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Using in vitrotooth germ cultures and analysis by confocal microscopy, ameloblasts treated with sodium fluoride were found to have elevated amounts of filamentous actin. Because this response is reduced by inhibitors of the Rho/ROCK signaling pathway, we generated mice that express dominant negative RhoA (RhoADN) in ameloblasts for in vivo analysis. Expression of the EGFP-RhoADN fusion protein was evaluated by RT-PCR and immunohistochemistry, and teeth were analyzed by scanning electron microscopy. The 3 strains expressed at either low (TgEGFP-RhoADN-8), intermediate (TgEGFP-RhoADN-2), or high (TgEGFP-RhoADN-13) levels, and the molar teeth from the 3 strains had enamel hypoplasia and surface defects. We conclude that RhoADN expressed in ameloblasts interferes with normal enamel development through the pathway that is induced by sodium fluoride.

Osteocalcin was purified by gel chromatography from a crude extract obtained after decalcification of rat incisors. The apparent molecular weight, as determined by 5-15% SDS-polyacrylamide gel electrophoresis, was 18,000, and amino acid analysis revealed 60 γ-carboxyglutamic acid residues per 1000. Antisera against osteocalcin, raised in rabbits, reacted specifically with osteocalcin when investigated by immuno-electroblotting of dentin crude extract. 4-μm cryosections of formaldehyde-fixed tooth germs showed positive immunocytochemical staining for osteocalcin in dentin and odontoblasts. The staining of the mantle dentin at the coronal sides of the tooth germs was more intense than that of the adjacent circumpulpal dentin, while the odontoblasts involved in the formation of mantle dentin showed stronger immunoreactivity than did odontoblasts involved in circumpulpal dentin formation. This marked difference was not observed on the root sides of the tooth germs. In 1-μm cryosections, osteocalcin immunoreactivity was found evenly distributed throughout the entire cell body, with the exception of the Golgi region, which was less intensely stained, while the nucleus and the cell process were negative. The positive staining reaction with anti-osteocalcin antiserum was found in dentin from the very onset of its formation in the fetus. ln conclusion, our results demonstrate the presence of osteocalcin in odontoblasts and dentin. Its immunocytochemical localization may be compatible with a distinct role in early dentinogenesis.

The relationship of the keratocyst antigen (KCA), the soluble component present in most keratocyst fluids, and keratin, was studied with immunofluorescence microscopy comparing their distribution in developing mouse embryonic teeth and in human ameloblastomas. In these tissues both molecules showed a strong codistribution in epithelial cells. In the embryonic teeth both molecules were present in the stratum intermedium cells between the stellate reticulum cells and ameloblasts, but the secretory ends of the ameloblasts showed fluorescent staining only for keratin. The relationship was further investigated by comparing the physicochemical characteriatics of KCA and keratin. Results on immunoblotting and two-dimensional gel electrophoresis showed that KCA existed in keratocyst fluid as a 60–68,000 dalton polypeptide with an isoelectric point of pi 6.8. Immunoblotting analysis of various isolated keratins revealed a typical polypeptide pattern of each keratin when anti-KCA antiserum was used for staining. These findings suggest that KCA and keratin arc related molecules and that KCA may be a soluble component of keratin.