Hippo Pathway in Submandibular Gland Development: Potential Interplay with N-glycosylation

Mouse submandibular gland (SMG) begins its development at embryonic day 11 when oral epithelium grows into the underlying mesenchyme. The metabolic pathway of protein N-glycosylation is critical for SMG development and partial inhibition of DPAGT1, the gene that initiates N-glycosylation, drives cytodifferentiation of ductal structures. We have shown that DPAGT1 is a target of the canonical Wnt signaling pathway and that DPAGT1 regulates E-cadherin-mediated cell-cell adhesion, required for the survival of differentiating duct cells. Recently, the Hippo pathway has been shown to be critical for tissue development by regulating Wnt signaling and establishing apical-basal polarity. TAZ is a Hippo pathway transcription factor and polarity enhancer that also serves as a mechanosensor of the extracellular matrix.

Objective: We investigated whether the Hippo pathway participated in SMG development and if it interacted with the metabolic pathway of N-glycosylation.

Method: SMGs were dissected from mice at embryonic day 13.5, epithelial rudiments were separated from the mesenchyme and grown in vitro in the presence and absence of siRNA to DPAGT1. After 48 hours, SMGs were processed for immunofluorescence staining for F-actin, ZO-1 and TAZ and analyzed by confocal microscopy.

Result: Confocal imaging showed colocalization of F-actin and ZO-1 extending further into the ductal region in DPAGT1-silenced glands compared to controls. TAZ was detected at the apical-lateral surfaces of differentiating duct cells, suggesting its role in polarity. Moreover, in DPAGT1 silenced glands, TAZ exhibited enhanced localization to intercellular junctions.

Conclusion: Our findings show that inhibition of N-glycosylation promotes establishment of apical domains in ductal progenitors by ZO-1. In addition, the Hippo pathway component, TAZ, is expressed in the embryonic SMG, localizing to intercellular junctions in ductal cells, which is enhanced by partial inhibition of DPAGT1. These data suggest an inverse relationship between N-glycosylation and the Hippo pathway.