Pax9 is a Key Modulator of Wnt Signaling During Palatogenesis

Objectives: We have recently shown that the Wnt signaling pathway is involved in Pax9-mediated signaling within the posterior palate. Such a molecular circuit is critical for the morphogenetic gradient that defines the medial-lateral (ML) axis. The functionality of this relationship is underscored by the genetic rescue of palatal clefts in mice that lack both Pax9 and Dkk1, a proven Wnt signaling antagonist. Exciting is the high fidelity correction of palatal clefts in Pax9^-/- embryos that received agonists through placental circulation(Development, 144, 3819-28, 2017). Although our data suggest that Pax9 functions upstream of the Wnt pathway, it is unclear whether Pax9 interacts directly with Wnt inhibitors or ligands. The objectives of these studies were to assess the molecular relationship between Pax9 and Wnt pathway genes so as to shed insight into their roles in palatogenesis.
Methods: Palatal cells were isolated from E13.5 murine posterior palatal shelves and transfected with a Myc-Pax9 expression construct using the Neon Transfection System. ChIP assay was performed using anti-Myc antibody or normal mouse IgG followed by qPCR analyses. Expression levels of Dkk1 and Dkk2 transcripts and palate-specific Wnt ligands, Wnt9b and Wnt3, were assessed by RT-qPCR in palatal shelves from E13.5 Pax9^-/- embryos and their control littermates. The spatial profiles of transcript distribution were finally assessed by in-situ hybridization approaches.
Results: Results of our ChIP-qPCR analyses revealed specific binding activities of Pax9 near the transcription start sites of Dkk1, Dkk2 as well as the intergenic region of Wnt9b and Wnt3. In pCMV-Pax9 transfected cells, expression levels of Dkk1 and Dkk2 were down-regulated while Wnt9b and Wnt3 were upregulated. The dual relationship of Pax9 and Wnt genes was underscored by data from the expression analysis that revealed an overlapping expression pattern between Pax9 and Wnt ligands. In contrast, the patterns of Dkk1 and Dkk2 expression was inversely related to that of Pax9.
Conclusions: Taken together, these results suggest that Pax9 modulates Wnt signaling activity through its dual role in inhibiting expression of Dkk1 and Dkk2 while increasing Wnt ligand levels during posterior palateogenesis.