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. 2018 May 15;8:7566. doi: 10.1038/s41598-018-25957-w

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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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Differential growth promotes cleft initiation. (A) The determination of morphological patterns of epithelial buds by the orientation of differential growth. (B) Spatial distribution of mitotic cells in eSMGs. n = 7 buds. (C) Maximum intensity projection images (Z-axis) of mitotic cells in eSMGs treated with 100 μM nifedipine or 10 μM U0126. pHis: phospho Histone H3. (D) Mitotic cell numbers (pHis positive cells) in developing eSMGs. n = 7 (E) Representative images of the cells under horizontal (left) and vertical (right) mitosis. (F) Distribution of mitosis angle in developing eSMGs. n = 82 cells (G) Distribution of mitosis direction in developing eSMGs. n = 12 (control) and 15 (+100 μM Nifedipine) buds. (H) Experimental scheme for live imaging of developing epithelial bud with selective staining of the peripheral nucleus (see Methods section). (I) Epithelial dynamics (Hoechst-stained; upper panels) and phase contrast images (lower panels) of a developing eSMG. Dotted region indicates the epithelial folding site. (J) Tracking images of the movement of the peripheral nucleus. Right: tracking results of a single cell in the dotted region. Tirangular points in the tracking results indicate the end position of the nucleus. Data are represented as mean ± SEM. Scale bars: 10 (E and J), 50 μm (C and I).