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. 2020 Sep 30;10:16075. doi: 10.1038/s41598-020-72828-4

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© The Author(s) 2020

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Generation of Pax6 cKO (Pax6^−/−) embryos. (A) Schematic of the breeding strategy used in the study to generate Pax6^−/− embryos after tamoxifen injection. (B) Pax6^fl/fl and Pax6^−/− embryos collected at E18 after tamoxifen injection at E15 display marked phenotypic differences. (C) Genotyping PCR to screen Pax6^fl/fl and Pax6^−/− animals based on the presence of cre. (D–F) DAPI staining of Pax6^fl/fl and Pax6^−/− embryos revealed a reduction in the thickness of the neuroretina of Pax6^−/− embryos (E) compared to the Pax6^fl/fl (D) embryos. Thickness of the retina is plotted as box plot using R program (F; t-test; p < 0.001). Scale bar—25 µm (G) Quantitative expression analysis for Pax6 in Pax6^fl/fl and Pax6^−/− retina showed a significant reduction in the expression level (p < 0.001). (H–S) Immunostaining with N-terminal Pax6 antibody showed a significant reduction in the number of Pax6 positive cells in both the central (I, L) and peripheral region (O, R) of Pax6^−/− retina compared to Pax6^fl/fl retina. As such, the numbers of Pax6 positive cells are lesser in the peripheral retina (O) compared to the central retina (I). Scale bar—25 µm. n = 3, at least 3 embryos each of Pax6^fl/fl /Pax6^−/− from 3 pregnant animals were collected. Error bars indicate SEM from three biological replicates.