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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. 5 — a Brightfield imaging of a representative D6.5 embryoid. np: neural plate. b Embryoid made of Sox1^GFP ESC expressing GFP in the neural plate. c–k In situ hybridization revealing expression of c Pax6 and d Scube2 in likely hindbrain (hb) and spinal cord (sc) and absence of an anterior forebrain (fb) expression domain, Rh3, Rh4 rhombomere 3 and 4. Expression of e En2 in likely midbrain (mb) and rhombomere 1 (rh1), f of fgf8 in an anterior stripe (likely the midbrain–hindbrain boundary, mhb) and tail bud (tb), tel: telencephalon. g Egr2 in 2 stripes in hindbrain, likely in rhombomere (rh) 3 and 5. h Hoxd4 in posterior hindbrain (post hb) and spinal cord, i Cdx2 in posterior spinal cord (post sc), j Hoxd9 in tail bud (tb), k Nkx1-2 in caudal epiblast (cle). Embryoids on side view, anterior (A) to the left, dorsal (D) to the top. Cartoons on the right show expression of the corresponding gene in the mouse embryo. Scale bars: 100 µm. (V): ventral, (P): posterior. Numbers of performed experiments and of biological samples analysed is provided in ‘Statistics and reproducibility’ in the Methods section. Variability in genes expression and measurement of the anterior-posterior extension for the expression of Hoxd4, Cdx2 and Hoxd9, is provided in Supplementary Fig. 13.