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Figure 4. Targeted disruption of the essential E-box in Barhl1 enhancer does not affect induction of EPL cells and otic progenitors from mESCs. (A) qRT-PCR analyses for expression of pluripotent cell-related marker Oct4, visceral endoderm-related marker AFP, mesoderm-related marker Brachyury, and primitive ectoderm-related marker Fgf5 in cell aggregates derived from WT and BEM mESCs on day 7. There were no significant differences in the mRNA expression levels of Oct4 and Fgf5 between two kinds of mESC-derived cells and no significant expressions of AFP and Brachyury in these induced cells, indicating the transition of both mESCs to EPL cells. Data are mean ± SEM (n = 3). (B) qRT-PCR analyses for expression of otic markers Dlx5, Eya1, Pax2, Pax8, and Six1 in WT-and BEM-derived cultures at day 21. No significant differences in the mRNA expression levels of Dlx5, Eya1, Pax2, Pax8, and Six1 between both kinds of mESC-derived cells were detected. Data are mean ± SEM (n = 3). (C,D) Immunostaining analyses of otic markers Pax2/Pax8 (C) and Pax8/Sox2 (D) in WT- and BEM-derived cultures at day 21. Scale bars, 20 μm. (E,F) The percentages of double-immunopositive cells for Pax2/Pax8 (E) and Pax8/Sox2 (F) in WT- and BEM-derived cultures at day 21. There were no significant differences in the percentages of double-immunopositive cells for Pax2/Pax8 and Pax8/Sox2 between these two mESC-derived cells. Data are mean ± SEM (n = 3). Image published in: Hou K et al. (2019) © 2019 by the authors. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View

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