Redox regulation of canonical Wnt signaling affects extraembryonic endoderm formation

Retinoic acid (RA) induces mouse F9 cells to form primitive endoderm (PrE) and increased levels of reactive oxygen species (ROS) accompany differentiation. ROS are obligatory for differentiation and while H2O2 alone induces PrE, antioxidants attenuate the response to RA. Evidence shows that ROS can modulate the Wnt/β-catenin pathway and in this study, we show that extraembryonic endoderm formation is dependent on the redox state of nucleoredoxin (NRX). In undifferentiated F9 cells, NRX interacted with dishevelled 2 (Dvl2) and while this association was enhanced under reduced conditions, it decreased following H2O2 treatment. Depleting NRX levels caused morphological changes like those induced by RA, while increasing protein kinase A activity further induced these PrE cells to parietal endoderm. Reduced NRX levels also correlated to an increase in T-cell-factors-lymphoid enhancer factors-mediated transcription, indicative of canonical Wnt signaling. Together these results indicate that a mechanism exists whereby NRX maintains canonical Wnt signaling in the off state in F9 cells, while increased ROS levels lift these constraints. Dvl2 no longer bound to NRX is now positioned to prime the Wnt pathway(s) required for PrE formation.