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. 2017 May 17;8:15206. doi: 10.1038/ncomms15206

Figure 6. In vivo> validation of YAP/TAZ as inhibitors of Notch signalling in epidermis.

Figure 6

(a–c) YAP induction inhibits epidermal differentiation and Notch signalling in vivo. (a) YAP overexpression in YAP-transgenic (Tg YAP) mice leads to the expansion of the basal layer and diminished terminal differentiation. IF on the back and tail skin of E20.5 embryos reveals increased thickness of the KRT14-positive basal layers (Bl) with concomitant reduction of the differentiated KRT10- and TGM1-positive suprabasal layers (Supr) compared to control littermates (Ctr). K14, keratin 14; K10, keratin 10. Scale bar: 20 μm. See also Supplementary Fig. 4a for additional data at E18.5. (b) YAP inhibits Notch signalling in epidermis. Immunohistochemistry on the back skin of Tg YAP at E20.5 embryos shows decreased Notch signalling in the epidermis compared to control littermates (Ctr), as visualized by the greatly reduced levels of the N1ICD fragment (top panel) and Hes1 (bottom panel). The insets (0.5 × ) show immunohistochemistry for Yap and nuclear counterstaining in Ctr and Tg YAP mice, as control of YAP overexpression. Scale bar: 20 μm. (c) qRT–PCR on skin biopsies of mice as in a,b. YAP overexpression inhibits the expression of both terminal differentiation markers of epidermis (Krt1, Ivl, Lor) and Notch transcriptional targets (Notch3, Nrarp), while it upregulates the YAP/TAZ target genes (Ctgf, Dll1). Relative mRNA expression values were normalized to an internal sample for each experiment and represented in arbitrary units (A.U.) as box plots (n=8 for Ctr and n=7 for Tg YAP, from three independent experiments. *P<0.0001, **P<0.01 compared to Ctr; Student's t-test). (d) YAP/TAZ double conditional knockout (cKO) induces terminal differentiation and Notch signalling in vivo. qRT–PCR analysis on the epidermis from E17.5 embryos shows an upregulation of both the terminal differentiation markers (Krt1, Ivl, Lor) and Notch transcriptional targets (Hes1, Notch3, Nrarp) in Yap/Taz cKO mice compared to controls (Ctr). The reduced YAP/TAZ transcriptional activity in cKO embryos is confirmed by the dowregulation of the target genes Ctgf and Dll1. Relative mRNA expression values were normalized to an internal sample for each experiment and represented in A.U. as box plots (n=5 for Ctr and n=4 for cKO, from three independent experiments. *P<0.05, **P<0.01 compared to Ctr; Student's t-test). Box plots in c,d show the median, interquartile range, minimum and maximum values.