Fig. 4. Wnt and Notch signaling regulate primitive erythroid development from Flk1⁺ cells. Flk1⁺ cells isolated from day 2.75 EBs generated from the indicated ES cell lines were cultured as aggregates in serum-free media containing VEGF (10 ng/ml) and either DKK1 (300 ng/ml), Dox (2 µg/ml), -secretase inhibitor (2.5 µM) or DMSO (2.5 µl/ml), as specified. (A) Expression of Axin2 and Wnt3 in aggregates at different times. Average expression normalized to Actb is shown. (B) Transient requirement for Wnt signaling in primitive erythroid specification of Flk1⁺ cells. Ep, primitive erythroid colonies; Def, combined macrophage, bipotential macrophage/erythroid, definitive erythroid and multipotential myeloid/erythroid colonies. Total colony numbers are shown. (C) Effect of Numb expression or addition of gSI on primitive erythroid development from Flk1⁺ cells. -Dox: doxycyclin-untreated group; +Dox: doxycyclin was added for the first 24 hours, and then washed away. (D) Effect of Notch expression on primitive erythroid development from Flk1⁺ cells. Dox was added as indicated above in C. (B-D) Error bars represent standard deviation of the mean number of colonies from n independent experiments (B, n=3; C, for the first three groups n=6, and last two groups n=3; D, n=6;^**P-value<0.01; ^***P-value<0.001).