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. 2019 Apr 1;9:5446. doi: 10.1038/s41598-019-41940-5

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© The Author(s) 2019

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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Over-proliferation and increased apoptosis at in the Tmem67^−/− cerebellum at E12.5. Mid-sagittal sections of the E12.5 cerebellar vermis stained for (a) active β-catenin; (b) proliferation (Ki67); (c) mitotic cells (P-H3, phospho-Ser10 histone H3; green, indicated by arrowheads), with bar graph on right to quantitate the number of P-H3⁺ cells per field of view for Tmem67^+/+ (+/+) and Tmem67^−/− (^−/−) cerebella; and (d) activated cleaved caspase-3, with bar graph on right to quantitate the intensity of IHC staining per field of view (AU; arbitrary units). In the wild-type embryonic cerebella, active β-catenin and Ki67 were largely restricted to the rhombic lip (arrowheads in a and b). Note the increase in canonical Wnt signalling (marked by active β-catenin), proliferation (Ki67) and apoptosis (active caspase-3), and the decrease in the number of mitotic cells (P-H3), in Tmem67^−/− sections. Asterisks indicate non-specific staining of red blood cells in (c). Scale bars = 50μm (a–c) and 20μm (d). Abbreviation: cp, choroid plexus; cv, cerebellar vermis; mb, mid-brain; rl, rhombic lip. Statistical significance of pairwise comparisons are indicated by: *p < 0.05; **p < 0.01; for Student’s t-test (paired, two-tailed). Error bars = s.e.m. for a minimum of 4 fields of view in n = 3 biological replicates.