Bra is required for an EMT event during mesendodermal differentiation. (A) Phase-contrast images from live imaging of Bra::GFP (Bra+/GFP) and Bra−/− mESCs in Act and Chi. Bra::GFP stills were shown in Figure 4A. In the absence of Bra, cells do not undergo a full EMT. (B) E14-Tg2A and Bra−/− mESCs after 48 h in Act and Chi, immunostained for E-cadherin, β-catenin and Bra and imaged by confocal microscopy. Scale bars indicate 50 μm. Hoechst stain marks the nucli. In the absence of Bra, E-cadherin and β-catenin are not effectively cleared from the membrane. (C,C') Instant velocities of Bra::GFP cells and Bra null cells in Act, Chi or Act/Chi. The thick black line in the individual graphs indicates the average velocity for each condition, and is displayed in greater detail in (C'). (D) Histograms of instant cell velocities as measured from frame-to-frame displacements for both Bra::GFP and Bra−/− mESCs. All the Bra::GFP live imaging data was displayed in Figure 4. AC, activin A + chiron; Act, activin A; Bra, brachyury; Chi, chiron CHIR99021; Eff., effective; EMT, epithelial-to-mesenchymal transition; FACS, fluorescence-activated cell sorting; GFP, green fluorescent protein; mESC, mouse embryonic stem cell.