for the Cx43 DN-T2A-mCherry transgene (control siblings: Ctr sib). Annotations are as in (A).

(E) Normalized GCamp6F levels in neutrophils positive (cx43 DN) or negative for the Cx43DN-T2A-mCherry transgene (Ctr sib). n = 7 cx43 DN transgenics and n = 5 control siblings from 3 experiments; Mann-Whitney test.

(F) Neutrophil radial speed over time post-wounding for neutrophils positive (cx43 DN) or negative for the Cx43DN-T2A-mCherry transgene (Ctr sib). n = 1,330–1,602 cell steps per bin from 5 control siblings and n = 2,573–3,050 cell steps per bin from 7 Cx43 DN larvae from 3 experiments; Mann-Whitney test.

Error bars represent SEM. ^∗p < 0.03, ^∗∗p < 0.002, ^∗∗∗p < 0.0002. See also Figures S4–S6 and Video S6.

Expression Data

Expression Detail

Antibody Labeling

Phenotype Data

Phenotype Detail

Acknowledgments

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