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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. 1 — a Experimental procedure (detailed in ‘Methods’) to generate embryoids by merging a large naive (untreated) ESC aggregate with a smaller aggregate in which expression of WNT3 and NODAL morphogens were induced following incubation with BMP4 protein. b A naive ESC aggregate labelled with DiO (green) placed in contact at D3 with a BMP4 instructed ESC aggregate labelled with DiI (red) had merged within 1 h and formed a spherical embryoid after 12 h. (D3.5). c Schematic summarizing the principle of instructing an ESC aggregate with a morphogen signalling centre. d, e In situ hybridization at D4 for d Nodal and e Wnt3. f Expression at D4 of Bra^GFP in an embryoid made of Bra-GFP ESC. g, h Experimental design to visualize the induction of Bra expression (i) in Bra-GFP ESC (naive aggregate, yellow) in response to morphogens secreted by the signalling centre (pink) made of unlabelled E14TG2 ESC. j, k Double colour in situ hybridization for Otx2 (blue) and Bra (red). l, m Expression of Sox1^GFP identifying the neurectoderm (l) and of Bra^GFP identifying the mesoderm (m). n Expression of Chrd (nl: notochord-like) in a D6 embryoid, right: drawing for Chrd expression in mouse embryo at E7.7. o D7 embryoid showing a large cavity (star) lined by an epithelium (arrows). p Expression at D6.5 of Wnt3a in a posterior primitive streak-like structure (psl) and in mouse at E8.5 (cartoon). q Double colour in situ hybridization showing expression of Otx2 (blue) anteriorly, of Bra (red) posteriorly and in notochord-like structure extending on top of a ventral cavity (star) in an embryoid at D6.5. Cartoon showing expression of Bra and Otx2 in the mouse embryo at E8.5 (fb-mb: forebrain-midbrain, n: notochord, ps: primitive streak). a: anterior, p: posterior, d: dorsal, v: ventral. Staging of embryoids (D: day) indicated for each panel. Numbers of performed experiments and of biological samples analysed is provided in ‘Statistics and reproducibility’ in the Methods section. Variability in genes expression is provided in Supplementary Fig. 4. Scale bars: 100 µm.