Fig. 1. Activation of canonical Wnt signaling during early ES cell differentiation. (A) Transient expression of Wnt8a and Wnt3 during ES cell differentiation. Semi-quantitative RT-PCR was carried out as described in the methods for Wnt8a, Wnt3 and Gapdh. Analysis was performed on ES cells (ESC) and on ES cells differentiated in SCM for the indicated number of days. (B) ES cells bearing a stably integrated SUPER8xTOPFlash luciferase reporter were differentiated in SCM either in the presence or absence of Dkk1, as indicated. Luciferase activity in undifferentiated ES cells (ESC) or in ES cells differentiating for the indicated number of days in SCM or SCM with addition of Dkk1 (SCM + Dkk1) was measured. SUPER8xTOPFlash luciferase activity is presented as a percentage of the activity measured in the same conditions treated with LiCl (20 mM) for 18 hours prior to harvesting. (C) SUPER8xTOPFlash transfected cells in B were differentiated in serum replacement medium alone (SRM) or with the addition of Dkk1 (SRM + Dkk1) for 2 and 4 days, and luciferase activity measured as in B.