Fine structure of the secretory and non-secretory ameloblasts in the frog. II. Fine structure of the non-secretory ameloblast.

The non-secretory ameloblasts present at the enamel-free surfaces of maxillary teeth in the frog Rana pipiens were examined by electron microscopy at different stages of tooth development. Their main fine structural features seem to reflect a transport function. During early tooth development, the non-secretory ameloblasts adjacent to odontoblasts and predentin exhibit extensive lateral surface specializations and numerous cytoplasmic vesicles. During late tooth development, the non-secretory ameloblasts adjacent to mineralizing dentin show numerous cellular junctions, well-developed intercellular channels with numerous interdigitating processes and labyrinthine configurations at their distal surfaces. An intact basal lamina is present between the non-secretory ameloblasts and the dentin surface until the dentin becomes fully mineralized. At this stage the adjacent cells no longer exhibit surface specializations. It is suggested that the non-secretory ameloblasts may participate in the mineralization of adjacent dentin at the enamel-free surfaces. This surface dentin becomes fully mineralized at a later stage of development than the underlying dentin.     

Fine structural changes and lysosomal phosphatase cytochemistry of ameloblasts associated with the transitional stage of enamel formation in the rat incisor.

Trimetaphosphatase (TMPase) and cytidine-5'-monophosphatase (CMPase) were used as lysosomal markers in the transitional ameloblasts (TA) to investigate the distribution of lysosomal structures and to correlate the cytochemical findings with the ultrastructural features of these cells. Of particular interest were the cytochemical and morphological changes which occur as the ameloblasts approach the maturation stage of enamel formation. The sequence of changes observed provides a basis for designation of three regions of the transitional zone (early and late TA and modulating ameloblasts). In the early TA region, the cells decreased in height and contained phagic vacuoles as well as numerous TMPase and CMPase reactive structures. Late transitional ameloblasts had invaginations at their distal ends as well as membrane-bound structures, both filled with fine granular material. Dense bodies, phagic vacuoles, and other elements of the lysosomal system were enzyme reactive. Modulating ameloblasts lacked the phagic vacuoles but exhibited large numbers of multivesicular bodies, vesicles, and secretory granules. Their distal ends were morphologically altered indicating a change towards ruffle- or smooth-ended varieties of maturation ameloblast. In the former, increased granular material was observed within cell membrane invaginations and associated membrane-bound structures. In the latter, intercellular spaces widened and were filled with granular material. The present cytochemical findings of an extensive lyosomal system in transitional ameloblasts confirm the function of those cells in reducing the secretory ameloblast population and in the selective elimination of their protein-synthesizing organelles. Furthermore, this extensive lysosmal system and the present morphological findings are consistent with a potential role for transitional ameloblasts in contributing to the marked loss of enamel protein known to occur during maturation.     

Ultrastructural morphometric analysis of ameloblasts exposed to fluoride during tooth development

Since a considerable amount of the world population is exposed to high doses of fluoride, it is of special concern to investigate its action mechanisms during dental enamel development. In this study, the toxicity of fluoride in ameloblasts during enamel development was evaluated by means of ultrastructural morphometric analysis. A total of 18 male Wistar rats were distributed into three groups. In Group I, the animals received deionized drinking water ad libitum (negative control) and in Groups II and III, they received sodium fluorided (NaF) drinking water at doses of 7 and 100 ppm ad libitum, respectively, for 6 weeks. Morphometric data were expressed as volume density of the most significant organelles present in the secretory and maturation phases of amelogenesis such as RER, granules, lysosomes, phagic vacuoles, microfilaments and mitochondria. The results showed that the volume density of mitochondria in the 100 ppm experimental group was 29% (P < 0.05) higher than the control group in secretory ameloblasts. No remarkable differences were found in maturation ameloblasts for all organelles evaluated. Taken together, these data indicate that NaF at high doses is able to induce cellular damage in secretory ameloblasts, whereas no noxious effect was observed during maturation stage of amelogenesis as depicted by ultrastructural analysis.     

Cytochemical calcium distribution in secretory ameloblasts of the rat in relation to enamel mineralization

Calcium distribution in secretory ameloblasts was studied in rat incisor enamel in which mineralization was temporarily disturbed by injection of either fluoride or cobalt. Pyroantimonate precipitates of calcium were analysed morphometrically in regions of the cell membranes, mitochondria and secretory granules. The disturbances in mineralization were characterized by accumulations of unmineralized enamel matrix at the secretory regions of Tomes' process within 1 h after injection. Fluoride-induced disturbances in mineralization were not accompanied by marked changes in calcium concentration and distribution. It may be that fluoride causes alterations in the synthesis and secretion of the organic matrix which affects its ability to mineralize. Secretory ameloblasts treated with cobalt showed a broad basis for interference with calcium, in particular that which is associated with cell membranes and secretory granules. Secretory ameloblasts may be actively controlling the availability of calcium to enamel by mechanisms involving the cell membrane as well as the secretory granules.     

Ameloblastin is a cell adhesion molecule required for maintaining the differentiation state of ameloblasts

Tooth morphogenesis results from reciprocal interactions between oral epithelium and ectomesenchyme culminating in the formation of mineralized tissues, enamel, and dentin. During this process, epithelial cells differentiate into enamel-secreting ameloblasts. Ameloblastin, an enamel matrix protein, is expressed by differentiating ameloblasts. Here, we report the creation of ameloblastin-null mice, which developed severe enamel hypoplasia. In mutant tooth, the dental epithelium differentiated into enamel-secreting ameloblasts, but the cells were detached from the matrix and subsequently lost cell polarity, resumed proliferation, and formed multicell layers. Expression of Msx2, p27, and p75 were deregulated in mutant ameloblasts, the phenotypes of which were reversed to undifferentiated epithelium. We found that recombinant ameloblastin adhered specifically to ameloblasts and inhibited cell proliferation. The mutant mice developed an odontogenic tumor of dental epithelium origin. Thus, ameloblastin is a cell adhesion molecule essential for amelogenesis, and it plays a role in maintaining the differentiation state of secretory stage ameloblasts by binding to ameloblasts and inhibiting proliferation.     

Enamel protein biosynthesis and secretion in mouse incisor secretory ameloblasts as revealed by high-resolution immunocytochemistry

Mouse secretory ameloblasts express a number of enamel proteins, which have been divided into amelogenin and enamelin subfamilies. We have used polyclonal antibodies to murine amelogenins to reveal enamel proteins in mouse ameloblasts using the protein A-gold immunocytochemical technique. Specific immunolabeling was detected over the extracellular enamel matrix and over the rough endoplasmic reticulum, the saccules of the Golgi apparatus, and the secretory granules of the ameloblasts. In addition, some lysosome-like granules were also labeled. Only background labeling was obtained over mitochondria, nuclei, cytosol, adjacent odontoblasts, and dentin. Quantitation of the intensity of labeling showed the presence of an increasing gradient along the secretory pathway, which may correspond to the concentration or the maturation of these proteins as they are processed by the cell. These findings indicate that the ameloblast displays an intracellular distribution of its secretory products similar to that of other merocrine secreting cells. The presence of enamel proteins in lysosomes suggests that crinophagy and/or resorption occurs in these cells.     

Immunological characterization, developmental pattern and vitamin-D-dependency of calbindin D-28 K in rat teeth ameloblasts

It has been suggested that vitamin D is involved in the process of cell differentiation and extracellular mineralization during tooth development. One of the best-defined molecular markers of the action of vitamin D is a calcium-binding protein of Mr 28,000 called calbindin D-28 K (CaBP 28 K). Since this protein is present in growing teeth, we have examined its synthesis in teeth from vitamin D-replete and -deplete rats by Western blotting and immunocytochemistry with an antiserum to CaBP 28 K purified from rat kidney. The CaBP 28 K present in the enamel organ is a single molecular species migrating near 30 k Da, similarly to the kidney protein. The differentiation and maturation of odontogenic cells were followed during early postnatal development (2-12 days) in rat molars. At the light-microscope level, CaBP 28 K was only found in a single cell-type, the ameloblasts. The expression of this protein appeared to be developmentally controlled, since its distribution varied with the cell stage and the functional steps of amelogenesis. The protein was localized in the basal compartment of ameloblasts from the presecretory stage. During the early secretory stage, the concentration of cytoplasmic CaBP 28 K formed a gradient from the apical to the basal pole of the ameloblasts. Staining appeared homogeneous in the cytoplasm of later secretory ameloblasts. CaBP 28 K was discontinuously distributed during the maturation stage. This discontinuity might be related to cyclical changes in mature ameloblasts. In all stages, ameloblasts from vitamin-D-deficient rats appeared depleted of CaBP 28 K.     

Cytochemical localization of Ca2+-Mg2+ adenosine triphosphatase in rat incisor ameloblasts during enamel secretion and maturation

A modified Wachstein-Meisel medium containing lead or cerium as capturing ions was used to localize Ca2+-Mg2+ adenosine triphosphatase (ATPase; EC 3.6.1.3) in rat incisor ameloblasts during enamel formation. Sections representing different developmental stages were processed for electron microscopic cytochemistry. Distribution and intensity of the observed reaction product, which was almost exclusively associated with cell membranes, varied according to the stage of enamel formation. During the secretory stage, intense reaction product was evident along the entire plasma membrane of ameloblasts and papillary cells. The early transitional ameloblasts showed reaction product on their proximal and lateral cell membranes, but not distally. In late transitional (pre-absorptive) ameloblasts, distal cell membranes exhibited intense reaction product. During enamel maturation, smooth-ended ameloblasts showed reaction product proximally and laterally, but not distally. Ruffle-ended maturative ameloblasts exhibited intense reaction product along their lateral and distal membranes. The intensity of the latter was decreased but not eliminated by levamisole. In the transition from smooth-ended to ruffle-ended cells, the reaction product became evident distally, concomitant with the appearance of cell membrane invaginations. These data are consistent with a possible role for Ca2+-Mg2+ ATPase in controlling calcium availability at the enamel mineralization front.     

Rat <Emphasis Type="Italic">wct</Emphasis> mutation prevents differentiation of maturation-stage ameloblasts resulting in hypo-mineralization in incisor teeth

A recent study provided genetic and morphological evidence that rat autosomal-recessive mutation, whitish chalk-like teeth (wct), induced tooth enamel defects resembling those of human amelogenesis imperfecta (AI). The wct locus maps to a specific interval of rat chromosome 14 corresponding to human chromosome 4q21 where the ameloblastin and enamelin genes exist, although these genes are not included in the wct locus. The effect of the wct gene mutation on the enamel matrix synthesis and calcification remains to be elucidated. This study clarifies how the wct gene mutation influences the synthesis of enamel matrix and its calcification by immunocytochemistry for amelogenin, ameloblastin and enamelin, and by electron probe micro-analysis (EPMA). The immunoreactivity for enamel proteins such as amelogenin, ameloblastin, and enamelin in the ameloblasts in the homozygous teeth was the same as that in the heterozygous teeth from secretory to transitional stages, although the homozygous ameloblasts became detached from the enamel matrix in the transitional stage. The flattened ameloblasts in the maturation stage of the homozygous samples contained enamel proteins in their cytoplasm. Thus, the wct mutation was found to prevent the morphological transition of ameloblasts from secretory to maturation stages without disturbing the synthesis of enamel matrix proteins, resulting in the hypo-mineralization of incisor enamel and cyst formation between the enamel organ and matrix. This mutation also prevents the transfer of iron into the enamel.     

Selachian tooth development: III. Ultrastructural features of secretory amelogenesis in Squalus acanthias

Ultrastructural features of secretory amelogenesis during selachian tooth development show several similarities to mammalian amelogenesis. However, the following critical differences were noticed: 1) subcellular organelles associated with merocrine-type protein synthesis and secretion were located in both the infranuclear as well as supranuclear regions of the selachian ameloblasts; 2) no evidence for Tomes process formation was found; 3) the basal lamina was not removed during epithelial differentiation into ameloblasts in the selachian model, and the structural features of the basal lamina were significantly altered during amelogenesis in rows III, IV, and VI; and 4) no dentine-enameloid junction was detected. It is suggested that enameloid is an extracellular matrix which is derived from the selachian inner enamel epithelium and appears to be secreted from both the lateral and apical surfaces of ameloblasts.     

Ultrastructure of secretory ameloblasts in the house musk shrew, Suncus murinus, Insectivora

Tooth germs from neonatal house musk shrews, Suncus murinus, were used for the study. The tooth morphogenesis was compared electron microscopically to that of Primates. In the tooth germ at the bell stage, the ameloblast was 3 x 50 microns in size, columnar in shape and had several tubular-type Golgi apparatus which were at the distal end of the cell. Most mitochondria were noted at the proximal end of ameloblasts. Tomes' processes were 1 micron in width, protruded 10 microns from the ameloblast and had many dense bodies and two kinds of vesicles. They were morphologically different from human ameloblasts and enamel rods.     

Development of larval and transformed teeth in Ambystoma mexicanum (Urodela, Amphibia): an ultrastructural study

Odontogenesis of early larval non-pedicellate teeth, late larval teeth with a more or less distinct dividing zone and fully transformed pedicellate teeth in Ambystoma mexicanum (Urodela) was studied to obtain insights into the development of differently structured teeth in lower vertebrates. Using transmission electron microscopy we investigated five developmental stages: (1) papilla; (2) bell stage (secretion of the matrix begins); (3) primordium (mineralization and activity of ameloblasts starts); (4) replacement tooth (young, old); and (5) established, functional tooth. Development of the differently structured teeth is largely identical in the first three stages. Mineralization takes place in apico-basal direction up to the (prospective) pedicel (early and some late larvae) or up to the zone that divides the late larval and transformed tooth in pedicel and dentine shaft (pedicellate condition). Mineralization starts directly at the collagen and by means of matrix vesicles. First odontoblasts develop small processes that extend to the basal lamina of the inner epithelial layer of the enamel organ. The processes are small and lack organelles in early larval teeth, but become larger, arborescent, and contain some organelles in late larval and transformed teeth. The processes are surrounded by unmineralized matrix (predentine). Odontoblasts at the basis of the teeth, at the pedicel, and in the zone of division do not develop significant cytoplasmic processes that extend into the matrix. Cells of the inner enamel epithelium differentiate to ameloblasts that secrete the enamel. In the early larval tooth they show an extensive basal labyrinth that becomes regressive when the enamel layer is completed. In late larval and transformed teeth, however, a large cavity arises between the basal ruffled border of ameloblasts and their basal lamina. This cavity appears to mediate amelogenesis. A small apical zone in early, but not in late larval teeth directly below the thin enamel layer consists of enameloid and is free of dentine channels.     

Maturation ameloblasts of the porcine tooth germ do not express amelogenin

 Amelogenins are the most abundant constituent in the enamel matrix of developing teeth. Recent investigations of rodent incisors and molar tooth germs revealed that amelogenins are expressed not only in secretory ameloblasts but also in maturation ameloblasts, although in relatively low levels. In this study, we investigated expression of amelogenin in the maturation stage of porcine tooth germs by in situ hybridization and immunocytochemistry. Amelogenin mRNA was intensely expressed in ameloblasts from the differentiation to the transition stages, but was not detected in maturation stage ameloblasts. C-terminal specific anti-amelogenin antiserum, which only reacts with nascent amelogenin molecules, stained ameloblasts from the differentiation to the transition stages. This antiserum also stained the surface layer of immature enamel at the same stages. At the maturation stage, no immunoreactivity was found within the ameloblasts or the immature enamel. These results indicate that, in porcine tooth germs, maturation ameloblasts do not express amelogenins, suggesting that newly secreted enamel matrix proteins from the maturation ameloblast are not essential to enamel maturation occurring at the maturation stage. Accepted: 14 January 1999     

The human homologue of the Aspergillus nuclear migration gene nudC is preferentially expressed in dividing cells and ciliated epithelia

An early event in apoptosis is exposure of phosphatidylserine, an aminophospholipid normally present in the inner leaflet of the plasma membranes, at the outer leaflet of the plasma membrane facing the extracellular space. Annexin V (Anx-V) is a 35-kDa protein with high affinity for phosphatidylserine, which can be applied to detect apoptosis. We injected biotin-labelled Anx-V intravenously in adult mice and examined the tissue distribution of Anx-V-labelled cells in dental and periodontal tissues using ABC-peroxidase histochemistry. In the continuously erupting incisors, strong and frequent immunostaining was observed in transitional stage and late maturation stage ameloblasts with less frequent staining in preameloblasts. Frequency of staining in odontoblasts and pulp cells was low but increased slightly at older stages of dentinogenesis. Labelling was also seen in phagocytic or phagocytic-like cells in the enamel organ and pulp. A positive staining was furthermore found in fibroblasts of the periodontal ligament in continuously erupting incisors and in fully erupted molar teeth. Staining intensity and the number of positive cells were enhanced by antigen retrieval using high-pressure cooking. We conclude that Anx-V-biotin labels dental cells in early stages of cell death and indirectly cells that have ingested labelled apoptotic cells during the course of the experiment. The data confirm that during amelogenesis most cell death occurs in transitional stage and late maturation stage ameloblasts. Thus, labelling with Anx-V is a useful marker for studying cell death and the dynamics of clearance of apoptotic cells during tooth development.     

Histochemical and electron probe analysis of secretory ameloblasts of developing rat molar teeth

Calcium was not found in secretory ameloblasts and stratum intermedium cells when treated with OsO4-pyroantimonate or when surfaces prepared by fracturing fresh, rapidly frozen, developing molar tooth germs were subject to electron probe X-ray analysis. Pyroantimonate reaction product, considered to be calcium, was found in mitochondria of enamel organ cells which were first placed in a bath containing calcium and potassium. The plasma membrane was disrupted in cells ehich showed mitochondrial localization of reaction product. The results provide no data which indicates that enamel organ cells have a direct, active role in the movement of calcium into the enamel. Rather, it is suggested that the secretory enamel organ might serve as a selective barrier in regulating the initial mineralization of enamel.     

Histochemical and electron probe analysis of secretory ameloblasts of developing rat molar teeth

Summary Calcium was not found in secretory ameloblasts and stratum intermedium cells when treated with OsO₄-pyroantimonate or when surfaces prepared by fracturing fresh, rapidly frozen, developing molar tooth germs were subject to electron probe X-ray analysis.Pyroantimonate reaction product, considered to be calcium, was found in mitochondria of enamel organ cells which were first placed in a bath containing calcium and potassium. The plasma membrane was disrupted in cells which showed mitochondrial localization of reaction product.The results provide no data which indicates that enamel organ cells have a direct, active role in the movement of calcium into the enamel. Rather, it is suggested that the secretory enamel organ might serve as a selective barrier in regulating the initial mineralization of enamel.     

Localization of calcium in differentiating odontoblasts and ameloblasts before and during early dentinogenesis and amelogenesis in hamster tooth germs

Potassium pyroantimonate-osmium tetroxide cytochemistry has been used to study the distribution of ionic calcium in hamster tooth germs during cell differentiation and during early dentinogenesis and amelogenesis. Before the onset of mineralization, pyroantimonate (PA) reaction product was found in the nucleus of differentiating preameloblasts and preodontoblasts. In the predentin, it was preferentially located along striated collagen fibrils, lying perpendicular to the basal lamina. At the onset of mineralization, a pronounced increase of PA reaction product was evident in the predentin and on the plasma membrane and in mitochondria of both preodontoblasts and preameloblasts opposite the mineralizing mantle dentin. During early enamel mineralization, PA reaction product was present in the "growing" crystal ends, while in the secretory ameloblasts, most of the PA reaction product was localized on the cytoplasmic side of the apical plasma membranes and in mitochondria. When Tomes' processes developed, PA reaction product, both cytoplasmic and membrane bound, was low or absent deep in the processes, but gradually increased toward the apical terminal web. A corresponding gradient of PA reaction product was observed on the opposing enamel crystallites. From this study we conclude that both preodontoblasts and preameloblasts seem to be involved in calcium acquisition necessary for the early stages of mantle dentin mineralization. Tomes' processes seem to regulate the entry of calcium into the enamel mineralization front.     

The ultrastructure of human secretory ameloblasts

Summary The ultrastructure of secreting ameloblasts of deciduous teeth from a human foetus (crown-rump length 195 mm) was investigated. The ameloblasts demonstrate a formation of granules in a juxtanuclear Golgi complex. In the Tomes' process the granules are released either through the lateral plasma membrane into the intercellular space between the Tomes' processes or directly through the apical plasma membrane into the enamel.The human ameloblasts differ from non-human ameloblasts in having a non-oriented vesicular granular endoplasmic reticulum. Further, the majority of mitochondria are situated in the apical part of the ameloblast adjacent to the Tomes' process.We would like to thank chief-surgeon A. Christensen, Bispebjerg Hospital, Copenhagen for his help in acquiring foetal material. For technical assistance we would like to thank M. Balslev and U. Eberth, Anatomy Department A.     

Selachian tooth development: II. Immunolocalization of amelogenin polypeptides in epithelium during secretory amelogenesis in Squalus acanthias

We have determined the distribution of amelogenin polypeptides in an order of elasmobranchs using indirect immunofluorescence with rabbit polyclonal antibodies prepared to purified murine amelogenins. We find that amelogenins are definitely present within the inner enamel epithelium prior to the production of the extracellular matrix component termed "enameloid" (row II developing tooth organs). During subsequent stages of selachian tooth development (row III tooth organs), immunofluorescence staining data indicated localization of amelogenin antigens within epithelium as well as the enameloid extracellular matrix. The results from these immunohistochemical studies suggest that the 16-20 kdalton amelogenins, which are characteristic of murine inner enamel epithelial cells undergoing terminal biochemical differentiation into secretory ameloblasts, may also be regarded as molecular markers for amelogenesis in developing teeth in the spiny dogfish, Squalus acanthias.     

Fine structure of the corpuscles of stannius in the toadfish.

The micro-anatomy of the corpuscles of Stannius of the toadfish, Opsanus tau, an aglomerular marine teleost, has been studied by light and electron microscopy. The corpuscles are composed of extensively anastomosed cords of epithelial cells which maintain intimate contact with blood capillaries. Most of the epithelial cells contain acidophilic granules which also show a positive reaction with the periodic acid-Schiff technique and aldehyde fuchsin. On the basis of fine structural criteria, three cell types can be recognized. The granular cells contain abundant quantities of granular endoplasmic reticulum, ribosomes, Golgi apparatus with prosecretory granules, coated vesicles, polymorphic mitochondria with lamellar cristae, filaments, microtubules, a cilium, a variety of lysosome-like dense bodies, glycogen particles, lipid droplets, secretory granules and intranuclear lipid-like inclusions. One variety of agranular cell (type I) is characterized by the total absence of secretory granules, but it contains large amounts of granular endoplasmic reticulum and ribosomes, conspicuous profiles of Golgi apparatus, coated vesicles and sometimes an abundance of glycogen. Another variety of agranular cell (type II) has poorly developed cytoplasmic organelles. The perivascular space between the capillary and parenchyma contains connective tissue cells and abundant nerve fibers. The different types of epithelial cells observed in the corpuscles of Stannius of this fish may represent functional stages of the secretory cycle in a single cell type.