Live microscopy of Bra::GFP and a β-catenin transcriptional reporter (TLC2) following mesendodermal differentiation. (A) Stills from live imaging of Bra::GFP mESCs in Act/Chi (Additional file 5: Movie M1); phase contrast (left) and fluorescence (right). (B,C) Cells were manually tracked (B) and their velocities (B', C middle), GFP expression (C, top) and distance travelled (C, bottom) were measured. Average velocities for all tracked cells in Act (blue), Chi (green) and Act/Chi (red) (B', bottom right) show that cells in Act/Chi have on average the highest peak velocities, which are reached earlier. (C) Each cell represented by a colour showing that all cells move, but only cells that express Bra move with a high velocity. There seems to be a relationship between the levels of Bra expression, velocity and distance travelled by individual cells. (D) Distribution of individual cell velocities under different conditions. Cells in Act/Chi have a higher proportion of fast movers. (E) Cell velocity increases with time. (F) Mean-squared displacement (MSD) curves representing the range of individual movements of all cells tracked over the whole experiment. (G) Effective diffusion coefficients at different time intervals. Coefficients were estimated by fitting straight lines to the MSD curves obtained when considering cells at different 10 h intervals. (H) Live imaging of the TLC2 Wnt/β-catenin reporter in Act/Chi (Additional file 6: Movie M2). Differentiation results in reporter up-regulation before cells initiate the EMT and down-regulation as cells leave the colonies. (I) FACS analysis of Bra::GFP and TLC2 reporters revealing activation of β-catenin transcriptional activity relative to activation of Bra within a population of cells. Average of three replicate experiments ± standard deviation. Scale bars represent 50 μm. AC, activin A + chiron; Act, activin A; Bra, brachyury; Chi, CHIR99021; FACS, fluorescence-activated cell sorting; Eff., effective; GFP, green fluorescent protein; MSD, mean-squared displacement.