Fig. 5. Canonical Wnt signaling is required for generation of ES cell-derived mesoderm. (A) ES cells were differentiated in SCM alone (no treatment) or treated with either Dkk1 or Noggin/Fc for 4 days as indicated. After 4 days, cells were washed and transferred to SCM without inhibitor for an additional 2 days. On day 6, cells were assayed for hematopoietic precursor potential using methylcellulose colony-forming assays as described in the methods. (B) ES cells described in A were treated as indicated, harvested at day 6 and assessed for expression of the indicated genes by RT-PCR. (C) ES cells were differentiated as in A, except that at day 4, cells were washed and transferred to gelatinized plates in SRM without inhibitor. At days 6, 7 and 8, cells were harvested and analyzed by RT-PCR for the indicated genes. (D) Cells were differentiated as in C and analyzed by fluorescence microscopy for expression of E-cadherin (green) or fibronectin (red) as described in the Materials and methods. Images of representative colonies, acquired using 10x objective magnification, are shown for unmanipulated (no treatment) and Dkk1-treated conditions.