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. 2018 Apr 3;8:5580. doi: 10.1038/s41598-018-23785-6

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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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In vivo, multi-color two-photon imaging. (a) Injection regimens and imaging time points for in vivo histomorphometry. (b) The coronal suture space (S) between frontal (fb) and parietal (pb) bones. (c) Animals were treated with either rmWnt3a (in PBS, 44 ng/day) or just PBS for 14 days. Maximum intensity projection of a 25-μm layer was used for demonstration. Images on Day0 show Prx1 + cells (Green), their progeny (Red or yellow, co-expression of tdTomato and EGFP). In addition, on Day 28, the initial bone structure was identified by calcein blue (Blue) and the new bone fronts were demarcated by tetracycline (purple), as indicated by blue and purple arrows in Fig. 1(d). Spontaneous recombination (white arrowheads) and incomplete recombination (green arrows) may be present. It is noted that a higher number of osteocytes originated from Prx1 + cells were embedded in lacunae (the red arrows and the inset) from rmWnt3a treatment. (d) Quantifications of bone morphometry and cellular dynamics were performed over a 50-μm layer, as shown in 3D reconstructed images.