Cxcl8/cxcr1 signaling is a positive regulator of HSPC colonization. (a) Transmitted light image of a 72 hpf embryo. HSCs and HSPCs were enumerated in the entire CHT (black box) using digital image analysis as described in the text. The red box shows the approximate area selected for the representative images below. Bar, 300 µm. (b–d) GFP or cxcr1 was overexpressed by heat shock induction at 36 and 48 hpf and HSPC colonization of the CHT was quantified at 72 hpf. Representative fluorescence images (b_i and c_i), digital image segmentation (b_ii and c_ii), and overlays (b_iii and c_iii) are shown. (d) GFP and CXCR1 groups were compared using Student’s t test (P = 0.020, n = 10 for GFP control; n = 16 for CXCR1). The experiment was repeated twice; combined results are shown. (e–g) GFP or cxcr1 was overexpressed as in panels b-d except that heat shock induction was performed at 48 and 60 hpf with imaging at 84 hpf (P = 0.023, Student’s t test, n = 15 for GFP control and n = 13 for CXCR1). The experiment was repeated twice; combined results are shown. (h–k) Runx1:mCherry;cxcl8^+/− animals were in-crossed to generate Runx1:mCherry;cxcl8^+/+ (h), Runx1:mCherry;cxcl8^+/− (i), and Runx1:mCherry;cxcl8^−/− (j) animals. Animals were imaged at 72 hpf and HSPC colonization of the CHT was quantified (k; P = 0.02 for the comparison of +/+ and −/− groups; Student’s t test; n = 17 for +/+, n = 9 for +/−, and n = 10 for −/− groups). The experiment was repeated four times; combined results are shown. Bars, 20 µm. (l) Expression of GFP or cxcr2 was induced by heat shock at 36 and 48 hpf, as before. Animals were imaged at 72 hpf, and HSPCs were enumerated in the CHT. Groups were compared using Student’s t test (p = NS; n = 14 for GFP control and n = 12 for cxcr2). The experiment was repeated twice with similar results; combined results are shown.