The Effects of Dkk1 Inhibitor Treatment on Wnt Signaling during Craniofacial Development

Objectives: PAX9 is known to be associated with human craniofacial abnormalities such as cleft palate and tooth agenesis. Homozygous mice lacking Pax9 die at birth with cleft palate and tooth developmental arrest at bud stage accompanied with decreased Wnt signaling activity. Recent studies in our lab demonstrated that Dkk1 inhibitor WAY-262611 treatment at specific embryonic stage could rescue the cleft palate defects in Pax9 mutant embryos, but the underlying mechanisms need further investigation. The objective of this study is to use Axin2^LacZ knock-in (KI) mice, which are widely used reporter strains for monitoring endogenous canonical Wnt signals, to analyze the molecular mechanisms of craniofacial defects rescued by WAY-262611 treatment.
Methods: The Pax9^+/- mice were mated with Axin2^LacZ KI mice to obtain Pax9^+/-;Axin2^LacZ mice. The Dkk1 inhibitor WAY-262611 was injected into pregnant Pax9^+/- mice which had been mated with Pax9^+/-;Axin2^LacZ males in the tail vein. The embryos were harvested at E13.5 followed by LacZ staining on frozen sections or whole mount embryo heads. Moreover, E13.5 palate shelves were micro-dissected from Pax9-deficient embryos and their control littermates that were untreated or treated with WAY-262611. Total RNA was extracted and the genome wide gene expression profiles were analyzed by RNA-seq approaches.
Results: LacZ staining in Axin2^LacZ embryos shows that canonical Wnt signals appears in the wildtype developing palate shelves with posterior to anterior gradient pattern at E13.5, while Wnt signals were decreased in Pax9 mutant palate. After WAY-262611 treatment, the endogenous canonical Wnt signaling activity was significantly increased in palatal shelves in wild type samples, especially in posterior region. In Pax9 mutants, the Wnt signaling activity in developing palate was partially restored by WAY-262611 treatment.

Conclusions: The rescue experiment suggests that WAY-262611 has the potential to be used as therapeutic drug for treating developmental abnormalities related to Wnt signaling deficiency. Our data indicate that WAY-262611 rescues the craniofacial defects in Pax9^-/- embryos partially by restoring canonical Wnt signaling activity in developing palate.