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. Author manuscript; available in PMC: 2016 Mar 7.
Published in final edited form as: Stem Cells. 2015 May 26;33(8):2596–2612. doi: 10.1002/stem.2044

Figure 5.

Figure 5

AM1241 mediates hematopoietic stem cell (HSC) expansion in the AGM, but not CHT, via PGE2-signaling. (A): Treatment with selective inhibitors to Ptgs2 (NS-398) or PKA/cAMP (H89, 5 μM) during niche specification (18–24 hpf) decreased runx1 expression and blocked AM1241-mediated inductions in hematopoietic stem and progenitor cell (HSPC) expression in the AGM (n≥45 per condition).(B): Qualitative phenotypic distribution of embryos from panel (A), scored with low, medium, or high runx1 expression in the AGM. (C): Treatment with NS-398 or H89 during hematopoietic stem cell (HSC) production (30–38 hpf) failed to block AM1241-mediated elevations in runx1;cmyb expression in the CHT (n≥75 per condition). (D): Qualitative phenotypic distribution of embryos from panel (C), scored with low, medium, or high runx1 expression in the CHT. (E): Absolute counts of Flk1:dsRed+;cMyb:GFP+ HSCs confirmed PGE2 production and signaling are required to mediate the effects of AM1241 in the AGM (DMSO: 5.8±0.3, AM1241: 9.1±0.5, NS-398: 4.9±0.3, AM1241+NS-398: 5.4±0.3, H89: 4.9±0.2, AM1241+NS-398: 5.0±0.2; *, p≤.05; ****, p<.0001, two-tailed t test, n≥10 per condition). (F): Absolute counts of CD41:GFP+ HSCs confirmed AM1241-mediated increases in the CHT are independent of PGE2 production and signaling (DMSO: 8.5±0.6, AM1241: 12.8±1, NS-398: 9.8±0.8, AM1241+NS-398: 11.4±1.1, H89: 9.4±1.0, AM1241+NS-398: 12.7±1.9, *, p≤.05; **, p≤.01, two-tailed t test, n≥9 per condition). (G): Embryos exposed to AM1241 during niche specification (18–24 hpf) exhibited an increased number of pH3+ (red) cells in the Flk1:GFP+ hemogenic vasculature of the AGM (dorsal aorta marked with white bracket) at 30 hpf, which was blocked by cotreatment with NS-398 (n≥9 per condition). (H): Absolute counts of pH3+ cells in the AGM, as described in panel (G) (DMSO: 5.0±0.27, NS-398: 4.22±0.28, AM1241: 7.15±0.45, AM1241+NS-398: 4.09±0.34; *, p≤.05; ***, p≤.001, two-tailed t test). (I): Embryos exposed to AM1241 during HSC production (30–38 hpf) exhibited a higher number of pH3+ (blue) cMyb:GFP+ HSPCs in the Flk1:dsRed vasculature of the CHT (CHT width marked with white bracket) at 38 hpf; this effect was independent of Ptgs2 activity (n≥9 per condition). (J): Absolute counts of cMyb:GFP+;pH3+ cells in the CHT, as described in panel (I) (DMSO: 3.55±0.57, NS-398: 3.78±0.94, AM1241: 6.67±1.33, AM1241+NS-398: 5.36±0.72; *, p<.05, two-tailed t test). Scale bars (A, C)=80 μm, (G, I)=100 μm. Abbreviations: AGM, aorta-gonad-mesonephros; CHT, caudal hematopoietic tissue; DMSO, Dimethyl Sulfoxide; GFP, green fluorescent protein; PH3, phospho-histone 3.