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. 2018 May 25;9:2073. doi: 10.1038/s41467-018-04447-7

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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. 1 — Aortic SMCs are polyclonal and highly migratory in development. a Cells of ROSA26R^(CreER/Rb) embryo induced with high dose 4-OH-T at E5.25 are expected to be of multiple colors at E5.5–7.25. If the aortic wall derives from a single cell (or instead from multiple polyclonal cells) present at ~E7.25 or thereafter, wall cells will be a single color (or multiple colors). b–g Sections of ROSA26R^(CreER/Rb) mice in transverse (b–e, g) or longitudinal axis (f) imaged with direct fluorescence of Rb channels. At E5.25, dams were induced with a single high 4-OH-T dose in b, c (150 µg) or, to label clonally, at a limiting dose in d, d′ (100 µg; Supplementary Table 1, clone ID1-1), e (20 µg; ID1-14), f (20 µg; ID1-18), and g (50 µg; ID1-10). Arrowheads indicate mOrange⁺ cells (d, e), with marked cells in d suggesting ventral migration of aortic progenitors. By the time of recombination, progenitors of aorta and lung mesenchyme (d, d′) or epithelium (g, g′) are differentially specified (h) (Supplementary Table 1). i, j Transverse sections of X^GFPX⁺ aorta stained for GFP at E14.5 and in the adult (5 weeks), representative of n = 4 embryos and 6 adults. k, l SMMHC-CreER^T2 embryos also carrying ROSA26R^(mTmG/+) or ROSA26R^(Rb/+) induced with tamoxifen 0.5 mg at E9.5 (when there is one SMC layer; Supplementary Fig. 1b, c) and analyzed at E16.5. In k transverse aortic section (left panel, section #30) of a 21-cell GFP-marked clone (Supplementary Table 2, clone ID2-12; Supplementary Fig. 3) after GFP staining. The middle panel shows positions of two marked cells in this section, and columnar clone schematic (right panel) is a compilation of transverse sections, among 60 consecutive sections analyzed, that contain marked cells. The first and 60th sections are included for reference. A 15-cell clone (ID2-10) is shown in l with the section stained for DAPI and imaged for Rb colors. The clones in k, l expanded radially, circumferentially, and longitudinally. Lu, aortic lumen; E, endothelial layer; 1–5, smooth muscle layers; A, adventitial layer. Scale bars, 100 µm (b, d, e, g, i) and 10 µm (c, f, j–l)