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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 7.

Figure 7

CNR2-signaling promotes thymic colonization via P-selectin activity. (A): MO-mediated knockdown of psgl-1 and selp reduced cmyb expression in the thymus and prevented AM1241 (30–96 hpf) from increasing thymic colonization (n≥50 per condition). (B): Qualitative phenotypic distribution of embryos from panel (A), scored with low, medium, or high cmyb expression in the thymus at 96 hpf. (C): MO-mediated knockdown of psgl-1 and selp, as described in panel (A), decreased rag1 expression in the thymus in the presence and absence of AM1241 (30–120 hpf) (n≥35 per condition). (D): Qualitative phenotypic distribution of embryos from panel (C), scored with low, medium, or high rag1 expression in the thymus at 96 hpf. (E): In vivo imaging of rag2:egfp embryos confirmed that psgl-1- and selp-MOs decreased the number of Rag2:GFP+ lymphoid progenitors in the thymus both with and without AM1241-treatment (n≥8 per condition). (F): Relative fluorescent intensity of the thymus in morphant embryos, as described in panel (E) (control/DMSO: 1.00±0.10 a.u., psgl-1 MO/DMSO:0.51±0.08, selp MO/DMSO: 0.64±0.29, Control/AM1241: 1.79±0.29 a.u., psgl-1 MO/AM1241: 0.91±0.09, selp MO/AM1241: 1.17±0.53; *, p≤.05; **, p≤.01, one-tailed t test). (G): Exposure to KF38789 (30–96 hpf) confirmed the role of P-selectin in normal and AM1241-enhanced thymic colonization, as indicated by Rag2:GFP at 96 hpf (DMSO: 1.00±0.18 a.u., AM1241: 1.76±0.20, KF38789: 0.73±0.20, AM1241+KF38789: 0.71±0.26; **, p≤.01; ****, p≤.0001, two-tailed t test, n≥7 per condition). (H): qPCR analysis showed that exposure to AM630 after the phase of HSC induction (48–96 hpf) altered the expression of psgl-1 and selp (psgl-1: 1.44-fold, selp: 0.72-fold, **, p≤.01; ****, p≤.0001, two-tailed t test). (I): Absolute counts of CD41:GFP+ cells revealed that exposure to AM630, as described in panel (H), increased the number of HSCs remaining in the CHT at 96 hpf (DMSO: 26.4±1.3, AM630: 35.6±1.4; ****, p≤.0001, two-tailed t test, n≥38,). (J): Relative fluorescent intensity of Rag2:GFP+ cells the thymus following exposure to AM630, as described in panel (G), indicated CNR2-signaling was required for thymic colonization (DMSO: 1.0±0.06 a.u., AM630: 0.77±0.05 a.u.; *, p≤.05, two-tailed t test, n≥14). Scale bars (A, C)=200 μm, (E)=175 μm. Abbreviations: DMSO, dimethyl sulfoxide; GFP, green fluorescent protein; MO, morpholino; psgl-1, p-selectin glycoprotein ligand-1; qPCR, quantitative polymerase chain reaction; sele, e-selectin.