Figure 3

Alteration in cervical tongue pattern by the lateral growth and the growth inhibition. (a) Epithelium of bell stage (E16) tooth germ was 3-D reconstructed from serial sections and is viewed from apical side. Dashed lines indicate frontal section level of cusps and of cervical tongue, which were depicted in panel b and c, respectively. (b,c) The localization of Ki67 protein, a nuclear marker for cell proliferation, is observed in most of inner dental epithelial cells at the level of cusps (b) and of cervical tongue (c). The total number of cells and the number of Ki67-positive cells were counted in the 50 µm * 50 µm square in the center of dental papilla (B, D) and in the lateral side of dental papilla (A, C). (d) The Ki67 proliferation index shows the significantly higher value in lateral side of dental papilla at the level of cusp than other area. (e) Single tooth germ cultured in vitro for 2 days from E14. (f,g) Single tooth germ cultured in vitro for 4 days from E14. From occlusal view, concave axial outlines can be observed in between each intercuspal region (white arrowheads). From apical view, whole-mount E-cadherin immunostaining reveals development of five sharp cervical tongues in tooth germ cultured in vitro (black arrowheads). (h) Three cervical tongues are observed in vivo E17 tooth germ (black arrowheads) from apical view. (i,j) Three E14 tooth germs were placed side by side and cultured for 4 days to mimic the in vivo-like lateral growth inhibition. After 4 days, from occlusal view, tooth germs, particularly the central tooth germ, show flattened outline where pressure was applied. E-cadherin immunostaining of tooth germs showed short and blunt three cervical tongues in central tooth germ. No cervical tongue is observed in between the buccal and lingual anteroconids, though the mesial side of the central tooth germ does not contact with other tooth germs. The surface of buccal anteroconid is contact with surface of adjacent tooth germ (white dotted line). (k–n) An E14 tooth germ is placed in between two glass barriers from occlusal view and cultured for 4 days (k and l). After 4 days, tooth germ shows flattened outline where pressure was applied by glass barriers. E-cadherin immunostaining of tooth germs shows short and blunt three cervical tongues from apical view. From mesial view, the buccal and lingual sides of tooth germ are observed flat. Scale bars: b,c, 100 µm and e–n, 200 µm. P < 0.05. Lin: Lingual, Buc: Buccal, Ocl: Occlusal, Apc: Apical.