(a) Z-stack of epiblast model composed of the SMAD1 reporter line 16 h after stimulation with 10 ng/ml BMP4 (from the same sample as in Fig. 6 with 16 individual 3D colonies). (b) IF staining 2 days after 1 ng/ml BMP4, showing examples with partial BRA expression and a heterogeneous pSMAD1 expression, with no apparent correlation between pSMAD1 and BRA expression. Plot shows no strong correlation between pSMAD1 and BRA IF intensity levels in partial BRA+ examples (n = 6 imaged 3D colonies showing partial BRA expression). (c) FACS sorting dot plot of dissociated RUES2-GLR epithelium grown on filters under pluripotency conditions (left) and after 24 h of 10 ng/ml BMP4 (centre). Right: gene expression analysis from Fig. 7b, c of sorted cells compared (in log2 fold change) to untreated cells. The measurement was done by pooling n = 10 individual samples (separate filters) and compared to n = 5 untreated samples, thus the variance was not measured. (d) Monitoring pluripotency maintenance of epiblast models made from the RUES2-GLR DKK1 KO line. Pie chart shows end-point patterning statistics. Snapshots show an example of live-cell imaging of the majority population under pluripotency conditions (n = 12 3D colonies for the majority population). Scale bar, 20 μm. (e) Patterning statistics and examples of WT RUES2 epiblast models stimulated with 2 μM IWP2 or 2 µM IWR1-endo along with 1 ng/ml BMP4 for 2 days. For each case, a representative example of a colony from the majority population is shown (that is, no differentiation). In both cases, n = 39 3D colonies were imaged. Scale bar, 20 μm. (f) Patterning of filter cultures, comparing RUES2 WT (3 independent experiments) and RUES2 DKK1 KO (2 independent experiments), stimulated for 24 h with 10 ng/ml BMP4 from the bottom compartment. Scale bar, 200 μm. (g) Filter colony stimulated with 1 ng/ml from the bottom compartment for 24 h (2 independent experiments). (h) Gene expression analysis from bulk cells at 4 h and 24 h, of filters stimulated with 10 ng/ml from the bottom compartment (n = 3 independent experiments, box centred at mean, error is s.e.m., each dot represents a mean of three technical replicas). (i) Differentiation of RUES2 NOGGIN KO model epiblasts with 1 ng/ml BMP4 for 2 days. Left: end-point statistics; right: IF stain of an example 3D colony from the majority population (total n = 19 imaged 3D colonies). Scale bar, 20 μm. (j) Checking for NODAL influence on symmetry breaking. Shown are stimulation with 1 ng/ml BMP4 for 2 days of RUES2 LEFTY1 & CERBERUS 1 (CER1) KO epiblast models (top) and the WT with the NODAL inhibitor SB (bottom). Pie charts show end-point statistics (n represents total imaged 3D colonies) and AP dipole quantification (the measured and the random population are statistically different, P = 0.008 calculated with the two-sided Mann-Whitney U test; n = 6 and n = 20 segmented BRA+/SOX2+ colonies for the LEFTY1/CER1 KO and WT with SB cases, respectively); right: IF stains of symmetry-broken 3D colonies. All scale bars, 20 μm.