Title: Pax9 Represses Wnt Inhibitors During Palate Development
Jing Zhou (Presenter)
University of Utah
Shihai Jia, University of Utah
Yinshen Wee, University of Utah
John Bonds, Texas A&M University
Pascal Schneider, University of Lausanne
Rena D'Souza, University of Utah
Objectives: Cleft palate is a common human birth defect with high public health impact and that can only be corrected surgically. Hence, it is imperative to development non-invasive therapies to prevent such defects. Our recent work demonstrated that Wnt agonist WAY-262611 or IIIc3a, which inhibit DKKs, could rescue the cleft palate in Pax9-/- mice, indicating Pax9 regulates Wnt signaling through Dkk inhibitors. The objective of this study is to verify the hypothesis by testing whether Pax9 can bind to the promoters of DKKs in palatal cells and regulate their expression during palate development.
Methods: Mouse palatal cells from posterior palatal tissue were transfected with Myc-Pax9 expression construct using Neon Transfection System. Then ChIP assay was carried out using anti-Myc antibody or normal mouse IgG followed by qPCR analyses. Further, we compared the distribution pattern by in-situ hybridization and expression level of Dkk1 and Dkk2 transcripts by RT-qPCR in the developing palate of E13.5 Pax9-/- embryos with their control littermates.
Results: ChIP-qPCR data demonstrated that the binding of Pax9 was 2-4 fold enriched near the transcription start sites of Dkk1 and Dkk2. In normal palatal tissue, Dkks and Pax9 showed complementary expression patterns, while in Pax9-deficient palatal mesenchyme, the expression of Dkk1 and Dkk2 was up-regulated.
Conclusions: Specific bindings of Pax9 near the transcription start sites of Dkk1 and Dkk2 and higher expression level of Dkk1 and Dkk2 in Pax9-/- palatal shelves confirmed that Pax9 regulates Dkk1 and Dkk2 by binding to their promoter regions. We will further test whether Pax9 can repress the expression of Dkk1 and Dkk2 by luciferase assay. Together, these results would provide the mechanisms through which Dkk inhibitors can rescue cleft palate caused by Pax9 deficiency.