Ameloblasts Research Articles

Single-cell census of human tooth development enables generation of human enamel.

Tooth enamel secreted by ameloblasts (AMs) is the hardest material in the human body, acting as a shield to protect the teeth. However, the enamel is gradually damaged or partially lost in over 90% of adults and cannot be regenerated due to a lack of ameloblasts in erupted teeth. Here, we use single-cell combinatorial indexing RNA sequencing (sci-RNA-seq) to establish a spatiotemporal single-cell census for the developing human tooth and identify regulatory mechanisms controlling the differentiation process of human ameloblasts. We identify key signaling pathways involved between the support cells and ameloblasts during fetal development and recapitulate those findings in human ameloblast invitro differentiation from induced pluripotent stem cells (iPSCs). We furthermore develop a disease model of amelogenesis imperfecta in a three-dimensional (3D) organoid system and show AM maturation to mineralized structure invivo. These studies pave the way for future regenerative dentistry.

Mitochondria modulate ameloblast Ca2+ signaling.

The role of mitochondria in enamel, the most mineralized tissue in the body, is poorly defined. Enamel is formed by ameloblast cells in two main sequential stages known as secretory and maturation. Defining the physiological features of each stage is essential to understand mineralization. Here, we analyzed functional features of mitochondria in rat primary secretory and maturation-stage ameloblasts focusing on their role in Ca2+ signaling. Quantification of the Ca2+ stored in the mitochondria by trifluoromethoxy carbonylcyanide phenylhydrazone stimulation was comparable in both stages. The release of endoplasmic reticulum Ca2+ pools by adenosine triphosphate in rhod2AM-loaded cells showed similar mitochondrial Ca2+ (m Ca2+ ) uptake. However, m Ca2+ extrusion via Na+ -Li+ -Ca2+ exchanger was more prominent in maturation. To address if m Ca2+ uptake via the mitochondrial Ca2+ uniporter (MCU) played a role in cytosolic Ca2+ (c Ca2+ ) buffering, we stimulated Ca2+ influx via the store-operated Ca2+ entry (SOCE) and blocked MCU with the inhibitor Ru265. This inhibitor was first tested using the enamel cell line LS8 cells. Ru265 prevented c Ca2+ clearance in permeabilized LS8 cells like ruthenium red, and it did not affect ΔΨm in intact cells. In primary ameloblasts, SOCE stimulation elicited a significantly higher m Ca2+ uptake in maturation ameloblasts. The uptake of Ca2+ into the mitochondria was dramatically decreased in the presence of Ru265. Combined, these results suggest an increased mitochondrial Ca2+ handling in maturation but only upon stimulation of Ca2+ influx via SOCE. These functional studies provide insights not only on the role of mitochondria in ameloblast Ca2+ physiology, but also advance the concept that SOCE and m Ca2+ uptake are complementary processes in biological mineralization.

Autoradiographic study of iron transport in rat incisor enamel.

An autoradiographic study using 55Fe was performed to investigate the distribution of iron in the ameloblasts and enamel of rat incisors. The study of methodological problems of 55Fe autoradiography revealed that in the undecalcified sections, the radiation by 55Fe within the ameloblasts and pigmented enamel produced grain scattering over the facing enamel surface and ameloblasts, respectively. The findings indicate that the autoradiographic observation of the enamel surface should be made using the incisors freed from the enamel organ and that the observation of the ameloblasts should be done in the region other than the zone of enamel pigmentation. After the 55Fe injection, 55Fe incorporation into the ameloblasts was observed in the maturation stage, reaching the maximum at 24 hours. The peak of 55Fe distribution in the ameloblasts moved from the early stage to the later stage of maturation between 24 hours and 7 days after the injection. At 3 and 7 days after the 55Fe injection, 55Fe was deposited markedly on the enamel surface at the end of maturation where the enamel pigmentation occurs. The present study established the utility of 55Fe autoradiography when applied on the undecalcified section of rat incisors and it was demonstrated that iron is incorporated into the maturation ameloblasts and secreted onto the enamel surface at the end of maturation.

Excessive fluoride inducing calcium overload and apoptosis of ameloblasts

To study the effect of excessive fluoride on calcium overload and apoptosis in cultured rat ameloblasts in vitro. Logarithmic-phase ameloblasts (HAT-7) were treated with 0, 0.4, 0.8, 1.6, 3.2, and 6.4 mmol · L(-1) sodium fluoride (NaF) solution. Cell activities were detected by using a Cell Counting Kit 8 (CCK-8) assay after 48 h of treatment. The effect of fluoride on cell apoptosis was analyzed by using flow cytometry. Excessive fluoride-induced calcium concentration and calreticulin expression changes in ameloblasts were detected by using laser scanning confocal microscopy, Western blot analysis, and real-time quantitative polymerase chain reaction. NaF inhibited ameloblast activity at 1.6, 3.2, and 6.4 mmol · L(-1) (dose-dependent) after 48 h of induction. The Ca2+ fluorescence intensity of HAT-7 cells incubated with 1.6 and 3.2 mmol · L(-1) NaF was higher than that in the control group. The fluoride-induced early-stage apoptosis of ameloblasts after 48 h of induction and the early-stage apoptosis rate was positively correlated with fluoride concentration. Calreticulin mRNA expression in HAT-7 cells was higher than that in the control group after 48 h of incubation with 0.8, 1.2, and 1.6 mmol · L(-1) NaF. Excessive fluoride-induced calcium overload in ameloblasts and further caused endoplasmic reticulum stress-mediated apoptosis.

Immunohistochemical expression of thymosin β4 in ameloblastomas and odontomas.

Ameloblastoma is regarded to be a benign odontogenic tumor, but it is destructive, locally invasive and presents a high rate of recurrence. Thymosin β4 (Tβ4) is closely associated with tooth germ development. Tβ4 also plays a role in malignant progression and invasion. However, little is known about the function of Tβ4 in odontogenic tumors. Thus, we investigated Tβ4 expression in ameloblastomas and compared it with odontomas. We immunohistochemically evaluated the expression of Tβ4, ameloblastin (AMBN), amelogenin (AMEL) and enamelin (ENAM) in 57 samples of ameloblastomas from 40 patients, and also assessed the expression of these molecules in 11 cases of odontomas, two of ameloblastic fibro-odontomas and one of tooth germ-like structures without the formation of enamel and dentin. Tβ4 signals were observed in almost all of the ameloblastomas. The signals were observed in both peripheral columnar cells and central polyhedral/angular cells. Similar findings were observed in tooth germ-like structures, and in the ameloblastomatous nests in the ameloblastic fibro-odontomas. These samples had negative results for AMBN, AMEL and ENAM. Meanwhile, Tβ4 signals were not seen in the odontomas, although immunolabeling for AMBN, AMEL and ENAM was observed in the enamel matrix and in some ameloblasts. Ectomesenhymal regions in the odontomas were negative for staining with the antibodies for AMBN, AMEL and ENAM. These results suggest that Tβ4 could be associated with morphogenesis and tumor invasion in the ameloblastoma, and that Tβ4 may play a role in the behavior of ameloblastoma.

Influence of oral zinc sulphate supplementation on the development of the teeth and jaws of growing rat (Histological study)

teeth and jaws on growing rat. Materials and methods: Fifteen western albino pregnant rats subjected to zinc deficiency diet from time of 16th of gestation till day 21 postnatal days. Oral Zinc supplement in different dose (o.25 and o.5 mg) were added to the normal zinc diet of the mothers from one day to 21 day postnatal periods). Neonate rats were sacrificed at the followings postnatal periods ( 7, 10, 14, 18 and21 day) .Histological evaluation for the development of 1st molar tooth with development of the jaws were estimated under light microscope .In addition ,serum zinc level in all studied periods were determined with atomic absorption spectrophotometer. Results: Histological examination for experimental group(0.5) showed acceleration in the growth of molar teeth and the bones of the maxilla and mandible illustrates in early apposition of dental hard tissue and bone trabeculea. Declination in zinc levels were observed approximately in all studied groups with the increment of the period of the growth. Conclusion: This study was illustrated that there are obvious effects of oral supplementing zinc on the growth of teeth and jaws of the rat suffered from zinc deficiency in gestation period and ameloblast showed to be the most affected dental cells.

Calcium Transport Protein and Amelogenin Expression in Ameloblastoma: Relationship to Normal Amelogenesis

The progressive maturation of the ameloblast epithelium, formation of enamel matrix, and the mineralization of that matrix characterize normal amelogenesis. This process is regulated, in part, by the amelogenins. The expression by ameloblasts of amelogenin proteins as well as calcium transport proteins parallels matrix formation and mineralization. Odontogenic tumors reproduce, to varying degrees, the morphological and inductive relationships found among various parts of normal tooth germ. In normal amelogenesis, amelogenin, plasma membrane Ca(superscript ++)-pump PMCA , and calbindin-28KDa calbindin proteins are minimally expressed in presecretory ameloblasts, but progressively increase in expression later as the enamel matrix forms and begins to mineralize. Ameloblastomas, however, do not form matrix or mineralize. In the present study, we demonstrate by immunohistochemistry, that amelogenin, PMCA and calbindin proteins are all expressed in the ameloblast-like palisading peripheral epithelial cells of ten cases of ameloblastoma. In serial sections processed by the von Kossa technique, we also found diffuse calcium in all tumors studied. Our study suggests that ameloblastomas recapitulate a stage of differentiation beyond the presecretory stage and that the mechanisms of extracellular matrix formation and mineralization associated with normal amelogenesis may be uncoupled in ameloblastomas.

Enamelin transcriptional expression in developing postnatal rat tooth germ

To observe the transcriptional expression of enamelin in developing postnatal rat first mandibular molar germs, for further studies of functions of enamelin in enamel development and mineralization. Tissue slices of first mandibular molar germ of rat 1, 3, 7, 10, 14 days after birth were prepared. The enamelin mRNA expression was identified by in situ hybridization. Enamelin mRNA was observed in both ameloblast and odontoblast in 1-10 day old rat postnatal first mandibular molar germs. Enamelin mRNA appeared very weakly at 1st day, and increased through 3rd day, reached the maximum at 7th day, and reduced at 10th day and became negative at 14th day postnatally; while the expression of enamelin mRNA in odontoblast maintained lower from 1st to 10th day and negative at 14th day postnatally. Enamelin gene transcriptional expression lasts from preameloblast to maturation ameloblast, which suggests that enamelin may participate in the development of enamel and mantle dentin.

The developmental biology of cementum.

In conclusion, we have reviewed an extensive literature on early cementogenesis and performed a detailed morphological and molecular analysis to illustrate and verify key issues in the current debate about epithelial and mesenchymal contributions to root cementum. We have demonstrated that prior to cementogenesis, Hertwig's epithelial root sheath disintegrates and dental follicle cells penetrate the epithelial layer to invade the root surface. Our studies confirmed that HERS became disrupted or disintegrated prior to cementum deposition. We visualized how mesenchymal cells from the dental follicle penetrated the HERS bilayer and deposited initial cementum, while immediately adjacent epithelial cells were separated from the root surface by a basal lamina and did not secrete any cementum. Human specimen from the Gottlieb collection indicated that HERS was removed from the root surface prior to cementum deposition. Our in situ hybridization and immolocalization data revealed that both amelogenin mRNAs and enamel proteins were restricted to the crown enamel and were absent from the root surface and from the cervical-most ameloblasts adjacent to the root margin. On Western blots, cementum protein extracts did not cross-react with amelogenin antibodies. Our studies in conjunction with our literature review together confirmed the classical theory of cementum as a dental follicle derived connective tissue that forms subsequent to HERS disintegration.

Nrf2 deficiency causes tooth decolourization due to iron transport disorder in enamel organ.

Rodents have brownish-yellow incisors whose colour represents their iron content. Iron is deposited into the mature enamel by ameloblasts that outline enamel surface of the teeth. Nrf2 is a basic region-leucine zipper type transcription factor that regulates expression of a range of cytoprotective genes in response to oxidative and xenobiotic stresses. We found that genetically engineered Nrf2-deficient mice show decolourization of the incisors. While incisors of wild-type mice were brownish yellow, incisors of Nrf2-deficient mice were greyish white in colour. Micro X-ray imaging analysis revealed that the iron content in Nrf2-deficient mouse incisors were significantly decreased compared to that of wild-type mice. We found that iron was aberrantly deposited in the papillary layer cells of enamel organ in Nrf2-deficient mouse, suggesting that the iron transport from blood vessels to ameloblasts was disturbed. We also found that ameloblasts of Nrf2-null mouse show degenerative atrophy at the late maturation stage, which gives rise to the loss of iron deposition to the surface of mature enamel. Our results thus demonstrate that the enamel organ of Nrf2-deficient mouse has a reduced iron transport capacity, which results in both the enamel cell degeneration and disturbance of iron deposition on to the enamel surface.

Proteomic analysis of enamel cells from developing rat teeth: big returns from a small tissue.

Poor understanding of the molecular links between disturbed calcium regulation in cells and disease remains a problem of considerable biomedical importance. Dental enamel cells might provide useful insights to this problem since they handle calcium in bulk without suffering its cytotoxic effects. Two practical challenges hindered investigation of calcium handling mechanisms in enamel cells--paucity of molecular information and limited availability of sample from developing rat teeth, the experimental system of choice. This paper outlines the microscale proteomic approaches we applied to overcome these difficulties and reviews several major findings that ensued from initial characterization of the enamel cell proteome. Enamel cells are now established as a powerful model for fundamental calcium research and have provided outcomes of broad biological relevance, including the discovery of a new endoplasmic reticulum protein. Future proteomic approaches that might benefit understanding of function are discussed.

Enamel Cell Biology Towards a Comprehensive Biochemical Understanding

Enamel cells ultimately determine the properties of dental enamel. Surprisingly little is known about enamel cell functions at the biochemical and molecular levels. Understanding of both normal and abnormal enamel formation should benefit from elucidation of this area. This paper reviews our recent efforts to establish microscale biochemical analyses of rat enamel cells, and the ensuing initial findings about their protein phenotype (i.e., proteome) and calcium-handling mechanisms. A perspective of the current status of enamel cell research, and where it might head, is also given.

Lectin binding patterns of odontogenic epithelium in the rat during various phases of molar tooth development.

Light and electron microscopic investigations of lectin binding patterns in rat tooth germs were undertaken in order to elucidate glycoconjugate localization in cells of the reduced enamel epithelia and their derivatives. It was found that Ulex europaeus agglutinin (UEA-1), peanut agglutinin (PNA), soybean agglutinin (SBA), and wheat germ agglutinin (WGA) exhibited variable reactivity patterns with different epithelia. UEA-1 was reactive with cells of the stratum intermedium and stellate reticulum in the tooth germ but unreactive with ameloblasts, outer enamel epithelial cells, and junctional epithelium at later stages. Reaction patterns of PNA in these cells differed from those of UEA-1. Results indicated that inner and outer cells of the reduced enamel epithelium are heterogeneous with regard to lectin binding patterns.

Abundant calcium homeostasis machinery in rat dental enamel cells. Up-regulation of calcium store proteins during enamel mineralization implicates the endoplasmic reticulum in calcium transcytosis.

Enamel cells handle large amounts of calcium, particularly during the developmental phase (termed maturation) when dental enamel is hypermineralized. The extent of intracellular calcium burden, and the nature of calcium homeostasis machinery used to accommodate it, are largely unknown. Here, the calcium-binding capacity of enamel cell cytosol was found to increase during development, in parallel with the putative transcellular flux of calcium. At maturation, the abundance of calcium-binding proteins in enamel cells exceeded that in brain and other established calcium-oriented tissues, which implies a large calcium burden. A search for likely cytosolic calcium transporters revealed only one high-affinity calcium-binding protein (12 kDa, distinguished from alpha-parvalbumin) that was up-regulated during maturation, but its low abundance (0.02% of soluble protein) precluded a major calcium transport or cytoprotective role. Two low-affinity calcium-binding proteins up-regulated during maturation (by 1.8-fold and 2.1-fold respectively) were identified as calreticulin and endoplasmin, both residents of the endoplasmic reticulum. Together, calreticulin and endoplasmin constituted an exceptionally high proportion (5%) of soluble protein during maturation, which gives an inferred calcium capacity 67-fold higher than that of the principal cytosolic calcium-binding protein. 28-kDa calbindin. Evidence that endoplasmin expression varied inversely with serum calcium concentration, and that the inositol trisphosphate receptor also was highly expressed during maturation, supported the novel hypothesis that non-mitochondrial calcium stores play a major role in transcellular calcium transport. (a) enamel cells contain a general high abundance of calcium homeostasis proteins, consistent with a heavy intracellular calcium burden; (b) the expression pattern (phenotype) of calcium-binding proteins varies with enamel cell function; (c) enamel cells appear to contain unusually large non-mitochondrial calcium stores; (d) contrary to the prevailing view that calcium passes mainly through the cytosol of calcium-transporting cells, the findings imply a route through the endoplasmic reticulum. This study gives novel information about how a highly calcium-oriented tissue avoids calcium toxicity, and provides a new focus for investigations into the mechanisms of transcellular calcium transport.

Calbindin28kDa and Calmodulin are Hyperabundant in Rat Dental Enamel Cells. Identification of the Protein Phosphatase Calcineurin as a Principal Calmodulin Target and of a Secretion-Related Role for Calbindin28kDa

Enamel cells are likely to experience heavy demands for intracellular calcium homeostasis during the secretion and hypermineralization of dental enamel. Here, the two major high-affinity calcium-binding proteins in rat enamel epithelium were identified as calbindin2gkDa and calmodulin, using a microscale approach. Both proteins were hyperabundant, totalling up to 2% of the soluble protein and surpassing the amounts in cerebellum, the benchmark tissue. Calbindin28kDa and calmodulin accounted for 26% of the total calcium-binding capacity in enamel cell cytosol, under near physiological conditions. Numerous calmodulin-binding proteins were detected with an overlay assay, indicating that calmodulin has multiple major targets in enamel cells. The calcium/calmodulin-regulated protein phosphatase, calcineurin, was identified as a principal calmodulin target constituting 0.1% of the soluble protein. Calmodulin and calcineurin were expressed constitutively, implying continued heavy usage of calcium/calmodulin-based and phosphorylation-based signalling events throughout enamel cell development. Calbindin28kDa, in contrast, was expressed at fourfold higher levels in secretion-phase cells than during the calcium-intensive hypermineralization phase, unexpectedly pointing to an important role associated with secretion. Supporting this notion, immunoblots revealed that 33 % of total (SDS-soluble) calbindin2SkDa was in the particulate fraction and predominantly associated with the Triton-insoluble cytoskeleton. Solubilisation of cytoskele-tal calbindin28kDa required high concentrations of NaCl or urea, indicating the existence of a high-affinity target ligand. The unusual abundance of calmodulin, calbindin28kDil and calcineurin demonstrated here provides the first molecular evidence that enamel cells possess a strong capability for intracellular calcium homeostasis. Since none of these proteins was up-regulated during enamel hypermineralization, it appears that other calcium-binding proteins are primarily involved in the putative transcellular passage of calcium.

Calbindin28kDa and calmodulin are hyperabundant in rat dental enamel cells. Identification of the protein phosphatase calcineurin as a principal calmodulin target and of a secretion-related role for calbindin28kDa.

Enamel cells are likely to experience heavy demands for intracellular calcium homeostasis during the secretion and hypermineralization of dental enamel. Here, the two major high-affinity calcium-binding proteins in rat enamel epithelium were identified as calbindin28kDa and calmodulin, using a microscale approach. Both proteins were hyperabundant, totalling up to 2% of the soluble protein and surpassing the amounts in cerebellum, the benchmark tissue. Calbindin28kDa and calmodulin accounted for 26% of the total calcium-binding capacity in enamel cell cytosol, under near physiological conditions. Numerous calmodulin-binding proteins were detected with an overlay assay, indicating that calmodulin has multiple major targets in enamel cells. The calcium/calmodulin-regulated protein phosphatase, calcineurin, was identified as a principal calmodulin target constituting 0.1% of the soluble protein. Calmodulin and calcineurin were expressed constitutively, implying continued heavy usage of calcium/calmodulin-based and phosphorylation-based signalling events throughout enamel cell development. Calbindin28kDa, in contrast, was expressed at fourfold higher levels in secretion-phase cells than during the calcium-intensive hypermineralization phase, unexpectedly pointing to an important role associated with secretion. Supporting this notion, immunoblots revealed that 33% of total (SDS-soluble) calbindin28kDa was in the particulate fraction and predominantly associated with the Triton-insoluble cytoskeleton. Solubilisation of cytoskeletal calbindin28kDa required high concentrations of NaCl or urea, indicating the existence of a high-affinity target ligand. The unusual abundance of calmodulin, calbindin28kDa and calcineurin demonstrated here provides the first molecular evidence that enamal cells possess a strong capability for intracellular calcium homeostasis. Since none of these proteins was up-regulated during enamel hypermineralization, it appears that other calcium-binding proteins are primarily involved in the putative transcellular passage of calcium.

The mandibular infected buccal cyst (paradental cyst). A radiographic and histological study

Mandibular infected buccal cysts occur on the buccal and distal aspects of the roots of partly or fully erupted mandibular molars with vital pulps. In the present investigation six cases associated with mandibular first and second molars were studied. The cysts were lined with a non-keratinised squamous epithelium with a mixed inflammatory cell infiltrate in the connective tissue. The clinical and microscopic features were similar to those of previously reported paradental cysts. The inflammation always present in these cysts may have an important role in their pathogenesis. It may induce periodontal pocketing, which may lead to hyperplasia and cyst formation in adjacent odontogenic epithelium, which may be either remnants of the reduced enamel epithelium or cell rests of Malassez.

Ultrastructural differentiation in the distal ends of ameloblasts from the presecretory zone to the early secretory zone.

Morphological differentiation of the distal ends of ameloblasts (AMs) from the late presecretory to early secretory zones of the rat upper incisor was studied by electron microscopy. Preameloblasts (PAs) showed a high columnar shape, with the nucleus located in the basal portion. The Golgi apparatus occupied the supranuclear region with type-1 vesicles, and microvilli were present at the distal cell membrane. Coarse-textured material was observed inside the type-1 vesicles and in the lateral intercellular spaces as well as along the distal cell membrane, whereas fine-textured material was found along the distal cell membrane. Near the region of initial enamel formation, large matrix islands were found in the lateral intercellular space. A thin electron-dense layer was observed on the dentin surface. This layer might have occurred as a result of diffusion after degradation of the coarse- or fine-textured material into a finer substance in the extracellular spaces. In the region of initial enamel formation, the distal cell membrane of AMs was flat, but shallow and narrow membrane invaginations were associated with the cell membrane close to the matrix islands. In the region of inner enamel formation, a cone-shaped Tomes' process was formed between large matrix islands which had developed in the intercellular spaces between the lateral portions of the distal ends of AMs. It was considered that the membrane invaginations which had existed at the distal end of PAs moved lower toward the distal terminal bar, thereafter becoming microvilli.

An electron microscopic study of ectomesenchymal contacts in rat incisors.

In order to clarify connections between epithelium and mesenchymal tissue in the early stage of odontogenesis, the formation of ectomesenchymal contacts and the presence of coarse-textured material at the interface between ameloblasts (AMs) and odontoblasts (ODs) on growing rat incisors were studied by electron microscopy. The presecretory zone was classified into five regions according to the ultrastructure of each preameloblast (PA). The region where collagen fibrils could be observed was designated PZ-2, the region where predentin existed PZ-3, the region where the basal lamina began to disappear PZ-4, and the region where the basal lamina had disappeared PZ-5. In PZ-2, the cytoplasmic processes of PAs penetrated the basal lamina and reached the dental papilla cells. In some locations, ectomesenchymal contacts were also observed, in which the cytoplasmic processes of the PAs were in contact with those of the preodontoblasts (POs). In PZ-3, the distal cytoplasm contained large amounts of type-1 vesicles, and secretory granules. In the distal membrane of the PA, membrane invaginations containing fibrillar structures were visible. Also in some areas, the cytoplasmic processes at the distal ends of the PAs invaded the predentin. In the predentin, a large amount of coarse-textured material, considered to be the precursor of the enamel matrix, was observed. In PZ-3, a large number of cytoplasmic processes extended into the predentin, while in PZ-4, microvillus-like processes extended from the distal ends of the PAs, showing a high frequency of ectomesenchymal contact. It was suggested that secretory activity of the PAs was induced after ectomesenchymal contact had been accomplished, but that the PAs did not undergo morphological change into secretory ameloblasts.

Calmodulin immunocytochemistry in rat incisor enamel organ through its life cycle.

The enamel organ of the growing rat incisor was perfusion-fixed with a mixture of formaldehyde and glutaraldehyde and processed for indirect immunogold labeling of calmodulin on post-embedded ultrathin sections. Throughout the zones of presecretion, secretion, and maturation of enamel, specific protein A-immunogold labeling was localized on polyribosomes and those attached to endoplasmic reticulum, mitochondria, nuclear chromatin, phagolysosomes, and cytoplasm adjacent to the plasma membrane, and tonofilaments associated with desmosomes of ameloblasts and cells of outer layer of enamel organ. Golgi membranes, condensing vacuoles, secretion granules, primary lysosomes, and micropinocytotic coated vesicles were hardly labeled. In the presecretion zone, the basal lamina of the preameloblasts and the matrix vesicles and collagen fibrils of the predentin matrix were not immunoreactive. Tomes' process of secretory ameloblast and adjacent enamel crystals were labeled. In addition to the above immunoreactive structures, some phagolysosomes, ferritin granules, and the cytoplasm of the ruffled border zone of maturation ameloblast contained immunogold particles. In control sections incubated with either protein A-gold complex alone, or antiserum preabsorbed with an excess of calmodulin and protein A-gold complex, only a few gold particles were observed to be randomly associated with the tissues. These results indicate that calmodulin is present in the cells of the enamel organ through all stages of amelogenesis. Its wide distribution is consistent with its involvement in various cytoplasmic functions.