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. 2016 Jul 1;143(13):2376–2388. doi: 10.1242/dev.137984

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© 2016. Published by The Company of Biologists Ltd

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

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Fig. 1. — Loss of SOX2 results in a reduction of RP progenitor proliferation. (A) Foxg1^Cre and Nkx3.1^Cre lineage-tracing analysis. Immunofluorescence for eYFP and SOX2. In Foxg1^Cre/+;R26R^eYFP/+ embryos at 10.5 dpc, the reporter displays a ubiquitous activity in RP. At 12.5 dpc in Nkx3.1^Cre/+;R26R^eYFP/+ embryos, eYFP is observed in the future IL. 69.6±6.5% (mean±s.d.) of SOX2-positive cells express eYFP (n=3) in Nkx3.1^Cre/+;R26R^eYFP/+ embryos at this stage. (B,C) Immunofluorescence for SOX2 on mutant embryos. SOX2 downregulation after Cre recombination is initially detectable at 10.5 dpc in Sox2^fl/fl;Foxg1^Cre/+ embryos (B) and 12.5 dpc in Sox2^fl/fl;Nkx3.1^Cre/+ embryos (C). Deletion of Sox2 using Foxg1^Cre results in formation of a hypomorphic pouch at 12.5 dpc, still attached to the oral ectoderm (arrow, B). Later deletion with Nkx3.1^Cre is initially associated with a thinner dorsal pouch at 14.5 dpc (arrow, C). (D,E) Analysis of cell proliferation after Sox2 deletion in RP. After a 1 h pulse at 12.5 dpc, the percentage of BrdU⁺; DAPI⁺ nuclei is lower in Sox2^fl/fl compared with Sox2^fl/+ embryos. BrdU incorporation is significantly reduced when Foxg1^Cre is used to delete Sox2 (Sox2^fl/+;Foxg1^Cre/+: 16.2±2%, n=4 compared with Sox2^fl/fl;Foxg1^Cre/+:7.7±2.0%, n=3; *P=0.03). Using Nkx3.1^Cre, proliferation is less affected (Sox2^fl/+;Nkx3.1^Cre^/+: 18.3±1.9%, n=4 and Sox2^fl/fl;Nkx3.1^Cre/+: 12.9±2.4%, n=4; ns). (F) Immunofluorescence for cyclin D1 at 12.5 dpc. Cyclin D1 delineates the dorsal proliferative region in Sox2^fl/+;Foxg1^Cre/+ RP; its expression is significantly reduced in homozygous Sox2^fl/fl;Foxg1^Cre/+ embryos. (G) TUNEL assay at 12.5 dpc. There is persistence of a patch of apoptotic cells in the hypoplastic Sox2^fl/fl;Foxg1^Cre/+ RP where it is still abnormally connected to the oral ectoderm. All sections are sagittal. Dotted outline indicates RP. Scale bar: 50 μm in A-C,G; 100 μm in F. RP, Rathke's pouch; VD, ventral diencephalon; Inf, infundibulum.