Figure 1
Figure 1. Etv2 expression in developing mouse embryos. (Aa) Etv2 is first detected in the posterolateral mesoderm around E7.0; panel i is the anterior and panel ii the posterior view. Scale bar indicates 50 μm. (b) Double immunostaining with anti-Flk-1 antibody revealed that Etv2 is expressed in a subset of Flk-1+ cells at this stage. Magnified view of the posterolateral mesoderm where Flk-1+ cells exists: Etv2-Green (i), Flk-1-Red (ii), and merged (iii). Scale bar indicates 50 μm. (Ba) Etv2 is expressed at E7.5 in the extra-embryonic mesoderm. Etv2 is almost colocalized with Flk-1 at this stage: (i) anterior view, (ii) posterior view, and (iii) lateral view. Scale bar indicates 200 μm. (b) At E8.5 expression was detected along the dorsal aorta, endocardium and allantois (al). Expression in the extra-embryonic yolk sac (YS) region was rapidly down-regulated. Scale bar indicates 500 μm. (c) Expression of Etv2 at E9.5. (d) Expression was detected along the vasculature. (e) Etv2 was barely detected in matured ECs incorporated into the vasculature (arrows). Scale bars in panels b and c indicate 500 μm; scale bars in panels d and e indicate 200 μm. (C) Flow cytometric analysis of Etv2 expression in developing embryos. (a) Flow cytometry of E7.5 and 8.5 Etv2-Venus+/− embryos. At E7.5, 76% of Flk-1+ cells were Etv2+. (b) At E8.5, Etv-2 expression was still restricted in the Flk-1+ population, with 56% of the Flk-1+ cells being Etv2-Venus+. (c) At E8.5, 79% of the VE-cadherin+ cells retained the Etv2 expression. Approximately 92% of the CD41+ HPCs were Etv2-Venus−, indicating the rapid down-regulation of Etv2 on HPC differentiation. (D) Etv2 expression in differentiating ESCs. Etv2-Venus ESCs were differentiated on OP9 cells. Mesoderm-inducing factors BMP4, FGF8b, and VEGF were added on day 3 of differentiation. On day 3, when Flk-1+ cells started to be seen, only 0.1% were Flk-1+/Etv2-Venus+ whereas 3.9% were Flk-1+/Etv2-Venus−. The addition of mesoderm-inducing factors promoted the generation of Etv2+ cells in 24 hours on day 4 (Flk-1+/Etv2-Venus+ cells from 0.6%-4.1%) and day 5 (Flk-1+/Etv2-Venus+ cells from 1.2%-4.8%). The majority of Etv2-Venus+ cells are present as Flk-1+.

Etv2 expression in developing mouse embryos. (Aa) Etv2 is first detected in the posterolateral mesoderm around E7.0; panel i is the anterior and panel ii the posterior view. Scale bar indicates 50 μm. (b) Double immunostaining with anti-Flk-1 antibody revealed that Etv2 is expressed in a subset of Flk-1+ cells at this stage. Magnified view of the posterolateral mesoderm where Flk-1+ cells exists: Etv2-Green (i), Flk-1-Red (ii), and merged (iii). Scale bar indicates 50 μm. (Ba) Etv2 is expressed at E7.5 in the extra-embryonic mesoderm. Etv2 is almost colocalized with Flk-1 at this stage: (i) anterior view, (ii) posterior view, and (iii) lateral view. Scale bar indicates 200 μm. (b) At E8.5 expression was detected along the dorsal aorta, endocardium and allantois (al). Expression in the extra-embryonic yolk sac (YS) region was rapidly down-regulated. Scale bar indicates 500 μm. (c) Expression of Etv2 at E9.5. (d) Expression was detected along the vasculature. (e) Etv2 was barely detected in matured ECs incorporated into the vasculature (arrows). Scale bars in panels b and c indicate 500 μm; scale bars in panels d and e indicate 200 μm. (C) Flow cytometric analysis of Etv2 expression in developing embryos. (a) Flow cytometry of E7.5 and 8.5 Etv2-Venus+/− embryos. At E7.5, 76% of Flk-1+ cells were Etv2+. (b) At E8.5, Etv-2 expression was still restricted in the Flk-1+ population, with 56% of the Flk-1+ cells being Etv2-Venus+. (c) At E8.5, 79% of the VE-cadherin+ cells retained the Etv2 expression. Approximately 92% of the CD41+ HPCs were Etv2-Venus−, indicating the rapid down-regulation of Etv2 on HPC differentiation. (D) Etv2 expression in differentiating ESCs. Etv2-Venus ESCs were differentiated on OP9 cells. Mesoderm-inducing factors BMP4, FGF8b, and VEGF were added on day 3 of differentiation. On day 3, when Flk-1+ cells started to be seen, only 0.1% were Flk-1+/Etv2-Venus+ whereas 3.9% were Flk-1+/Etv2-Venus−. The addition of mesoderm-inducing factors promoted the generation of Etv2+ cells in 24 hours on day 4 (Flk-1+/Etv2-Venus+ cells from 0.6%-4.1%) and day 5 (Flk-1+/Etv2-Venus+ cells from 1.2%-4.8%). The majority of Etv2-Venus+ cells are present as Flk-1+.

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