Regulation of WNT signaling activity by BMP, WNT, and NODAL.

(A, E) Snapshots of GFP-β-catenin hESCs from time-lapse imaging movies at 46 h post BMP treatment. Time between BMP4 and LDN or IWP2 addition is indicated above each image. Boxes show the region represented at higher magnification below each colony image. (B, F) Average nonmembrane β-catenin levels as a function of radial position. Control represents samples with no inhibitor addition. The timing of LDN or IWP2 addition after BMP4 treatment for each curve is shown in the legend, while the time being analyzed in shown above the plot. Error bars represent standard error. N = 3 colonies for the samples LDN at 0 h (LDN@0h), LDN at 11 h (LDN@11h); for all other samples N ≥ 5. (C, G) Temporal evolution of the position and intensity of peak signaling (defined by maximal nonmembrane β-catenin intensity). (D, I) Temporal evolution of the front of the domain of active signaling. In (C, D, G, H, and I) at time points earlier than the first one in each curve, signaling was below threshold signaling at all positions. (H) Temporal evolution of average nonmembrane β-catenin levels in a narrow ring inside the region of mesodermal differentiation (distance from edge: 134–150 μm) and at the center (distance from edge: 277–350 μm) of the colony. (J) Images of GFP-β-catenin hESCs at 46 h post treatment with either BMP4 (control) or BMP4 and SB. Average nonmembrane β-catenin levels as a function of radial position at 46 h. Error bars represent standard error. N ≥ 6. Underlying data can be found in S5 Data. BMP, Bone Morphogenic Protein; GFP, Green Fluorescent Protein; hESC, human embryonic stem cell; LDN, LDN193189.