Supplementary Figure 5: EMT events in ETX embryos.

a. ETX embryos at day 5; white box indicates magnified field showing nuclei of T/Brachyury-positive cells changing from a plane perpendicular (asterisks) to parallel (arrowheads) to the basal membrane of the ES-derived compartment. Representative of 6 ETX embryos, 2 experiments. Bar=20μm. Bar in zoomed image=10μm. b. Still images from time-lapse movie of live ETX embryo built from CAG:GFP ES cells presented in Fig. 4d. White dashed lines outline XEN layer; purple dashed lines, embryonic/extra-embryonic boundary. White arrowhead indicates change in cell shape on the boundary of prospective posterior side. Representative of 3 separate time-lapse movies of 7 ETX embryos. c. Transverse sections from embryonic compartments of E6.75 embryo (top) (3 embryos) and day 5 ETX embryos (bottom) (3 ETX embryos). E-cadherin immunostaining reveals change in orientation of T/Brachyury-expressing mesenchymal cells (white arrowheads). 3 experiments. Bar=20μm. d. Immunostaining of E6.75 embryo (top) and day 5 ETX embryo (bottom) to reveal phosphorylated Histone 3 (H3S10-P) (magenta) – there is no increase in mitotic cells in the mesoderm, white boxes. ETX embryo presented in Fig. 4f re-stained for T/Brachyury in the same channel as GM130. Non-nuclear anti-T/Brachyury VE/XEN fluorescence is non-specific. Proportion of H3S10-P positive cells within and outside boxed region. LE, lateral epiblast; S, streak; R1, region 1; R2, region 2. n=3 E6.75 embryos; n=6 ETX embryos. Two-sided Student’s t-test. Means ± SD. Bar=20μm e. Day 5 ETX embryo and E6.75 embryo immunostained for N-cadherin (cyan) and T/Brachyury (magenta). Magnified images below show up-regulated N-cadherin in re-oriented T/Brachyury expressing cells that identify mesoderm formation (white arrowheads). Non-nuclear anti-T/Brachyury VE fluorescence is non-specific. Representative of 4 E6.75 embryos; 3 ETX embryos. Bar on the zoomed images=5μm f. XZ sectioned orthogonal views from the ES-derived embryonic compartment of ETX embryo also presented in Fig. 4h demonstrating laminin break-down on the T/Brachyury expressing side. Yellow arrows indicate break in laminin. g. Oblique section of an ETX embryo at day 5 showing break in laminin in T/Brachyury expressing posterior domain. Dashed lines outline the TS-derived extra-embryonic compartment. Representative of 3 ETX embryos, 2 experiments. h. Quantification of ETX embryos expressing T/Brachyury with and without DKK1 treatment (200ng/ml) for 24h presented as a bar chart. Contingency table used to perform statistical test. Two-sided Fisher’s exact test, total of 20 structures scored per group from n=2 separate experiments. The number of structures scored in each independent experiment is reported in Supplementary Table 3.