Immunofluorescence was carried out as described previously 38 (link). Briefly, embryos were fixed in 4% paraformaldehyde, washed in PBS-T (PBS containing 0.1% Tween) and permeabilized with 0.5% Triton X-100 for 20 minutes. Embryos were incubated with primary antibodies in 3% BSA (Sigma) in PBS-T overnight at 4°C. The embryos were then washed twice in PBS-T and incubated with secondary antibodies (Invitrogen, AlexFluor; 1:400 in 3% BSA) for 2 hours before final washes in PBS-T and M2 and imaging in drops of M2 on glass-bottomed dishes. Primary antibodies used: goat anti-Sox17 (R&D Systems), mouse anti-Cdx2 (Biogenex), rabbit anti-Nanog (2B Scientific), goat anti-Gata4 (Santa Cruz), goat anti-Gata6 (R&D Systems), mouse anti-Oct4 (Santa Cruz), rabbit anti-pSmad1 (Cell Signaling) and rabbit anti-cleaved Caspase 3 (R&D Systems). DAPI was used as the nuclear counterstain in all experiments and all primary antibodies were used at a dilution of 1:200. Multi-channel images were acquired for multiple sections using a Leica SP5. Image processing and cell counting were performed with Fiji software (http://fiji.sc).