β-catenin activation in Wnt-responsive Axin2 + intercalated duct cells drives early epithelial and stromal remodelling in the adult murine submandibular gland

Abstract Adult salivary glands depend on a branched ductal network for secretion, yet the identity of Wnt-responsive ductal cells that support adult homeostasis and their relevance to early salivary gland lesion formation remain unclear. Using Axin2 CreERT2/+ ; R26 mTmG/+ mice for lineage tracing, together with TCF/Lef: H2B-GFP reporter mice and validation by Axin2 RNA in situ hybridisation (RNAscope), we identified canonical Wnt-responsive cells predominantly within intercalated ducts of the adult murine submandibular gland. Axin2-labelled intercalated duct cells showed partial overlap with keratin 5, keratin 14 and p63, markers previously associated with salivary gland stem/progenitor populations, supporting heterogeneity within this compartment. Lineage tracing and proliferation analyses indicated that these Axin2 + cells cycle infrequently but contribute to maintenance of intercalated and downstream ductal compartments. Notably, this homeostatic Wnt-responsive state was diminished with age, with reduced Wnt ligand expression and fewer Axin2-labelled intercalated duct cells, consistent with age-related niche remodelling and reduced abundance of a ductal progenitor-associated compartment. Functionally, conditional deletion of Wntless (Wls) in pCAG CreERT2/+ ; Wls fl/fl mice reduced ductal proliferation and intercalated duct-associated markers, supporting an important role for Wnt secretion in adult duct homeostasis. In contrast, stabilised β-catenin activation in Axin2 CreERT2/+ ; Ctnnb1 lox(ex3)/+ ; R26 mTmG/+ mice induced rapid expansion of solid and cystic lesion-like epithelial nests accompanied by fibrosis and inflammatory infiltration. Axin2-lineage cells showed an early proliferative burst followed by reduced proliferation and appearance of senescence-associated markers, whereas neighbouring non-lineage epithelial clusters showed increased proliferation and β-catenin accumulation, consistent with lesion-associated epithelial remodelling. Together, these findings identify Axin2-positive intercalated duct cells as a Wnt-responsive population contributing to adult duct maintenance. They further implicate this compartment as a permissive epithelial context for early Wnt-driven lesion formation and epithelial–stromal remodelling in the adult murine submandibular gland.