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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).
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