Abstract

The functional regeneration of the dentin-pulp complex is pivotal for tooth preservation, yet the molecular mechanisms governing odontoblast differentiation remain poorly understood. In the current study, we revealed a distinct NKD1 + subpopulation exhibiting secretory odontoblast characteristics, which was specifically induced in dental pulp stem cells (DPSCs) by Wnt3a, but not by Wnt5a or Wnt10a through single-cell transcriptomic profiling. We then found that the NKD1 + subpopulation was functional conservation, which were consistently identified in the odontoblast layers of developing tooth germs in both murine and miniature pig models, as well as within the apical open area in human molars. This conserved spatial distribution and co-localization with DSPP strongly indicates that NKD1 + cells were active dentin-secreting odontoblasts. Analysis of gene regulatory networks using SCENIC identified MSX1 as a key transcription factor regulating the specification of NKD1 + lineage. Mechanistically, Wnt3a orchestrates a tripartite cascade: upregulating NKD1/MSX1 expression, triggering NKD1 membrane detachment, and facilitating direct NKD1-MSX1 interaction to promote MSX1 nuclear translocation. CUT&Tag analysis demonstrated MSX1 occupancy at promoters of odontogenic regulators, establishing its necessity for odontogenic gene activation. Murine pulp exposure models validated that Wnt3a-activated NKD1-MSX1 signaling significantly enhances reparative dentin formation. This study delineates an evolutionarily conserved Wnt3a-NKD1-MSX1 axis that resolves stem cell heterogeneity into functional odontoblast commitment, providing both mechanistic insights into dentin-pulp regeneration and a foundation for targeted regenerative therapies.