Genes regulated by Etv2. (A) Gene-expression profiling of Flk-1⁺Etv2⁺ and Flk-1⁺Etv2^– populations generated from differentiated of Etv2-Venus^+/− ESCs for 5 days on OP9. Flk-1⁺Etv2⁺ or Flk-1⁺Etv2⁻ cells were analyzed by (a) microarray and (b) Q-PCR. Genes important for EC and HPC differentiation were preferentially expressed in Etv2⁺ Flk-1⁺ cells. Genes that showed significant changes between Flk-1⁺Etv2⁺ and Flk-1⁺Etv2⁻ populations are listed in supplemental Table 1. (B) (a) Gene-expression analysis of Flk-1⁺ PDGFRα⁺ cells derived from Etv2^+/− and ^−/− ESCs. After 5 days of differentiation, Flk-1⁺PDGFRα⁺cells were subjected to microarray analysis. Genes that showed significant changes between Etv2^+/− and ^−/− populations are listed in supplemental Table 2. (b) Expression levels of key EC and HPC genes were analyzed by Q-PCR in Etv2^+/−and Etv2^−/− populations. Genes important for EC and HPC development were down-regulated in Etv2^−/− cells. In Q-PCR experiments (Ab and Bb) PCR reactions were performed in triplicate. Data for each gene was normalized by GAPDH and are shown as the -fold change compared with Flk-1⁺Etv2⁻ (Ab) or Etv2^+/− (Bb) populations. (C) Expressions of Scl, Fli1, GATA2 (a), Runx, and GATA1 (b) in E7.5 Etv2^+/− or Etv2^−/−embryos. Scl, Fli1, and Runx: in situ hybridization; GATA1: immunostaining. Unlike expression analysis in ESC-derived Flk-1⁺ cells, Runx1 was undetectable in Etv2^−/− embryos. GATA1 and GATA2 were down-regulated but detectable in differentiated Etv2 ^−/− cells but totally undetectable in Etv2^−/− embryos. Scale bar indicates 300 μm.