Fig 2: Phenotypes of maxillary incisors in the F2 generation of 3-month-old Cebpb-/-Runx2+/- mice. All sections were decalcified. (A–G) Correspond to the same mouse, H corresponds to another animal, and (I–K) correspond to the other animal. (A) Three-dimensional image of the skull. Scale bar: 10 mm. (B) Micro-computed tomography (CT) axial image at the level of the maxillary incisor. (C) Sagittal hematoxylin-eosin (H&E)-stained section of the upper incisor. A box E indicates epithelial pearls. An arrowhead indicates a developing tooth. Developing teeth are characterized by odontoblasts, dentin, ameloblasts derangement, and surface roughness of the enamel matrix (hypocalcification site). Scale bar: 100 µm, 100×. (D) Amelogenin immunostaining (red) and Hoechst nuclear staining (blue). An arrowhead indicates epithelial pearls. Scale bar: 100 µm, 100×. (E) High-magnification image of epithelial pearls. Scale bar: 100 µm, 400×. (F) High-magnification image of epithelial pearls. Scale bar: 50 µm, 1000×. (G) Ameloblasts derangement and surface roughness of the enamel matrix are visible in an opposite incisor. Scale bar: 100 µm, 400×. (H) Two mature teeth and a developing tooth are visible. Scale bar: 1 mm, 40×. Inset: Higher magnification image of a developing tooth showing odontoblasts, ameloblasts, and dentin 400×. (I) Developing and mature teeth are visible. Scale bar: 1 mm, 40×. (J) A multipartite mature tooth (arrowhead) is observed in the dental pulp. Scale bar: 100 µm, 100×. (K) Many developing teeth (arrowheads) are visible near an extra mature tooth. Scale bar: 100 µm, 200×.

Fig 3: Decorin immunostaining in the incisors of 3-month-old Cebpb-/-Runx2+/- mice (F2 generation). (A,C) Wild-type (WT)/WT mice Scale bar: 1 mm, 40×. (B,D,E) Cebpb-/-Runx2+/- mice. (B) Positive staining of cells in the dental pulp, except for cementum-like hard tissue, and the extracellular matrix. Scale bar: 1 mm, 40×. (D) Positive staining in the enamel epithelial pearl and extracellular matrix near the supernumerary tooth in the periapical region. Scale bar: 100 µm, 400×. (E) Positive staining in the extracellular matrix surrounding the cementum-like hard tissue in the dental pulp. Scale bar: 100 µm, 400×.

Fig 4: Schematic representation of the role played by Cebpb and Runx2 in odontogenic epithelial stem cells (OESCs). Cebpb acts upstream of SRY (sex determining region Y)-box 2 (Sox2). In cooperation with Oct 3/4 protein (Pou5f1), Sox2 inhibits genes related to differentiation and maintains stemness in OESCs. Cebpb and Runx2 prevent epithelial-mesenchymal transition (EMT) of OESCs via snail family zinc finger 2 (Snai2). Both also inhibit biglycan (Bgn), decorin (Dcn) and Bmp4. EMT promotes expression of Sox2 and thus stemness of OESCs. EMT enables disengaged OESCs to develop supernumerary teeth by interaction with various mesenchymal stem cells such as dental MSCs.

Fig 5: Analysis of differentiated ameloblasts in maxillary incisors of 3-month-old mice (F2 generation). All sections were decalcified. These insets in (A–C,E) are 1000×. (F–H) N-cadherin immunostaining. All scale bars: 200 µm, 400×. (A) Highly polarized secretory ameloblasts are visible in wild-type mice. (B) Differentiated ameloblasts are seen to have lost their cell polarity in Cebpb+/+Runx2+/- mice. (C) Non-polarized differentiated ameloblasts and surface roughness of the enamel matrix are visible in Cebpb-/-Runx2+/+ mice. (D) Amelogenin immunostaining (red) and Hoechst nuclear staining (blue). Free blobs containing amelogenin are observed in Cebpb-/-Runx2+/+ mice. (E) Derangement of ameloblasts and surface roughness of the enamel matrix. in Cebpb-/- Runx2+/- mice. (F) Positive staining in ameloblasts containing non-polarized differentiated ameloblasts and labial cervical loop epithelium in Cebpb+/+Runx2+/- mice incisor. Scale bar: 500µm, 100×. The inset: High-magnification image of non-polarized differentiated ameloblasts. 1000×. (G) Ectopic enamel formation and cementum-like hard tissue within the dental pulp in the periapical region of Cebpb-/- Runx2+/- mice incisor. Scale bar: 2 mm, 40×. (H) Positive staining of the cell component surrounding ectopic enamel formation and cementum-like hard tissue are visible. Scale bar: 200 µm, 400×. The inset: High-magnification image of the cementum-like hard tissue 1000×.