Cxcr1 signaling alters the structure of the CHT. (a–c) Overexpression of GFP (control) or cxcr1 was induced in microinjected zebrafish by heat shock at 36 and 48 hpf. (a and b) Representative WISH images for kdrl, marking endothelial cells, at 72 hpf are shown. Bars, 100 µm. (c) Blinded scoring of kdrl staining in the CHT (Wilcoxon rank sum test, P = 0.041; n = 17 for GFP control; n = 23 for cxcr1). The experiment was repeated twice with similar results; a representative experiment is shown. (d–f) Isosurface rendering of the CHT using Imaris. A representative kdrl:mCherry transgenic fish, imaged at 72 hpf, is shown (d). A three-dimensional isosurface rendering of the CHT and overlay are shown (e and f). Only the yellow portion of the isosurface was included in the volumetric analysis. Bars, 100 µm. (g–j) CHT volume was measured in kdrl:GFP or kdrl:mCherry reporter zebrafish at 72 hpf. CHT volume is plotted in µm3. (g) All zebrafish were injected with hsp70l:cxcr1 DNA and gene expression was induced in one half of the animals by heat shock at 36 and 48 hpf (Wilcoxon rank sum test, P = 0.02, n = 15 for uninduced controls and n = 15 for heat shock–induced embryos). The experiment was repeated three times with similar results, a representative experiment is shown. (h) Kdrl:mCherry transgenic fish were treated with the CXCR1/2 inhibitor SB225002 (SB) or DMSO control from 48 to 72 hpf (Student’s t test, P = 0.012; n = 9 for untreated controls; n = 7 for treated embryos). The experiment was repeated twice with similar results; a representative experiment is shown. (i) Kdrl:mCherry zebrafish were injected with hsp70l:cxcr1 DNA or hsp70l:GFP as a control followed by heat shock at 36 and 48 hpf. A time series plot showing the relative change in CHT volume from 52 to 72 hpf compared with baseline is shown. Colored bands represent 95% confidence intervals. n = 5 for GFP control and n = 5 for hsp70l:cxcr1. The experiment was repeated twice with similar results; a representative experiment is shown. (j) The CHT volume of kdrl:mCherry;kdrl:cxcr1 zebrafish and kdrl:mCherry (WT) clutchmates is shown (Student’s t test, P = 0.02; n = 9 for WT; n = 7 for kdrl:cxcr1). The experiment was repeated twice, with similar results. A representative experiment is shown. (k–p) Representative images of a lyve1b:GFP;kdrl:cxcr1 transgenic are shown. (k–m) Low power views showing expression of the lyve1b:GFP reporter transgene predominantly in the CHT. GFP expression in the heart is driven by a secondary marker transgene (cmlc:GFP) for kdrl:cxcr1. Bars, 500 µm. (n–p) High power views of the CHT in the same embryo. (n) GFP expression in the CHT and caudal vein (CV). (o) Three dimensional isosurface of the lyve1b:GFP-expressing tissues. Only the yellow portion of the isosurface is used for quantifying CHT volume. (p) Overlay of the isosurface and lyve1b:GFP expression. Bars, 100 µm. (q) The CHT volume of lyve1b:DsRed (WT) and kdrl:cxcr1;lyve1b:DsRed (kdrl:cxcr1) transgenics was measured at 72 hpf as before (P = 0.0006, Wilcoxon rank sum test; n = 23 for WT; n = 20 for kdrl:cxcr1). The experiment was repeated twice with similar results; a representative experiment is shown.
