Fig. 4. Early inhibition of canonical Wnt signaling enhances long-term neuronal differentiation. (A) ES cells were differentiated in SCM alone or with addition of 1x Dkk1. At day 4, cells were washed and transferred to SCM without inhibitor. At days 6-8, RNA was isolated and examined for expression of indicated genes by RT-PCR. (B-D) Dkk1-treated cells described in A were transferred at day 9 to fibronectin-coated slides in SRM and stained for neuronal ßIII-tubulin (green), nestin (red) or Pax6 (D, red) expression at day 13. Nuclei are stained with Hoechst 33342 (blue) and images were acquired using 10x (B,D) and 20x (C) objective magnification. Untreated cells exhibited negligible staining for neuronal markers.