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. 2017 Nov 7;8:1339. doi: 10.1038/s41467-017-01105-2

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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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Fig. 2 — FGFR/Mek signaling is required for the differentiation of Fgf5⁺ epiblast-like cells. a Quantification of fgf5 and six3 expression via RT-qPCR from days 0 to 5 of embryonic stem cell (ESC) culture. D, day. b Montage of images taken from Supplementary Movie 3, showing upper panel; merged image of Six3::Venus and Fgf5::Turq (monomeric Turquoise2-HA-NLS), middle panel; Six3::Venus, lower panel; pseudo color of Fgf5::Turq. c Time-lapse imaging of Six3::Venus⁺ cells. Each color dot indicates a cell that is a component of each Six3⁺ small cluster. Broken lines (cyan, magenta and yellow) indicate each Six3⁺ small cluster. White solid line indicates a large cluster. d Immunohistochemistry against Venus and HA epitope (monomeric Turquoise2-HA-NLS) was performed on cryosections of day-3.3 aggregates, showing Six3::Venus and Fgf5::Turq signals. Six3::Venus signals were enhanced by the auto contrast function of ImageJ to see a dim expression. e Diagram of pharmacological inhibition of FGFR and Mek (FGFRi and Meki) by PD173074 and PD0325901, respectively. f, h Merged images of transillumination (Trans) and Fgf5::Turq (f) or Six3::Venus (h). g Quantification of Fgf5::Turq-HA-NLS positive cells via ImageJ analysis (See Methods). Total cells were counted by DAPI signals. i Quantification of Six3::Venus⁺ cells via FACS. Error bars indicate s.e.m of each experiment (a, g, i). Significance was determined using Dunnett’s test (g, i). *P < 0.05; **P < 0.01; ***P < 0.001. Cont, control. Scale bars, 100 µm (b, d, f, h). These images were one of n = 3 experiments (b, c, d, f, h)