Cxcl8/cxcr1 signaling in endothelial cells induces gene expression changes favoring HSPC colonization. (a) Kdrl:cxcr1;kdrl:mCherry zebrafish and kdrl:mCherry clutchmates (WT) were dissociated at 72 hpf, and mCherry⁺ endothelial cells were FACS sorted. Quantitative PCR for cxcr1 is shown. The experiment was repeated three times with similar results. (b–d) Kdrl:cxcr1;Runx1:mCherry zebrafish were imaged at 72 hpf for HSPC colonization of the CHT (a and b). Bars, 20 µm. (d) Plot showing increased HSPC colonization in kdrl:cxcr1 animals (P = 0.001, Wilcoxon’s rank sum test; n = 35 for WT control; n = 28 for kdrl:cxcr1) The experiment was repeated twice with similar results; combined results are shown. (e) Mpx:GFP (WT) and kdrl:cxcr1;mpx:GFP zebrafish were imaged at 72 hpf, and neutrophil numbers in the CHT were quantified (p = NS, Student’s t test; n = 47 for WT control; n = 35 for kdrl:cxcr1). The experiment was repeated three times with similar results. Combined results are shown. (f–k) Kdrl:cxcr1 and WT clutchmates were fixed at 72 hpf and WISH was performed for cxcl12a (f–h) and cxcl12b (i–k). Bar, 100 µm. h and k show the results of blinded semiquantitative scoring of CHT staining for each probe (cxcl12a: P = 0.03, Wilcoxon’s rank sum test, n = 26 for WT control and n = 34 for kdrl:cxcr1; cxcl12b: p = NS, Wilcoxon’s rank sum test, n = 19 for WT control and n = 22 for kdrl:cxcr1). The experiment was performed three times with similar results; combined results are shown. (l) HUVECs were serum starved for 12 h, and then treated with 10 ng/ml rhCXCL8 or vehicle control. Quantitative RT-PCR was performed for expression of CXCL12, CXCL8, and survivin and VEGFA. (m) RNA sequencing was performed on HUVEC RNA. IPA analysis identifying the top enriched molecular and cellular functions is shown. The HUVEC experiments were performed with biological duplicates.