Continuous WNT and NODAL signaling synergize to achieve maximal mesodermal differentiation.

(A, B, D) Images of samples immunostained for fate markers after 44 h of BMP treatment. The time between BMP4 and chemical inhibitor (LDN/IWP2/SB) addition is indicated above the image. Quantification represents average nuclear intensities of indicated markers normalized to DAPI as a function of radial position. Error bars represent standard error. N ≥ 10. (C) (Left image) Image of a colony immunostained for BRA and DAPI after 47 h time-lapse imaging of BMP-treated GFP-β-catenin hESCs. (Right image) Snapshot from time-lapse imaging for the same colony at 47 h. (Left plot) Quantification represents average intensity levels for BRA and nonmembrane β-catenin at 47 h, normalized by DAPI, plotted as a function of radial position. Error bars represent standard error of the mean. N = 9. (Right plot) Average BRA intensity levels as a function of β-catenin levels, color-coded by distance from the colony edge (μm). Scale bar: 100 μm. Underlying data can be found in S9 Data. BMP4, Bone Morphogenic Protein; BRA, BRACHYURY; GFP, Green Fluorescent Protein; hESC, human embryonic stem cell.