The tooth is a highly vascularized organ. During odontogenesis, blood vessels enter the forming tooth through the dental papilla and surround the dental epithelium from the cap stage. We show that during the late bell stage, endothelial cells invade the outer enamel epithelium (OEE) and migrate through the stellate reticulum to vascularize the forming ameloblast layer. This migration was evident in both mouse and human tooth germs and is likely to represent a conserved mechanism. Migration was coordinated by dynamic changes in Vegf expression in the dental epithelium, with expression at the OEE at the bell stage shifting toward the ameloblast layer. Invasion through the OEE involved loss of integrity of the basement membrane, downregulation of tight junctions, and apoptosis of some OEE cells. Changes in the OEE were dependent on Hedgehog signaling, with a failure to invade in K14creSmoothenedfl/fl mice, where epithelial cells cannot respond to Hedgehog signaling. The impact of failed migration through the OEE was followed in Cdh5creERT2 Vegfr2fl/fl mutant mice, where the endothelial cells cannot respond to vascular endothelial growth factor (VEGF). Failure of OEE invasion resulted in differentiation defects and extensive cell death of the ameloblast layer, highlighting the essential requirement for vascularization for development of this layer. Our results reveal the essential role of Hedgehog and VEGF signaling in correct vascularization of the tooth germ epithelial layers, allowing breakdown of the OEE and targeting endothelial cell migration into the epithelium. A better understanding of the molecular regulation of endothelial cells will help decipher how this cell population interacts with different cells of the enamel organ and will aid in attempts to revascularize teeth.

Centre for Craniofacial and Regenerative Biology Faculty of Dentistry Oral and Craniofacial Sciences King's College London London UK

Institute of Histology and Embryology 1st Faculty of Medicine Charles University Prague Czech Republic

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