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. 2019 Apr 11;9:5912. doi: 10.1038/s41598-019-42040-0

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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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TGFβ pathway expression in the inner ear during early development. (A) Expression of Tgfβ2, Tgfβ3, Tgfβr1, Tgfβr2, Tgfβr3 and Eng mRNA in the inner ear was measured by RT-qPCR at the indicated developmental stages. Eukaryotic 18s rRNA was used as an endogenous control gene. Gene expression was calculated as 2^−ΔΔCt and normalized to the HH17 levels. The results are expressed as mean ± SEM from four independent experiments. (B) TGFβ pathway components (Tgfβ2, Tgfβr2 and pSMAD2) expression in inner ear sections. At HH18 Tgfβ2 was expressed in the dorsal area of the otic epithelium, where the endolymphatic duct will emerge (first panel, arrow). Tgfβr2 was expressed predominantly in the dorsal area of the otic epithelium, including the endolymphatic duct anlagen (second panel, arrow). The presence of active TGFβ signalling was assessed with immunodetection of the phosphorylated downstream effector protein SMAD2 (pSMAD2), which showed a nuclear localization throughout the otic epithelium and the developing endolymphatic duct (third panel, arrow). (C) Tgfβ2 was strongly expressed in the developing endolymphatic duct at later developmental stages, HH20 and HH25 (arrows). Representative microphotographs are shown from at least three embryos per condition. Orientation: A, anterior; D, dorsal; L, lateral; M, medial; R, rostral. Scale bars: 100 μm.