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. 2018 Jul 19;9:2816. doi: 10.1038/s41467-018-05189-2

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© The Author(s) 2018

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. 4 — Wnt5a-Ror2 signaling regulates SM morphogenesis by synchronizing the radial SM-cell polarity. a SM morphology in Wnt5a^−/− trachea and control at E12.5. Whole tracheas were stained for SMA (red), TOPRO-3 (blue). Coronal (upper panel) and transversal (lower panel) sections. b Array of SM cells. Apical view (upper panel) and lateral view (lower panel). See also Supplementary Movie 3. c, d Polarity of Ror2-positive SM cells, determined by the Golgi-apparatus position relative to the nucleus. Sections were stained for GM130 (white), Ror2 (green), and DAPI (blue). White-dotted lines show the contours of cell membranes. Quantifications in three embryos are shown below. c Developmental time-course analysis in wild type (E10.5; n = 115, E11.5, n = 214, E12.5; n = 236). d In Wnt5a^−/− trachea at E11.5 (control; n = 99, Wnt5a^−/−; n = 73). e Polarity of Ror2-negative or positive (arrowheads) SM cells in Foxg1^Cre; Ror2^flox/flox trachea at E11.5: the lower panel shows magnified images of the upper panel. f, g Mosaic targeting of SM cells using Tagln1^Cre line. GFP (green), SMA (red), DAPI (blue) in control (f) and Ror2^flox/flox (g). Arrowheads indicate Ror2-negative SM cells. h Time-lapse sequence of epithelial sphere (red) and SM cells (magenta, cyan, gray, yellow, blue, and green) in Matrigel. Cells were labeled by Image J. ^*Cells keeping at a distance from spheres. See also Supplementary Movie 4. i Approaching distance of SM cells to sphere in h. Numbers represent means (control; n = 23, Wnt5a^−/−; n = 29). j Distribution of angles reflecting directionality to sphere in h. Scale bar = 100 μm (e, left), 10 μm (a–c; upper, d; upper, e; right upper, g; upper, h), 3 μm (c; lower, d; lower, e; lower, g; lower panels)