Fig. 5

Low expression of FGF family genes accounted for the reduced ERK activity in the neuroectoderm. a Activity of ERK1/2 in the E7.5 neuroectoderm (NE) and primitive streak (PS) was detected by western blot analysis. b Secreted signals were responsible for regulation of ERK activity. Left panel shows manipulation of cells. Right panel shows western blot analysis results. Lanes 1–3 show EpiSCs, primitive streak (PS-like cells) induced by Activin A (AA) and neuroectoderm (NE-like cells) induced by SB431542 (SB). After 24 hours of differentiation, cells were washed with N2B27 twice, and refilled with fresh N2B27 medium for 1 hour. Supernatants were collected to stimulate NE-like cells. Lanes 4–6 show SB-induced NE-like cells stimulated for 1 hour with the supernatant from EpiSCs (lane 4), PS-like cells (lane 5), and NE-like cells (lane 6). c Heatmap showing relative mRNA levels of FGF family members detected by microarray in the NE and PS. Heatmap was generated by Cluster 3.0. d Expression of main Fgf family members in ESCs, E6.5 epiblasts, NE, and PS examined by real-time PCR. Expression patterns classified into three categories characterized by high expression in ESCs, epiblasts, and PS, respectively. e Phospho-ERK1/2 of EpiSCs detected by western blot analysis after treatment with DMSO, 5 ng/ml FGF2, 1 μM PD0325901, or 2 μM SU-5402 in N2B27 for 6 hours. f FACS showed that FGFR inhibitor SU-5402 treatment on Sox1-GFP EpiSCs increased the proportion of GFP-positive cells. **p < 0.01. AA Activin A, DMSO dimethyl sulfoxide, E embryonic day, EpiSC epiblast stem cell, ESC embryonic stem cell, ERK extracellular signal-regulated protein kinase, SOX2 sex determining region Y-box 2, T brachyury, FGF fibroblast growth factor, GFP green fluorescent protein