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Figure 5. Adam13 induces pcdh8l via AP2α.(A, C, D) Relative expression of AP2α and pcdh8l in naïve ectoderm (animal cap) by real-time PCR. One-cell stage embryos were injected with 1 ng of the various adam13 constructs and morpholinos and embryos were collected at stage 9 to dissect animal cap cells (AC). AC were grown in 0.5X MBS until sibling embryos reached stage 20 to 22. Messenger RNA was extracted from 10 AC for gene expression analysis. Expression was normalized using GAPDH and compared to the expression in non-injected AC (NI). (A) Real-time PCR data show that adam13 can induce AP2α by more than four fold. Both proteolytic activity and the presence of the cytoplasmic domain are essential for full activation. (B) Luciferase activity of AP2α promoter in Hek293T cells shows induction by adam13 and ADAM19 but not ADAM9. For each transfection the luciferase values were normalized to the Renilla values driven by the CMV promoter. In these assays, the absence of proteolytic activity (A13E/A) reduced AP2α induction only slightly, while the deletion of the cytoplasmic domain (ΔCyto) prevented the activity. (C) Induction of AP2α by adam13 was prevented by the KD of AP2α (10 ng of MOAP2α) but not β-catenin (20 ng of Moβ-catenin). (D) Expression of AP2α in response of adam13 also depends on AP2α but not β-catenin. Error bars represent standard error to the mean (Mean ±S.E.M). One-way ANOVA was performed to determine statistical significance. Statistically significant at **p<0.01, ***p<0.001.10.7554/eLife.26898.015Figure 5—source data 1. Source data for Figure 5. Larger Image Printer Friendly View

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