Fig. 2. Nodal-expressing ES cells differentiate into definitive endoderm and mesoderm at the expense of neuroectoderm. (A) Western blot showing the phosphorylation of Smad2, an effector of Nodal signaling, and the phosphorylation of Smad1/5/8, in each condition. (B) The expression of downstream transcriptional targets of Nodal at stage 2 by RT-PCR analysis. (C) Quantitative real time RT-PCR analysis of transcriptional targets at stage 2. (D,E) RT-PCR analysis showing the expression of definitive endoderm and mesoderm gene markers (D), and anterior visceral endoderm and neuroectoderm markers (E) at stage 2. (F,G) Immunofluorescent microscopy image of pan-neuronal marker (β-tubulinIII) in Tet-on/Nodal-off ES cells (F) or Tet-off/Nodal-on ES cells (G). The default pathway to neuroectoderm from ES cells is repressed in the Nodal-expressing ES cells. The original magnification was 400x. (H) Flow cytometric analysis of Nodal-expressing ES cells for the definitive endoderm progenitor marker (CXCR4) and mesoderm progenitor markers (VEGFR2 and PDGFR). The CXCR4⁺GFP⁺, VEGF2⁺GFP⁺, and PDGFR⁺GFP⁺ cell population increased more in stage 2 Nodal-expressing ES cells than in Nodal-off ES cells. (I) The populations of progenitor cells in each culture condition. Notably, the largest populations of cells were definitive endoderm and mesoderm progenitor cells in Nodal-on ES cells at stage 2. ^*P<0.01 compared with wild-type ES cells, ^**P<0.05 compared with 100 ng/ml activin A-treated ES cells, ^***P<0.05 compared with Nodal on ES cells.