Macrophages Promote Vascular Maturation to Support Tooth Germ Morphogenesis

Macrophages are increasingly recognized as key regulators of organogenesis, yet their contributions to tooth germ development remain poorly understood. Here, we integrated analysis of publicly available single-cell RNA sequencing datasets with multiplex immunohistochemistry to investigate the presence, distribution, and activation states of macrophages during tooth development. Macrophages were enriched in the dental follicle and dental papilla, expanded through local proliferation, and displayed a predominant tissue-resident macrophage (TRM)-like and M2-like phenotype. Spatial and transcriptomic analyses further revealed that macrophages preferentially localized adjacent to developing blood vessels and established a localized provascular niche enriched for vascular regulatory signaling pathways. Functional depletion of macrophages, both in vivo using Cx3cr1-dependent DTA mice and ex vivo with clodronate liposomes, impaired tooth germ growth and morphogenetic progression, accompanied by reduced tooth germ size, abnormal cusp morphology, increased apoptosis, and impaired vascular maturation. Notably, macrophage depletion reduced vascular maturation without markedly affecting overall vascular density. Given the well-established requirement for vascular maturation in tooth development and the close spatial association between macrophages and developing vessels, we examined whether macrophages contribute to the immune-vascular interactions required for morphogenesis. Macrophages expressed multiple vascular regulatory factors, including VEGFA, TGF-β, and IL1B, while inhibition of vascular maturation phenocopied the developmental abnormalities caused by macrophage depletion and was associated with attenuated bone morphogenetic protein-associated signaling. Together, these findings identify macrophages as important upstream regulators of tooth germ morphogenesis, acting in part by modulating vascular maturation, and provide new insights into immune-vascular crosstalk during tooth development.