Depletion of macrophages during early postnatal development leads to disrupted tooth root development and altered Gli1⁺ MSC trajectory

Mammalian tooth development progresses through two principal stages-crown formation and root development-orchestrated by intricate interactions between the oral epithelium and neural crest-derived mesenchyme. After crown formation, Hertwig's epithelial root sheath (HERS) directs root development. In this phase, Gli1⁺ mesenchymal stem cells (MSCs) give rise to dental pulp, dentin, cementum, and the periodontal ligament (PDL). The root anchors the tooth to the alveolar bone via PDL fibers, forming a dynamic occlusal buffer that mediates mechanosensation and nutrient supply. Although previous work has shown that macrophages are abundant in the dental pulp and follicle, the functional importance of macrophages in tooth development has not been well characterized. Here, we investigated the spatiotemporal dynamics of macrophage populations (identified by CD68, F4/80, CD206, and other markers) in molars and surrounding tissues during postnatal root development in mice. Importantly, Macrophage depletion via clodronate liposomes resulted in shortened root, impaired PDL elongation and retarded alveolar bone shooting surrounding the root. Gli1⁺ MSCs exhibited increased proliferation but impaired osteo/odontogenic differentiation upon macrophage depletion. Single-cell RNA sequencing and in vitro co-culture experiments support a model in which macrophage-derived TGF-β acts on mesenchymal TGF-β receptors to direct MSC fate and thereby regulate root morphogenesis. Collectively, these findings establish macrophages as critical niche components that orchestrate tooth root development through immune-mesenchymal crosstalk.