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. 2017 Nov 9;7:15136. doi: 10.1038/s41598-017-15427-0

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© The Author(s) 2017

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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Development of E4.5 ICMs. (A) ICM immediately after IS, and (B) 6 hrs after IS, (C) ICM treated with cytochalasin D (CD) 6 hrs after IS, (D–F) ICMs 24 hrs after IS: (D) surrounded by single layer of PE, (E) containing cavity, (F) surrounded by two layers of PE separated by cavity, (G) E4.5 blastocyst labelled with DiI dye, (H) E4.5 ICMs with DiI-marked PE layer immediately after IS and (I) 24 hrs after IS. Blue: Oct4 or Cdx2 or F-actin, green: Gata4, red: DiI, white: nuclei_; right panel shows merged pictures; yellow (*) indicates blastocyst cavity, (J) Time-lapse imaging of E4.5 ICM immediately after isolation, after 12 hrs and after 24 hrs of in vitro culture (single optical sections), (K) The scheme of the ultimate PE origin: (a) Pdgfrα ^H2B-GFP-expressing cells, which maintained their outside position, (b) Pdgfrα ^H2B-GFP-expressing cells, which translocated from inside to outside, (c) Pdgfrα^H2B-GFP-negative cells, which up-regulated Pdgfrα ^H2B-GFP expression during culture, (d) placed outside Pdgfrα^H2B-GFP-negative cells, which up-regulated Pdgfrα on the surface of ICM, (L) The scheme of the fate of all Pdgfrα ^H2B-GFP-expressing cells: (a’) cells which were localised on the surface of ICM, (b’) cells which changed their localisation from inside to outside, (c’) cells which down-regulated Pdgfrα ^H2B-GFP, (d’) cells which underwent apoptosis. (*) statistically significant difference compared to corresponding cell groups of E3.5 ICMs (p < 0.05). Scale: 20 μm.