Osterix is Obligatory for Odontoblast-Ameloblast Maturation but Not Tooth-Morphogenesis

Runx2 and its downstream target Osterix are essential for skeletogenesis, and mutations of these genes in humans are associated with CCD and osteogenesis imperfecta. Tooth formation in Runx2-null mice arrests at the cap stage.  However, these mice completely lack alveolar bone, whose presence is considered a pre-requisite for tooth development. Osx-null model provide unique opportunity to reveal Osterix role in odontogenesis, and distinguish between contribution of alveolar bone and Runx2 in tooth-formation.  Objective:   Investigate function of Osterix during tooth development.  Methods: Odontogenesis in Osterix-null mice was evaluated by histological, biochemical, and molecular approaches.  Results: Osterix-null mice exhibited failed skeletogenesis and absence of alveolar bone.  Newborn heads were sectioned sagitally to evaluate tooth development.  Surprisingly, homozygous mutants showed normal tooth morphogenesis.  Incisors and multi-cusped 1st and 2nd molar were noted in WT and homozygous littermates.  These data demonstrate that alveolar bone is not a prerequisite for tooth morphogenesis.  Despite normal shape and number, Osx-null tooth-organs were significantly smaller in size.  BrdU-labeling assays at E18 revealed Osterix role in regulating cell proliferation.  We next followed differentiation of epithelial-ameloblasts and mesenchymal-odontoblasts.  Impaired matrix synthesis and collagen production was noted in Osx-null incisors.  WT incisors exhibited well-polarized, and fully mature odontoblasts/ameloblasts. However, Osx-null showed only cuboidal and aggregated cells in epithelial and mesenchymal portions of teeth. To better understand if this finding reflects a developmental arrest or a delayed differentiation, we compared expression of stage/cell-specific markers.   DSPP/DMP1 were undetectable in Osx-null odontoblast, and only a weak Amelogenin signal was noted in ameloblasts.  Analysis of WT and Osx-null incisor mRNA further confirmed lack of maturation, with reduced expression of key odontoblast/ameloblast markers by 4-8 fold.  Conclusions: We report for the first time that alveolar bone is not essential for tooth morphogenesis, and show Osterix is obligatory for odontoblast and ameloblast maturation, but not their commitment.