An EMT is associated with mesendodermal differentiation of E14-Tg2A. (A) Phase-contrast images from live-cell imaging of wild-type (WT) mESCs following Act/Chi. At the start of differentiation, cells begin to loosen up within the colonies, adopt a more motile morphology and become migratory over time. (B,C) Differentiating mESCs were treated with Act/Chi and stained for E-cadherin (B) or brachyury (C and Additional file 2: Figure S2D) together with fibronectin and phalloidin. Panels (a) and (b) in (B) show two separate colonies and their corresponding magnified regions (i and ii) in different phases of an EMT. The decrease in E-cadherin correlates with the appearance of filopodia and the laying down of fibronectin basally. This is clear at the edge of the colony, where these changes are associated with Bra expression (C and Additional file 2: Figure S2C); 320x13.75 μm sections through the colony, indicated by yellow hashed lines illustrate this (C, top). A region of the colony (C' white hashing) shows the EMT phases with corresponding section through the colony (C"m single yellow horizontal line). (D) 3D rendering of E14-Tg2A mESCs in Act/Chi, stained for fibronectin (green), phalloidin (red) and E-cadherin (white). Cells emerging from the colony secrete high levels of fibronectin and have altered F-actin architecture. E-cadherin is obscured due to the rendering process used to generate the 3D image. Individual channels are shown in (ii) illustrating membrane location of E-cadherin within the central colony; EMT initiation results in E-cadherin loss from the membrane. Fibronectin is observed on the basal surface of the colony. Scale bars represent 50 μm in (A) and 100 μm in (B,C,D). Hoechst marks the nuclei in all images. AC, activin A + chiron; Act, activin A; Bra, brachyury; Chi, chiron CHIR99021; E-cad, E-cadherin; EMT, epithelial-to-mesenchymal transition; mESC, mouse embryonic stem cell; WT, wild type.