Fig. 6

P-selectin mediates the effect of CNR2-stimulation in the caudal hematopoietic tissue (CHT).

(A): qPCR analysis revealed expression of p-selectin (selp) and its ligand (psgl-1) were significantly upregulated by AM1241- but not PGE2-exposure (30–36 hpf) (psgl-1: dmPGE2, 1.06-fold; AM1241, 1.29-fold; selp: dmPGE2, 0.96-fold; AM1241, 1.62-fold; sele: dmPGE2, 1.34-fold; AM1241, 1.10-fold; *, p d .05; **, p d .01, two-tailed t test, n e 7).

(B): qPCR analysis using FACS-sorted populations from flk1:dsRed;cmyb:GFP embryos at 48 hpf showed that psgl-1 is expressed at a higher level in HSCs (Flk1:dsRed⁺;cMyb:GFP⁺: 28.1 a.u.) than in the vasculature (Flk1:dsRed⁺; cMyb:GFP: 1.86 a.u.) (normalized to 18s).

(C): Knockdown of psgl-1 and selp by MO injection reduced runx1;cmyb expression in the CHT and inhibited the effect of AM1241 (30–38 hpf) on hematopoietic stem and progenitor cell production (n e 60 per condition).

(D): Qualitative phenotypic distribution of embryos from panel (C), scored with low, medium, or high runx1;cmyb expression in the CHT at 38 hpf.

(E): In vivo imaging of cd41:egfp embryos showed that P-selectin knockdown decreased the number of CD41:GFP⁺ HSCs in the CHT (arrowheads) at 38 hpf and prevented AM1241-stimulated expansion (n e 7 per condition).

(F): Absolute counts of CD41:GFP⁺ cells confirmed P-selectin activity was required for increased hematopoietic stem cell (HSCs) numbers in the CHT mediated by AM1241 (control/DMSO: 11.9 ± 0.7, psgl-1 MO/DMSO: 8.6 ± 1.1, selp MO/DMSO: 9.9 ± 0.7 control/AM1241: 15.8 ± 1.2, psgl-1 MO/AM1241: 9.0 ± 1.7, selp MO/AM1241: 10.7 ± 0.8; *, p d .05, two-tailed t test).

(G): Exposure (30–38 hpf) to a selective inhibitor of P-selectin-mediated cell adhesion (KF38789, 0.5 µM) confirmed the requirement of P-selectin activity for normal and AM1241-enhanced CD41:GFP⁺ HSC numbers in the CHT (n e 18).

(H): Absolute counts of CD41:GFP⁺ HSCs in the CHT after exposure to KF38789 and/or AM1241 (DMSO: 12.2 ± 1.0, AM1241: 16.8 ± 1.4, KF38789: 6.2 ± 0.5, AM1241+KF38789: 6.9 ± 0.7; *, p d .05; ****, p d .0001, two-tailed t test).

(I): qPCR analysis showed that expression of selp and psgl-1 was significantly upregulated during HSC expansion and secondary niche colonization (30–72 hpf) (psgl-1: 1.31-fold; selp: 1.40-fold; sele: 1.51-fold; *, p d .05, two-tailed t test, n = 4 replicates). Scale bars (C, E, G) = 100 µm. Abbreviations: DMSO, Dimethyl Sulfoxide; FACS, fluorescence-activated cell sorting; MO, morpholino; PGE2, prostaglandin E2; psgl-1, p-selectin glycoprotein ligand-1; qPCR, quantitative polymerase chain reaction; selp, p-selectin; sele, e-selectin.