OVOL2 enhances ER71-mediated FLK1⁺ cell generation, as well as hematopoietic and endothelial cell lineage development. (A) Expression of Er71, Ovol2, and Flk1 in ESC differentiation. The expression in each day point was normalized against Gapdh and 100% denotes the level where its maximum expression was reached. (B) VENUS⁺ cells and VENUS⁻ cells from E8.5 ER71-VENUS embryos were subjected to qRT-PCR analysis. (C) E8.5 embryos were sectioned and subjected to immunohistochemical analysis of OVOL2 (green) and ER71 (red). DAPI (blue) was used for nucleus staining. Scale bar, 100 µm. (D) Synergistic activation of Flk1 promoter by ER71 and OVOL2. Expression constructs were transfected into 293T cells with pGL3 or pGL3-Flk1 promoter-luciferase reporter plasmids. The relative luciferase activity was gained by normalizing Firefly luciferase activity to Renilla luciferase activity at 48 hours posttransfection (***P < .001). (E-F) iER71, iOVOL2, and iER71-OVOL2 ESCs were differentiated either in a serum-free medium for 4 days (E), or in a serum-containing medium for 3.5 days (F), and subjected to fluorescence-activated cell sorter (FACS) analysis. Numbers in the plots denote the percentages of FLK1⁺ cells. DOX was added at D2 and D1 in a serum-free or a serum-containing differentiation medium, respectively. (E’) The percentage of FLK1⁺ cells in iER71, iOVOL2, and iER71-OVOL2 differentiated in a serum-free condition in the absence (-D) or presence (+D) of DOX. Results are mean ± SEM from 3 replications (**P < .01). (F’) Fold change of the generation of FLK1⁺ cells in ESCs after DOX treatment (+DOX/-DOX) in a serum-containing media. Results are mean ± SEM from 3 replications (**P < .01). (G, G’) FACS analysis for CD31/PECAM1 and VE-Cad in D6.5 EBs. iER71, iOVOL2, and iER71-OVOL2 ESCs differentiated in a serum-containing medium were treated with DOX at D3.5, followed by FACS analysis for CD31 and VE-Cad at D6.5 (**P < .01). (H) Hematopoietic replating assay. iER71, iOVOL2, and iER71-P2A-OVOL2 ESCs differentiated in a serum-free condition were treated with DOX at D3 and subjected to hematopoietic replating assay at D6. Colonies were counted 4 days later. B4, BMP4; V, VEGF-A. Results are mean ± SEM from 3 replications (**P < .01). (I) Gene expression profiling in D3.5 EBs overexpressing either ER71 (iER71) or OVOL2 (iOVOL2), or both ER71 and OVOL2 (iER71-P2A-OVOL2) in a serum-containing medium. DOX was added at D1 and RNA was prepared at D3.5. Expression of each gene was normalized against Gapdh, and the fold change of its expression level (+DOX/-DOX) was calculated. (J-L) iER71 ESCs infected with lentiviral shRNA particles against Ovol2 or Gfp control were differentiated in a serum-containing medium, and subjected to FACS analysis for FLK1 (J), and qRT-PCR for Ovol2 (K) (***P < .001). (L) Gene expression analysis in D3.5 EBs that had been infected with lentiviral particles of Ovol2 shRNAs (**P < .01; ***P < .001).