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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. 6. — Deletion of p27 in Sox2^fl/fl;Nkx3.1^Cre/+ embryos result in a rescue of IL cell proliferation, but melanotroph cell fate acquisition is not restored. (A) Analysis of cell proliferation in IL. The percentage of EdU⁺;DAPI⁺ nuclei in IL was quantified at 18.5 dpc after a 1 h pulse. There is a significant (*P=0.026) decrease in the percentage of cells that incorporate EdU between wild-type (11.4±1.2%, n=4) and Sox2^fl/flNkx3.1^Cre/+ embryos (7.1±2.4%, n=3). In contrast, in double mutants, percentages of EdU-positive nuclei (12.03±3.1%, n=3) are not significantly different from wild-type (ns, P=0.76). Therefore, ablation of p27 restores normal levels of proliferation in mutants. (B) Analysis of cell fate acquisition in IL. 72±5.5% (n=3) of cells in the IL are POMC-positive in wild-type embryos. Both Sox2^fl/flNkx3.1^Cre/+ and p27^−/−;Sox2^fl/flNkx3.1^Cre/+ embryos display significantly lower percentages (15±3%, n=3, ***P=0.0001 and 20.3±1%, n=4, ****P<0.0001) but there is a significant difference between single and double mutants (*P=0.019) demonstrating a slight rescue of the differentiation defect. Results presented as mean±s.d. (C) Immunofluorescence for POMC and PAX7 at 18.5 dpc. PAX7 and POMC appear normally expressed in the IL of p27^−/−;Sox2^fl/fl;Nkx3.1^+/+ embryos. In contrast, expression of both proteins is downregulated in Sox2^fl/fl;Nkx3.1^Cre/⁺ embryos, as previously observed, and also in p27^−/−;Sox2^fl./flNkx3.1^Cre/+ embryos. (D) Immunofluorescence for GR and SOX2 at 18.5 dpc. GR is not present in the IL of p27^−/−;Sox2^+/+;Nkx3.1^Cre/+ but it is still ectopically expressed in p27^−/−;Sox2^fl/fl;Nkx3.1^Cre/+ embryos. There is a noticeable increase in SOX2 staining in p27^−/−;Sox2^+/+;Nkx3.1^Cre/+, in agreement with previous data demonstrating upregulation of Sox2 in absence of p27 (Li et al., 2012). Scale bars: 50 μm. IL is outlined.