Fig. 5. Interaction of Wnt and Notch signaling in modulating primitive erythropoiesis 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 hVEGF (10 ng/ml) and either Wnt3a (100 ng/ml), DKK1 (300 ng/ml), Dox (2 µg/ml), -secretase inhibitor (gSI; 2.5 µM) or DMSO (2.5 µl/ml), as specified. Hematopoietic potential of the aggregates was assayed as described in the Materials and methods. (A) Synergistic effects of Numb and Wnt3a on primitive erythroid development from Flk1⁺ cells. (B) Synergistic effects of -secretase inhibitor and Wnt3a on primitive erythroid development from Flk1⁺ cells. (C) Effect of Wnt3a on Notch1-IC-induced suppression of primitive erythroid development. (D) Effect of Numb expression on DKK1-induced block in primitive erythropoiesis. DKK1 was added to the reaggregation media after 6 hours of culture to allow time for Numb protein to accumulate. (A-D) Error bars represent the standard deviation of the mean of the number of colonies from n independent experiments (A, n=6; B, n=4; C, n=5; D, n=3) (^**P-value<0.01; ^***P-value<0.001). (E) The TOP/FOPflash reporter assay demonstrates interaction between Wnt and Notch pathways. Error bars indicate standard deviation of TOP/FOP values of triplicate electroporations. Numbers above each bar represent actual TOP/FOP values.