Fig. 3. Wnt and Notch signaling affect blast colony formation from the hemangioblast. (A) Role of Wnt signaling in blast colony development. Flk1⁺ cells (4x10⁴) isolated from the day 2.75 EBs generated from either sβ-cat, Numb or Notch1-IC ES cells were cultured in M10 serum-free hemangioblast media in the presence or absence of doxycyclin (Dox; 2 µg/ml) and DKK1(DKK; 300 ng/ml) as indicated. Dox was added at 0 hours of culture, whereas DKK1 was added at 6 hours of culture. Colonies were washed with media at 24 hours to deplete DKK1 and Dox, and replated back into fresh M10. Blast and core colonies were scored after 4 days of culture. (B) Effect of DKK1 treatment on blast and core colony development. (C) Hematopoietic potential of DKK1-treated colonies. Sixteen-hundred colonies of each group were picked at day 3 of culture, pooled, dissociated and replated into hematopoietic methylcellulose cultures. Ep, primitive erythroid colonies; Def, combined macrophage, bipotential macrophage/erythroid, definitive erythroid and multipotential myeloid/erythroid colonies. (D) Role of Numb in blast colony development. gSI, -secretase inhibitor (2.5 µM). (E) Role of Notch signaling in blast colony development. (A-E) Error bars represent standard deviations of the mean of number of colonies from n independent experiments (A, n=3; B, n=3; C, n=3; D, n=4; E, n=4; ^**P<0.01; ^***P<0.001)