Fig. 2. Wnt signaling is required for generation of Flk1⁺ mesoderm. (A) Differentiating ES cells in SCM were treated either with Dkk1 or Noggin/Fc as indicated, or left unmanipulated (no treatment). Embryoid bodies were harvested at day 3, 4 and 5, and analyzed for Flk1 expression as described in the Materials and methods. Shown are dot plots gated on live cells. Numbers represent the percentage of Flk1⁺ within the indicated region. (B) ES cells were differentiated in SCM supplemented with the indicated protein, and Flk1 expression determined on day 4 as described above. (C) Dkk1 was added to differentiating cultures at day 0, 1.0, 1.5, 2.0, 2.5 or 3.0. The frequency of Flk1-expressing cells at day 4 was determined as described in A. The data are represented as the percentage of inhibition, measured by dividing the frequency of Flk1⁺ cells in each Dkk1 treated culture by the frequency of Flk1⁺ cells in parallel untreated cultures.