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. 2021 Jun 2;12:3277. doi: 10.1038/s41467-021-23653-4

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — a–c Immunodetection of NANOG and GATA6 from D3.0 to D4.5. d, e DBA labelling of visceral endoderm (ve) at d D4.0 and e at the surface of the embryoid at D5.5. f, g Clumps of endoderm cells expressing f Gata6 or g Sox17. At D5.5 h Sox17 was expressed around small cavities bordered i, j by a polarized epithelium positive for Acetylated Tubulin (Ac. Tub). k Immunodetection of SOX17 identifies the endodermal-like epithelium. l In situ hybridization for Sox17 at D6.0 after the merging of small cavities into a single one. m Mosaic of DBA(+) (ve cells) and DBA(−) cells (definitive endoderm: de) in the endoderm epithelium. n Patchy expression of Pyy (de marker). o, p Expression of Sox17 (red) and Pyy (blue) showing that the endoderm epithelium was a mosaic of de and ve. o Superficial view of the epithelium. p Optical cross-section of the epithelium at higher magnification. q–s In situ hybridization for q Cldn4 in the whole gut epithelium, r Apela in the ventral foregut (vfg) and hindgut (hg), s Rnf128 in the hg. t Folding of the primitive-like gut showing a presumptive fg (pfg) followed dorso-posteriorly by likely presumptive midgut (pmg), presumptive hindgut (phg), presumptive yolk stalk (pyst) and presumptive yolk sac (pys). u Phalloidin (red) and Hoechst (nuclei, blue) labelling of primitive gut tube. v Combined images of a double colour in situ hybridization of pgt at D7.5 showing expression of Pyy (red) in pfg, Nepn (blue) in pmg (phg and pyst were unlabelled). sc: spinal cord. Scale bars: 100 µm except for k, p, u, v: 50 µm. Embryoids (e–v) in lateral views, anterior to the left, dorsal to the top. Numbers of performed experiments and of biological samples analysed is provided ‘Statistics and reproducibility’ in the Methods section. Variability in genes expression is provided in Supplementary Figs. 9, 10.