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. 2017 Jun 22;7:4086. doi: 10.1038/s41598-017-04315-2

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© The Author(s) 2017

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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SOX2 is required for the downstream expression of NeuroD1. (A) Sox2 ^+/+ control section showing the antereoventral portion of the otic vesicle from which neuroblasts delaminate. Boxed region refers to the area in which cell counts were performed. (A’ and A”) show a higher magnification of the neuroblasts contained within the boxed region. Note the extensive colabeling of SOX2 (red) and NEUROD1 (green). Dotted line shows the boundary between the neurosensory epithelium and the mesenchyme. (B) Representative section showing the absence of SOX2 and NEUROD1 protein expression in a Sox2-deficient mutant. (B’ and B”) Show a higher magnification of the boxed region from (B). Note the absence of immunofluorescence for both SOX2 and NEUROD1. To note, the handful of green cells seen in the mesenchyme did not meet inclusion criteria for neuroblasts and were not counted. (C) The total number of NEUROD1 and SOX2-positive neuroblasts was quantified, and both markers were found to be significantly decreased in Sox2 ^CreER/fl mice (n = 4) compared to Sox2 ^+/+ controls (n = 3) (****p < 0.0001) (Student’s t test). (D) Total NEUROD1 cell counts were separated based on either being located in the neurosensory epithelium, or migrating into the mesenchyme; in both cases the number of NEUROD1-positive cells were significantly reduced in the Sox2 ^CreER/fl mice (epithelial NEUROD1: ****p < 0.0001 migrating NEUROD1: ***p = 0.0002). Error bars represent SEM. OV: Otic Vesicle; CVG: Cochleovestibular ganglion; FG: Facial ganglion. Scale bars: 100 μm.