Fig. 1: Formation and characterization of BELAs and VE cysts.

a Schematic of mouse embryonic development from E3.0 to E5.5 (top) and GATA4-inducible embryonic stem cell (ESC) system to model interactions between epiblast (Epi) and extraembryonic endoderm (bottom). b Stills from a movie of ESC-derived Epi and primitive endoderm (PrE) cells seeded on a low adhesion substrate in N2B27 medium. See also Supplementary Movie 1. One representative out of n = 3 independent experiments shown. c, d Orthogonal views (c) and 3D volume rendering (d) of a bilayered aggregate imaged with light sheet microscopy. POU5F1 (green) marks Epi identity and GATA6 (magenta) marks PrE/VE identity. See also Supplementary Movie 2. One representative out of n = 5 structures shown. e–g Immunostainings of bilayered aggregates for the PrE/VE markers GATA6 ((e), (f)) or SOX17 (magenta, g), the apical markers PODXL (orange) and pERM (blue) (e), the basement membrane and adhesion markers LAM (orange) and ITGB1 (blue) (f), and the epithelial markers CDH1 (orange) and ZO-1 (blue) (g). One representative out of at least n = 7 structures shown. Arrows in (g, inset) mark punctate ZO-1 staining characteristic for tight junctions. h Schematic of experimental protocol to differentiate pure populations of PrE cells. i VE cysts formed in N2B27 supplemented with FGF4 on a low adhesive substrate. One out of n = 3 independent experiments shown. j Diameters of detached BELAs and VE cysts grown for 3 days on a low adhesive substrate. n = 72 (BELAs) and n = 36 (VE cysts); bars indicate mean ± SD. k–m Immunostainings of VE cysts for the same markers as in e–g. One representative out of at least n = 7 structures shown. Scale bars: 50 µm in (b–g, i (inset) and k–m), 10 µm in g (inset), 200 μm in i. Source data for j are provided in the Source Data file.