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. 2020 Sep 8;11:4477. doi: 10.1038/s41467-020-18185-2

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© The Author(s) 2020

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, b Two-photon imaging of myosin II-RLC GFP in a representative WT (a) or tre1^−/− (b) cluster. Myosin II-RLC is pseudocolored with the indicated color bar on top (intensity ranges: 2319–4005 (a) and 2203–3803 (b)). Different Z slices with indicated depth relative to the first slice are shown. Cyan arrows indicate regions of myosin II-RLC accumulation. Representative images are from n = 15 embryos in (a) and n = 13 embryos in (b). c, d Quantification of polarized myosin II RLC intensity (c) and myosin II RLC orientation (d) in WT (n = 123 from 15 embryos) or tre1^−/− (n = 127 from 13 embryos) PGCs. Data are presented as mean values ± SD. Statistical comparisons are from a Mann–Whitney test in (d) with p < 0.0001. ***p < 0.001. Two-photon timelapse imaging of a representative WT (e) or tre1^−/− (f) PGC cluster expressing lifeact-tdTomato and myosin II RLC-GFP for 18 min prior to endoderm EMT. Myosin II-RLC is pseudocolored with the indicated color bar above (a) (intensity ranges: 1072–2171 (e), 3198–7726 (f)). A single Z slice is shown. Representative images are from n = 6 embryos in (e and f). g Schematic of how intensity vs. distance from cluster centroid is calculated. h Quantification of the average myosin II RLC intensity vs. distance from the cluster centroid over time in WT (n = 6 embryos) and tre1^−/− (n = 6) PGC clusters. i Two photon timelapse imaging of a representative WT PGC cluster expressing lifeact-tdTomato and myosin II RLC-3xGFP over time. Myosin II-RLC is pseudocolored with the indicated color bar on top of (a) (intensity range: 2560–5624). A single Z slice is shown. Cyan arrows indicate regions of myosin II RLC accumulation from the same pair of cells. Representative images are from n = 5 embryos. j Quantification of myosin II RLC-3xGFP intensity at the posterior over time. Grey curves show single cells while the red curve indicates the mean. n = 13 PGCs from 5 embryos. All scale bars, 10 μm. All times are in minutes. Source data in (c, d, h and j) are provided as a Source Data file.