<i>Notum</i> Regulates Tooth Root Morphogenesis by Modulating Wnt Signaling

Molar root morphogenesis, particularly the formation of the furcation that determines root number, is a complex process governed by precise epithelial-mesenchymal interactions. Although Wnt signaling is critical for tooth development, the mechanisms that spatially restrict its activity to ensure proper root architecture are not fully understood. In this study, to investigate the role of the secreted Wnt antagonist Notum in regulating molar root furcation, we analyzed <i>Notum</i> knockout (KO) mice. Temporospatial expression analysis revealed that <i>Notum</i> is specifically and transiently expressed in the differentiating odontoblasts of the apical mesenchyme during furcation formation. <i>Notum</i> KO mice exhibited a severe failure of molar root furcation with an enlarged pulp chamber. Mechanistically, particularly at the molar furcation region, <i>Notum</i> deficiency led to impaired extension of Hertwig's epithelial root sheath (HERS) and dysregulated signaling in the underlying mesenchyme, characterized by an upregulated Wnt pathway and, consequently, increased cell proliferation of apical pulp cells and reduced odontoblast differentiation. Notably, inducible deletion of <i>Wntless</i> (<i>Wls</i>) in the dental epithelium phenocopied the furcation defect and led to a dramatic loss of <i>Notum</i> expression, demonstrating that epithelial Wnt signaling is required for <i>Notum</i> induction at the mesenchymal apex of furcation region. These findings identify Notum as a critical regulator of molar root furcation. We propose a model in which HERS-derived Wnt signaling induces <i>Notum</i> expression in the adjacent mesenchyme, which, in turn, antagonizes the Wnt signal to orchestrate the switch from cell proliferation to odontoblastic differentiation, thereby driving the formation of the root furcation. This study provides novel insights into the molecular mechanisms underlying the negative feedback loop between Wnt and <i>Notum</i> through epithelial-mesenchymal interactions, contributing to a deeper understanding of tooth development.