IADR Abstract Archives
Pax9 plays a dual role in controlling Wnt signaling during murine palatogenesis
Objectives: Despite the strides made in understanding the complex network of key regulatory genes and cellular process that drive palate morphogenesis, patients suffering from these conditions face treatment options that are limited to complex surgeries and multidisciplinary care throughout life. Hence, a better understanding of how molecular interactions drive palatal growth and fusion is critical for the development of treatment and preventive strategies for cleft palates in humans. Our previous work demonstrated that Pax9-dependent Wnt signaling is critical for the growth and fusion of palatal shelves. These studies further explored the nature of the molecular relationship between Pax9 and Wnt signaling in palate development.
Methods: To verify the direct target of Pax9 in Wnt signaling pathway, we first confirmed their genetic interaction using compound mutant backcrosses of Pax9-/-Dkk1f/+;Wnt1Cre mice. The phenotype of embryos with inducible Dkk1 expression was also evaluated to confirm the function of Dkk1 during palate formation. To investigate whether Pax9 can directly modulate Wnt pathway gene expression via the paired box DNA binding domain, we transfected the mouse palatal cells from posterior palatal tissue with Myc-Pax9 expression constructs for ChIP-qPCR analyses.
Results: Our data show that the genetic reduction of Dkk1 during palatogenesis corrected secondary palatal clefts in Pax9-/- mice with restoration of Wnt signaling activities. In contrast, genetically-induced overexpression of Dkk1 mice pheno-copied the defects in tooth and palate development visible in Pax9-/- strains. Results from ChIP-qPCR assays showed that Pax9 can bind to regions near the transcription start sites of Dkk1 and Dkk2 as well as the intergenic region of Wnt9b and Wnt3 ligands that are down regulated in Pax9-/- palates.
Conclusions: Taken together, these data suggest that the molecular mechanisms underlying Pax9’s role in modulating Wnt signaling activity involve the inhibition of Dkk expression as well as the control of Wnt ligands during palatogenesis. Such information provides the framework for designing novel therapeutic approaches for the treatment of cleft defects in humans.
Methods: To verify the direct target of Pax9 in Wnt signaling pathway, we first confirmed their genetic interaction using compound mutant backcrosses of Pax9-/-Dkk1f/+;Wnt1Cre mice. The phenotype of embryos with inducible Dkk1 expression was also evaluated to confirm the function of Dkk1 during palate formation. To investigate whether Pax9 can directly modulate Wnt pathway gene expression via the paired box DNA binding domain, we transfected the mouse palatal cells from posterior palatal tissue with Myc-Pax9 expression constructs for ChIP-qPCR analyses.
Results: Our data show that the genetic reduction of Dkk1 during palatogenesis corrected secondary palatal clefts in Pax9-/- mice with restoration of Wnt signaling activities. In contrast, genetically-induced overexpression of Dkk1 mice pheno-copied the defects in tooth and palate development visible in Pax9-/- strains. Results from ChIP-qPCR assays showed that Pax9 can bind to regions near the transcription start sites of Dkk1 and Dkk2 as well as the intergenic region of Wnt9b and Wnt3 ligands that are down regulated in Pax9-/- palates.
Conclusions: Taken together, these data suggest that the molecular mechanisms underlying Pax9’s role in modulating Wnt signaling activity involve the inhibition of Dkk expression as well as the control of Wnt ligands during palatogenesis. Such information provides the framework for designing novel therapeutic approaches for the treatment of cleft defects in humans.