FGF and TGF-β/Nodal/activin signaling pathways are involved in BMP-4-initiated mesoderm induction. hES cells were differentiated in SFM in the presence of BMP-4 alone (B25), or BMP-4 and Dkk1 (B + Dkk1), or BMP-4 and SB431542 (B + SB), or BMP-4 and SU5402 (B + SU) for 2 days. Cultures were collected for (A) expression analysis of brachyury, WNT3, and MIXL1 by RT-PCR, or (B) brachyury expression at day 1 of differentiation by immunocytochemistry. (C-D) The inhibitor SB431542 (SB) or SU5402 (SU) was added at different time points of BMP-4 treatment (C), or cells were treated with BMP-4 in the presence of the inhibitor for different periods of time, washed, and then exposed to BMP-4 without inhibitors (D). Then cells were collected at 24 hours of differentiation and analyzed for brachyury expression by Q-PCR. hES cells treated by BMP-4 alone for 24 hours (B25) were used as a positive control. Numbers on the x-axis indicate time points when the inhibitors were added (C) or removed (D); numbers on the y-axis indicate relative gene expression level normalized to that of GAPDH. Error bars indicate SD. BRACH indicates brachyury. Scale bar represents 50 μm.