During late stages of mouse development, FoxO6 is expressed specifically in craniofacial tissues and FoxO6-/- mice undergo expansion of the face, frontal cortex, olfactory component and skull. Enlargement of the mandible and maxilla and lengthening of the incisors in FoxO6-/- mice are associated with increases in cell proliferation. In vitro and in vivo studies demonstrated that FoxO6 activates Lats1 expression, thereby increasing Yap phosphorylation and activation of Hippo signaling. FoxO6-/- mice have significantly reduced Hippo Signaling caused by a decrease in Lats1 expression and decreases in Shh and Runx2 expression, suggesting that Shh and Runx2 are also linked to Hippo signaling. In vitro, FoxO6 activates Hippo reporter constructs and regulates cell proliferation. Furthermore PITX2, a regulator of Hippo signaling is associated with Axenfeld-Rieger Syndrome causing a flattened midface and we show that PITX2 activates FoxO6 expression. Craniofacial specific expression of FoxO6 postnatally regulates Hippo signaling and cell proliferation.
Together, these results identify a FoxO6-Hippo regulatory pathway that controls skull growth, odontogenesis and face morphology.
Pitx2 activates FoxO6 expression – A) Heat map from gene expression microarray showing FoxO6 is increased in Krt14-PITX2 overexpression mouse incisor dental epithelium compared to WT. DNA microarray analyses of five different E18.5 mouse dental epithelial tissue samples reveals an increase in FoxO6 expression in the PITX2 over-expression incisor. B) Real-time PCR demonstrates increased endogenous FoxO6 transcripts in LS-8 cells and MDPC cells that over-express PITX2 compared with empty vector. C, D) LS-8 cells (C) and MDPC cells (D) were infected with Pitx2 or scrambled lentivirus and probed for Pitx2 and FoxO6 expression by Western Blot. β-Tubulin served as a loading control.