Rat Incisor Ameloblasts Research Articles

Localization of Core Planar Cell Polarity Proteins, PRICKLEs, in Ameloblasts of Rat Incisors: Possible Regulation of Enamel Rod Decussation.

To confirm the possible involvement of planar cell polarity proteins in odontogenesis, one group of core proteins, PRICKLE1, PRICKLE2, PRICKLE3, and PRICKLE4, was examined in enamel epithelial cells and ameloblasts by immunofluorescence microscopy. PRICKLE1 and PRICKLE2 showed similar localization in the proliferation and secretory zones of the incisor. Immunoreactive dots and short rods in ameloblasts and stratum intermedium cells were evident in the proliferation to differentiation zone, but in the secretion zone, cytoplasmic dots decreased and the distal terminal web was positive for PRICKLE1 and PRICKLE2. PRICKLE3 and PRICKLE4 showed cytoplasmic labeling in ameloblasts and other enamel epithelial cells. Double labeling of PRICKLE2 with VANGL1, which is another planar cell polarity protein, showed partial co-localization. To examine the transport route of PRICKLE proteins, PRICKLE1 localization was examined after injection of a microtubule-disrupting reagent, colchicine, and was compared with CX43, which is a membrane protein transported as vesicles via microtubules. The results confirmed the retention of immunoreactive dots for PRICKLE1 in the cytoplasm of secretory ameloblasts of colchicine-injected animals, but fewer dots were observed in control animals. These results suggest that PRICKLE1 and PRICKLE2 are transported as vesicles to the junctional area, and are involved in pattern formation of distal junctional complexes and terminal webs of ameloblasts, further implying a role in the formed enamel rod arrangement.

Planar Cell Polarity Protein Localization in the Secretory Ameloblasts of Rat Incisors

The localization of the planar cell polarity proteins Vang12, frizzled-3, Vang11, and Celsr1 in the rat incisors was examined using immunocytochemistry. The results showed that Vang12 was localized at two regions of the Tomes' processes of inner enamel-secretory ameloblasts in rat incisors: a proximal and a distal region. In contrast, frizzled-3 was localized at adherens junctions of the proximal and distal areas of inner enamel- and outer enamel-secretory ameloblasts, where N-cadherin and β-catenin were localized. frizzled-3 was also localized in differentiating inner enamel epithelial cells. Vang11 was localized sparsely in differentiating preameloblasts and extensively at the cell boundary of stratum intermedium. Celsr1 was not localized in ameloblasts but localized in odontoblasts extensively. These results suggest the involvement of planar cell polarity proteins in odontogenesis.

Immunocytochemical Localization of Claudin-1 in the Maturation Ameloblasts of Rat Incisors

Claudin-1 is a tight junction transmembrane protein. Its localization in the maturation ameloblasts of rat incisors was examined by immunofluorescence microscopy. Distal junction area of ruffle-ended ameloblasts (RA) and the Golgi apparatus of a sub-population of smooth-ended ameloblasts (SA) and RAs stained positive with anti-claudin-1 antibodies. Since it has been shown that ameloblasts repeatedly alternate between an SA and an RA morphology during enamel maturation, the presence of claudin-1 in the Golgi cisterns may indicate the presence of tight junction precursors before transportation to the junctional area.

Reuptake of Extracellular Amelogenin by Dental Epithelial Cells Results in Increased Levels of Amelogenin mRNA through Enhanced mRNA Stabilization

Amelogenin is an extracellular matrix protein secreted by ameloblasts and is a major component of enamel matrix. Recently, in addition to their role in enamel formation, the biological activity of enamel proteins in the process of cell differentiation has recently become widely appreciated. In this study, we examined the biological activity of amelogenin on ameloblast differentiation. Recombinant mouse amelogenin (rm-amelogenin) enhanced the expression of endogenous amelogenin mRNA in a cultured dental epithelial cell line (HAT-7), despite a lack of increased amelogenin promoter activity. To solve this discrepancy, we analyzed the effects of rm-amelogenin on the stability of amelogenin mRNA. The half-life of amelogenin mRNA is extremely short, but in the presence of rm-amelogenin its half-life was extended three times longer than the control. Furthermore, we showed the entry of exogenous fluorescein isothiocyanate-conjugated rm-amelogenin into the cytoplasm of HAT-7 cells. It follows from our results that exogenous amelogenin increases amelogenin mRNA levels through stabilization of mRNA in the cytoplasm of HAT-7 cells. Here we speculated that during differentiation, dental epithelial cells utilize a unique mechanism for increasing the production of amelogenin, the reuptake of secreted amelogenin.

Transient increase in anti-p-ATF2 immunoreactivity in the late secretion ameloblasts apical to the transition zone of rat incisors.

Activating transcription factor 2 (ATF2) was localized in the ameloblasts of rat incisors by immunohistochemistry. A specific antibody against phosphorylated ATF2 (p-ATF2), which is an activated form of ATF2, was detected from the proliferation zone to maturation ameloblasts just after the transition. In the secretion zone, a transient increase in p-ATF2 was observed in the late secretion ameloblast nuclei, where a stronger reactivity of p-ATF2 extended from 1 mm apical to the transition to the transition zone, whereas ameloblast nuclei in most of the maturation zone exhibited either weak or no reactivity. A similar pattern was also observed in the case of c-Jun immunohistochemistry, except for in most of the maturation zone, where strong c-Jun reactivity was detected. Thus, ATF-2 and c-Jun are deeply involved in amelogenesis and, in particular, ATF2 is related to the proliferation, differentiation, secretion and transition zones.

Immunohistochemical Localization of SERCA2 in the Ameloblasts of Rat Incisors

Sarcoplasmic or endoplasmic reticulum Ca2+ ATPase (SERCA2) was localized in the ameloblasts of rat incisors by immunohistochemistry. Secretion ameloblasts and maturation ameloblasts, including both ruffle-ended and smooth-ended cells, were labeled with anti-SERCA2 antibodies, with maturation ameloblasts showing stronger binding than secretion ameloblasts. The pattern of labeling was cytoplasmic and diffuse. Papillary layer cells were not labeled with anti-SERCA2 antibodies. These results suggest that the SERCA2 in ameloblasts is intimately involved in mineralization during amelogenesis.

Colchicine-induced apoptosis and anti-Fas localization in rat-incisor ameloblasts.

Apoptosis of ameloblasts were examined by the TdT-mediated dUTP-biotin nick end-labelling method and electron microscopy 8 h after injection of colchicine. The results showed that extensive apoptosis occurred in ameloblasts of secretion to maturation zones. To determine the possible involvement of stimulators in ameloblast apoptosis, Fas, Fas ligand, tumor-necrosis-factor alpha, and tumor-necrosis-factor receptor 1 were examined utilizing immunohistochemistry and Western blotting analysis. Only Fas was consistently detected in the secretion, transition and maturation ameloblasts and overlying enamel organ epithelia. These results suggest that ameloblasts could undergo apoptosis by colchicine and that one of the ameloblast apoptosis mediators would be the Fas receptor.

Localization of Transcription Factor AP-1 Family Proteins in Ameloblast Nuclei of the Rat Incisor

We examined by immunocytochemistry the localization of the AP-1 family proteins c-Jun, JunB, JunD, c-Fos, FosB, Fra-1, and Fra-2 in rat incisor ameloblasts. Most of the antibodies against AP-1 family proteins, except for c-Fos-specific antibody, labeled ameloblast nuclei. The labeling intensity of the c-Jun, JunD, and Fra-2 antibodies was stronger than that of JunB, FosB, and Fra-1. Antibody reactivities of c-Jun, JunD, and Fra-2 were greatly enhanced during or after the transition zone. Furthermore, c-Jun antibodies labeled maturation ameloblasts in a cyclic pattern, which was correlated with ameloblast modulation. Disruption of ameloblast modulation by colchicine injection resulted in greatly decreased reactivity of the c-Jun antibody in the ameloblast nuclei of the maturation zone. Phospho-specific antibodies to c-Jun labeled ameloblast nuclei only weakly throughout the secretion, transition, and maturation zones. These results suggest that the stage-specific localization of AP-1 in ameloblasts is closely related to tooth enamel formation.

Internalization of amelogenin by dendritic cells of the papillary layer during transition and early maturation stages

The existence of amelogenin in the papillary layer facing transition and early maturation zones of ameloblasts of rat incisors and the role of dendritic cells were examined by light microscopic immunocytochemistry and immunoelectron microscopy using anti-porcine 25 kDa amelogenin antibodies and anti-MHC class II antibodies. At the light microscopic level, no overall relationship was observed between anti-class II-positive dendritic cells and anti-amelogenin-positive materials located intercellularly: anti-amelogenin-positive dots were scattered in papillary layers and ameloblasts. Anti-MHC class II-positive cells were present in the papillary layer at the transition and maturation stages. Under electron microscopy, however, the dendritic cells occasionally endocytosed anti-amelogenin-positive materials and formed small vesicles. The results suggest that the dendritic cells play a role in eliminating amelogenin from the enamel organ.

Expression and localization of ferritin mRNA in ameloblasts of rat incisor

At the maturation stage, the ameloblasts of the rat incisor incorporate iron, supplied through the bloodstream, and deposit it into the surface layer of the enamel. In this unique iron transport system, ferritin functions as a transient iron reservoir in the cells. Here the expression of ferritin mRNA and its localization in the rat enamel organ was examined. Among various tissues, the enamel organ showed the highest expression for both ferritin H- and L-chain mRNA, as quantified by reverse transcription–polymerase chain reaction. In situ hybridization using digoxigenin-labelled cRNA probes for each chain demonstrated that both ferritin H- and L-chain mRNA were abundantly expressed in presecretory and secretory ameloblasts. The intensity of the positive hybridization signal gradually decreased toward the incisal direction. Differing from the mRNA localization, ferritin protein was immunologically undetectable in presecretory or secretory ameloblasts but was found in ameloblasts at the maturation stage, into which iron is known to be incorporated from the bloodstream. Thus, the expression of ferritin mRNA precedes the protein expression in the developmental stages of rat incisor ameloblasts, and the translation of ferritin and its half-life are probably controlled by the iron entry, as has been reported for other cell types.

Endocytotic functions of ameloblasts and odontoblasts: immunocytochemical and tracer studies on the uptake of plasma proteins.

Biochemical, (immuno)cytochemical, and radioautographic data accumulated over several years have lead to the view that ameloblasts carry out both secretory and degradative functions throughout amelogenesis. Whereas it has been assumed that maturation stage ameloblasts endocytose aged enamel proteins from the enamel layer, the origin of the newly formed ones detected in the endosomal/lysosomal compartment of ameloblasts from all stages remains to be elucidated. One possible source is from secretory products released ectopically along basolateral surfaces. To test this hypothesis, we have investigated, using colloidal gold immunocytochemistry, whether plasma proteins (albumin and alpha 2HS-glycoprotein) found in the interstitial fluid are endocytosed by rat incisor ameloblasts and other cells from hard and soft tissues. Rat albumin, tagged with dinitrophenol, was injected intravenously to trace the movement of this protein. Plasma proteins were immunodetected along the baso-lateral surfaces and in multivesicular bodies of ameloblasts where enamel proteins were also found. By 2 hours following intravenous administration of dinitrophenylated albumin, the tracer had left the blood and diffused into the enamel organ and between odontoblasts and osteoblasts. The tracer was also found in multivesicular bodies of all cells examined. The uptake of albumin by many different cell types suggests that this process is not restricted to ameloblasts and likely occurs in a nonselective manner. Hence, baso-lateral uptake in ameloblasts may play a role not only in the continuous removal of plasma proteins leaking from the blood, but also of enamel proteins 'dumped' laterally between these cells. Likewise, odontoblasts may use the same mechanism to internalize some of the plasma proteins and any enamel protein that diffuse toward them.

Amelogenin Gene Expression in the Ameloblasts of Rat Incisors suffering from Experimental Enamel Hypoplasia

Amelogenin Gene Expression in the Ameloblasts of Rat Incisors suffering from Experimental Enamel Hypoplasia

Alternative splicing of amelogenin mRNA from rat incisor ameloblasts.

Amelogenin proteins are a major component of the developing enamel matrix, and are likely to have a key role in the control of enamel biomineralization. The heterogeneity of amelogenin is in part due to alternative splicing of the amelogenin RNA transcripts. Several patterns of alternative splicing have been described in mouse, bovine, porcine, and human enamel, with all alternatively spliced products having homologous 5' and 3' sequences within the coding regions. In these studies, we have used anchored PCR to identify alternatively spliced amelogenin cDNA sequences in the rat. We found amelogenin cDNAs that could be divided into two groups based on their 3' sequence. Group 1 cDNAs had a novel terminal sequence that has not been previously identified, while group 2 cDNAs were similar to those previously identified in other animal species. We identified a sequence, identical to the novel 3' amelogenin cDNA sequence, in rat genomic DNA downstream from the previously identified exon 7. The putative amelogenin proteins predicted for the two groups differed in their predicted isoelectric points, with the novel group 1 proteins having a more basic isoelectric point.

Gene expression of epimorphin in rat incisor ameloblasts

Epimorphin has been recently identified as an important factor in the morphogenesis of epithelial cells. A cDNA encoding epimorphin from skin of newborn mice was cloned by the polymerase chain-reaction technique before the preparation of digoxigenin-labelled cRNA probes. In situ hybridization of longitudinal sections of rat incisors revealed a distinct pattern of expression of epimorphin mRNA in the ameloblast layer. Epimorphin mRNA was detected from the presecretory stage up to the beginning of the maturation stage of amelogenesis. With the identification of this expression by epithelial-derived cells, i.e. ameloblasts, it is thought likely that epimorphin is one of the factors that modulate the differentiation cascade of ameloblasts in the course of amelogenesis.

Correlated alkaline phosphatase histochemistry and quantitative backscattered electron imaging in the study of rat incisor ameloblasts and enamel mineralization

The different functional conditions of pre-ameloblasts, secretory ameloblasts, and maturation ameloblasts in 9 day rat incisors were recognized using high resolution light microscopic alkaline phosphatase histochemistry: Digital backscattered electron imaging was performed using the block surfaces from which thin sections were taken for histochemical study. It was possible to correlate exact locations in histochemical sections with positions in the block face at all stages of enamel mineralization from early secretion through late maturation. The first steep increase in the rate of mineralization of completed enamel matrix occurs after the first transition from smooth ended ameloblasts to ruffle ended ameloblasts. In the 9 day rat incisors used for this purpose, there are only two smooth to ruffle cyclical transitions, and the width of successional smooth ended bands of ameloblasts in the maturation cycling process is always narrow. Nevertheless, there seems to be a good correlation between mineralization increase and the acquisition of the high alkaline phosphatase activity in the deeply enfolded distal cytoplasm of the ruffle-ended maturation stage ameloblasts.

A method for light microscopy examination of cellular and structural interrelations in undemineralized tooth specimens.

This study examines ameloblasts and odontoblasts in relation to mineralizing enamel and dentin in thin undemineralized tooth sections from unerupted and partly erupted human third molars. The material comprised 12 freshly removed third molars. After fixation for 24 h the specimens were dehydrated and embedded in hydroxyethylmethacrylate. Undemineralized sections 10-15 microns thick were prepared with a cutting-grinding system, and radiomicrographs were taken. Postsecretory ameloblasts were seen in different morphologic stages ranging from ruffle- and smooth-ended ameloblasts to cuboidal and squamous cells adhering to incomplete and fully mineralized enamel. The former occurred in the most coronal tooth part, whereas the latter were seen cervically. It was not possible to identify systematic modulation cycles as seen in postsecretory rat incisor ameloblasts. The well-defined subodontoblastic region with cell-free and cell-rich layers was established before tooth eruption. The function of the subodontoblastic region is not fully understood, but our findings suggest that the establishment of the region plays a role in maintaining the specific odontoblast phenotype in the coronal region possibly linked to progressive dentin mineralization.

Differential expression of calbindin‐D 28 kDa in rat incisor ameloblasts throughout enamel development

Calbindin-D 28 kDa (CaBP 28 kDa), a vitamin D-dependent calcium-binding protein, has been associated with calcium handling by cells. We have investigated the expression of this protein in the rat incisor enamel organ, an epithelium interposed between a mineralizing matrix and connective tissue rich in blood vessels, by radioimmunoassay (RIA), Western blotting, and quantitative protein A-gold immunocytochemistry with antibodies to rat kidney CaBP 28 kDa. RIA of cytosolic extracts showed that enamel organs contained relatively high concentrations of CaBP 28 kDa (compared to kidney; see review by Christakos S., C. Gabrielides, and W.B. Rhoten 1989 Endocr. Rev., 10:3-25). Immunoblotting of proteins extracted from enamel organ strips revealed an intensely-stained band near 28 kDa throughout amelogenesis following ameloblast differentiation. Immunocytochemically, CaBP 28 kDa was localized exclusively within ameloblasts. The density of labelling increased from the presecretory stage to the secretory stage and fluctuated across the maturation stage in relation to ameloblast modulation. Ruffle-ended ameloblasts consistently showed the most intense immunoreaction. Gold particles were present throughout the cytoplasm and nuclei of ameloblasts but regions rich in rough endoplasmic reticulum or cell webs showed a higher immunolabelling. Some gold particles were also associated with the external face of the rough endoplasmic reticulum. Multivesicular bodies in maturation stage ameloblasts were occasionally immunoreactive. These data suggest that the intracellular concentration of CaBP 28 kDa is regulated throughout amelogenesis reflecting a stage-specific control of calcium homeostasis in ameloblasts.

Histochemical, ultrastructural, and electron microprobe analytical studies on the localization of calcium in rat incisor ameloblasts at early stage amelogenesis

The enamel organs of rat incisors were separated from the enamel surface and processed for rapid freezing and freeze-substitution. A histochemical stain for calcium (GBHA) of thick Epon sections revealed intense calcium reactions in the secretory ameloblasts, exclusively in the tubulovesicular structures extending throughout their distal cytoplasm. Electron microscopy revealed a thin layer of amorphous material with clusters of electron-dense granules along the distal surface of secretory ameloblasts. In young secretory ameloblasts without typical Tomes' processes, a considerable number of mitochondria were located in the distal cytoplasm and contained numerous electron-dense granules. Similar dense granules as well as fine ribbon-like electron-dense figures, all containing significant amounts of calcium, were observed in some of the tubulovesicular structures at the distal end of these cells. A putative exocytotic figure of such dense granules was also observed. The electron-dense granules were rare in more differentiated ameloblasts with elongated Tomes' processes, which occasionally displayed ribbon-like figures in some of the tubulovesicular structures in the process region. No significant calcium peak was detected in the extracellular amorphous material, secretory granules, or along the lateral plasma membranes. These observations may imply high calcium concentrations in mitochondria and tubulovesicular structures in the distal cytoplasm of secretory ameloblasts relative to that of the cytosol and support the possible contribution of these organelles in secretory ameloblasts to cellular calcium regulation at least in the early stage of amelogenesis.

Immunocytochemical Localization of Amelogenin in Rat Incisor Ameloblasts Using Ultrathin Frozen Sections

The localization of amelogenin, an enamel matrix protein, was examined by ultrastructural immunocytochemistry using unembedded ultrathin frozen sections of undecalcified rat incisor ameloblasts. Antibody against bovine amelogenin labeled Golgi complexes, secretory granules, and lysosomal structures in the preameloblasts and inner enamel-secretory ameloblasts as well as the enamel. The antibody also labeled dentin matrix facing preameloblasts. These results support the findings in previous reports using conventional epon embedded specimens. However, rough-surfaced endoplasmic reticulum failed to be labeled by this antibody.

Cytochemical localization of calcium and Ca2+,Mg2(+)-adenosine triphosphatase in colchicine-altered rat incisor ameloblasts.

Colchicine is known to affect secretory, transport, and degradative functions of ameloblasts. The effects of colchicine on membrane-associated calcium and Ca2+,Mg2(+)-ATPase in secretory and maturation ameloblasts were investigated cytochemically. The pyroantimonate (PPA) method was used for localizing calcium and a modified Wachstein-Meisel medium was used to localize Ca2+,Mg2(+)-ATPase. Sections representing secretory and early maturation stages were examined by transmission electron microscopy. Morphological changes induced by colchicine included dislocated organelles and other well-established reactions to such anti-microtubule drugs. Calcium pyroantimonate (Ca-PA) deposits in most ameloblast types were markedly reduced, with the greater reduction occurring in those cells more severely altered morphologically. However, the cell membranes of both control and experimental smooth-ended maturation ameloblasts were essentially devoid of Ca-PA. The normal distribution and intensity of Ca2+,Mg2(+)-ATPase was not affected by colchicine. Because the observed reduction of membrane-associated calcium is apparently not mediated by Ca2+,Mg2(+)-ATPase in this case, other aspects of the calcium regulating system of ameloblasts are apparently targeted by colchicine.