Etv2 is dispensable for the generation of Flk-1⁺ mesoderm but is required for the generation of HPCs/ECs. (A) Etv2^+/− cells differentiated on OP9 could generate 1.88% VE-cadherin⁺ or 0.148% CD41⁺ cells (top). Etv2^−/− ESCs generated Flk-1⁺ cells but failed to differentiate into VE-cadherin⁺ (0%) nor CD41⁺ cells (0.0025%) (bottom). Flk-1⁺ cells from Etv2^−/− ESCs were arrested as PDGFRα⁺ cells. Etv2^+/− Flk-1⁺ cells could down-regulate PDGFRα. (B) In contrast to Etv2^+/− (top), E8.5 Etv2^−/− cells (bottom) failed to generate Flk-1⁺VE-cadherin⁺ cells (leftpanels). Most of the Flk-1⁺ cells remained as PDGFRα⁺ in Etv2^−/− embryos, failing to become Flk-1 single-positive cells (right). (C) Double immunostaining of Etv2^+/− (i) and Etv^−/− (ii) E7.75 mouse embryos by anti-Flk-1 (red) and anti-PDGFRα (green) Abs (iii). In the Etv2^−/− embryos, Flk-1 staining in extra-embryonic mesoderm was missing, whereas Flk-1⁺ cells in the cardiac crescent (cc) and allantois (al) were still present. Scale bar indicates 100 μm. (D) Rescue of Etv2^−/− cells by reexpression of Etv2 transgene. (a) Diagrams of Etv2 transgene constructs. PB-based tTA and Tet-inducible promoter-driven transgene vectors were transfected into Etv2-null ESCs with transgene expression monitored by CD90.1 staining. (b) HPCs (CD41⁺ and CD45⁺) and ECs (VECAD⁺ and Flk-1⁺) were generated in Etv2-rescued Etv2^−/− cells (bottom), whereas none were generated in cells transfected with empty vector (top).