Abstract
Tooth germ development is a precisely orchestrated process dependent on integrated cellular interactions and molecular signals, yet its regulatory mechanisms remain incompletely defined. Here, we constructed a high-resolution cellular atlas of miniature pig tooth germs using 10× single-cell RNA sequencing to investigate the molecular mechanisms underlying tooth mineralization. By leveraging cellular heterogeneity and dynamic gene expression trajectories in epithelial and mesenchymal populations, we identified microfibril-associated protein 5 (MFAP5) as a previously unrecognized regulator of the odontogenic program. Functional assays demonstrate that MFAP5, an extracellular matrix component, is indispensable for mesenchymal differentiation and matrix mineralization in vitro. Mechanistically, MFAP5 engages Integrin alpha-5 (ITGA5) to activate Extracellular Signal-Regulated Kinase/Mitogen-Activated Protein Kinase (ERK/MAPK) signaling in odontoblast-lineage cells, thereby promoting odontoblast differentiation and dentin deposition. Collectively, our single-cell-resolved analyses uncovered a MFAP5-ITGA5-ERK/MAPK signaling axis that operates in a cell-state-specific manner during tooth germ mineralization, providing new mechanistic insights into odontogenic differentiation and a potential molecular basis for dental tissue regeneration strategies.