Induction of Bra expression by activin (Act) and CHIR99021 (Chi) during the differentiation of mESCs. (A) Bra::GFP mESCs were treated with Act, Chi or Act/Chi and dimethyl sulfoxide (DMSO) (i), Act and Chi with the Porcupine inhibitor IWP3, which inhibits the secretion of Wnt proteins (ii), with the tankyrase inhibitor XAV939, which reduces active β-catenin (iii), the nodal/Act receptor inhibitor SB431542 (SB43) (iv) or the BMP inhibitor dorsomorphin (v). A control for long-term pluripotency growth, leukaemia inhibitory factor (LIF) and BMP (LB), is included (vi). Notice that the robust expression of Bra induced by Act and Chi, is suppressed by inhibition of Wnt/β-catenin or nodal/Act signalling but not by BMP inhibition. Measurements were made with GFP-positive cells daily by FACS (±standard deviation from at least three replicates). Single and double asterisks denote P < 0.05 and P < 0.01, respectively, versus DMSO. (B) E14-Tg2A mESCs were grown in LB, Act, Chi or Act/Chi for the indicated durations prior to RNA extraction and quantitative real-time reverse-transcription-polymerase chain reaction (RT-qPCR) analysis for the indicated genes. The average expression level of the RT-qPCR replicates relative to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) are shown for a representative experimental replicate. Error bars indicate absolute error of the normalized mean. Notice the transient expression of Bra in Chi and dual Act and Chi compared with the delayed expression in Act. (C) Quantification of Bra::GFP v Bra expression by immunostaining indicates a high correlation (Pearson coefficient of 0.773). Scale bar denotes 50 μm. A + C or AC, activin A + chiron; Act, activin A; AFU, arbitrary fluorescence units; Bra, brachyury; Chi, chiron CHIR99021; DM, dorsomorphin; DMSO, dimethyl sulfoxide; FACS, fluorescence-activated cell sorting; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFP, green fluorescent protein; LB, leukaemia inhibitory factor and bone morphogenetic factor; RT-qPCR, quantitative real-time reverse-transcription-polymerase chain reaction.