Podoplanin, E-cadherin, ß-catenin, and CD44v6 in recurrent ameloblastoma: their distribution patterns and relevance.

BACKGROUND: Ameloblastoma is a benign but locally infiltrative odontogenic epithelial neoplasm with a high risk for recurrence. Podoplanin, a lymphatic endothelium marker, putatively promotes collective cell migration and invasiveness in this neoplasm. However, its role in the recurrent ameloblastoma (RA) remains unclear. As morphological, signaling, and genetic differences may exist between primary and recurrent tumors, clarification of their distribution patterns is of relevance. MATERIALS AND METHODS: Podoplanin was examined immunohistochemically in conjunction with E-cadherin, ß-catenin, and CD44v6 in 25 RA. Immunostaining according to tumor area, cellular type, and location, and relationship of these proteins were analyzed. Findings were compared with 25 unrelated primary ameloblastomas (UPA). RESULTS: All four proteins were detected in RA and UPA samples. Expression rates for each protein were not significantly different between these two groups. RA demonstrated significant upregulation of podoplanin at the invasive front (P < 0.05), whereas upregulation of ß-catenin and CD44v6 and downregulation of E-cadherin at this site were not statistically significant (P > 0.05). Immunolocalization for all four proteins was predominantly membranous and less frequently cytoplasmic. Pre-ameloblast-like cells were podoplanin(+) /CD44v6(-), while stellate reticulum-like cells were podoplanin(-)/CD44v6(+). Acanthomatous, granular cell, and desmoplastic variants in both RA and UPA were podoplanin(-/low) but stained weak-to-moderate for E-cadherin, ß-catenin, and CD44v6. Stromal fibroblasts and lymph channels were variably podoplanin-positive. CONCLUSIONS: Podoplanin, ß-catenin, and CD44v6 upregulation at the tumor invasive fronts in RA and UPA supports a differential regulatory role by these molecules in mediating collective cell migration and local invasiveness. E-cadherin downregulation suggests altered cell adhesion function during tumor progression.

Consequences for enamel development and mineralization resulting from loss of function of ameloblastin or enamelin.

Although the nonamelogenin proteins, ameloblastin and enamelin, are both low-abundance and rapidly degrading components of forming enamel, they seem to serve essential developmental functions, as suggested by findings that an enamel layer fails to appear on teeth of mice genetically engineered to produce either a truncated form of ameloblastin (exons 5 and 6 deleted) or no enamelin at all (null). The purpose of this study was to characterize, by direct micro weighing, changes in enamel mineralization occurring on maxillary and mandibular incisors of mice bred for these alterations in nonamelogenin function (Ambn(+/+, +/-5,6, -5,6/-5,6), Enam(+/+, +/- ,-/-)). The results indicated similar changes to enamel-mineralization patterns within the altered genotypes, including significant decreases by as much as 50% in the mineral content of maturing enamel from heterozygous mice and the formation of a thin, crusty, and disorganized mineralized layer, rather than true enamel, on the labial (occlusal) surfaces of incisors and molars along with ectopic calcifications within enamel organ cells in Ambn(-5,6/-5,6) and Enam(-/-) homozygous mice. These findings confirm that both ameloblastin and enamelin are required by ameloblasts to create an enamel layer by appositional growth as well as to assist in achieving its unique high level of mineralization.

Immunocytochemistry of matrix proteins in calcified tissues: functional biochemistry on section.

The organic matrix of calcified tissues comprises collagenous and/or noncollagenous matrix proteins (NCPs). Identification and precise mapping of these matrix components is essential for determining their function, formulating coherent hypotheses on their mechanism(s) of action, and developing novel therapeutic approaches based on biologics. Fibrillar collagen can be readily identified by its conspicuous structure, however, NCPs, in general, do not individually exhibit characteristic structural features that permit to identify them and morphologically determine their localization. To address this limitation, we have used immunocytochemistry, a form of "biochemistry on section", to correlate composition with structure. For cytochemical characterizations, including immunolabeling, our laboratory has opted for colloidal gold labelings and pioneered their application to calcified tissues because they yield high spatial resolution and are quantitative. Over the years, this approach has been applied to identify and map various NCPs in bone and teeth and, in this review of our work, we will emphasize some selected studies that highlight it application to also achieve functional information.

Subcultured odontogenic epithelial cells in combination with dental mesenchymal cells produce enamel-dentin-like complex structures.

We showed in a previous study that odontogenic epithelial cells can be selectively cultured from the enamel organ in serum-free medium and expanded using feeder layers of 3T3-J2 cells. The subcultured odontogenic epithelial cells retain the capacity for ameloblast-related gene expression, as shown by semiquantitative RT-PCR. The purpose of the present study was to evaluate the potential of subcultured odontogenic epithelial cells to form tooth structures in cell-polymer constructs maintained in vivo. Enamel organs from 6-month-old porcine third molars were dissociated into single odontogenic epithelial cells and subcultured on feeder layers of 3T3-J2 cells. Amelogenin expression was detected in the subcultured odontogenic epithelial cells by immunostaining and Western blotting. The subcultured odontogenic epithelial cells were seeded onto collagen sponge scaffolds in combination with fresh dental mesenchymal cells, and transplanted into athymic rats. After 4 weeks, enamel-dentin-like complex structures were present in the implanted constructs. These results show that our culture system produced differentiating ameloblast-like cells that were able to secrete amelogenin proteins and form enamel-like tissues in vivo. This application of the subculturing technique provides a foundation for further tooth-tissue engineering and for improving our understanding of ameloblast biology.

The dentin sialoprotein (DSP) expression in rat tooth germs following fluoride treatment: an immunohistochemical study.

UNLABELLED: Fluoride is known to alter expression of dentin matrix proteins and affect their posttranslational modifications. OBJECTIVE: The objective of our study was to examine dentin sialoprotein (DSP) expression in the early and late bell stages of development of the first molar tooth germs in rats treated with fluoride. DESIGN AND METHODS: Pregnant dumps were divided into three groups. They were fed a standard diet and from the fifth day of pregnancy, each group received either tap water (with trace amounts of fluoride), tap water with a low concentration of fluoride, or tap water with a high concentration of fluoride. Changes in DSP expression and distribution were visualized by immunohistochemistry. RESULTS: Immunoreactivity for DSP was detected in the cervical regions of the early bell stage in tooth germs of the 1-day-old animals. The earliest reaction was visible in the control group and the group supplemented with the low fluoride concentration (F(L)) but not in the group supplemented with the high fluoride concentration (F(H)). In early bell stages across all experimental groups, the immunoreactivity to DSP was observed in the cusp tip regions and was localized to preameloblasts, young and mature odontoblasts, dental pulp cells, predentin, and dentin. Generally, more intense positive staining for DSP was detected in animals supplemented with the high fluoride concentration. In the late bell stage found in the 4-day-old control group and the group supplemented with the low fluoride concentration, immunoreactivity for DSP was less intense compared with younger animals. However, immunoreactivity was greater in the group treated with the high dose of fluoride. In this group, the positive immunostaining for DSP, especially in young ameloblasts, was prolonged and relatively strong. CONCLUSIONS: Fluoride supplementation causes changes in the developmental pattern of DSP expression and its distribution in rat tooth germs.

Expression pattern of Dlx3 during cell differentiation in mineralized tissues.

The present study was designed to compare the expression pattern of Dlx3 in four different mineralized tissues because of: 1-its role in skeleton patterning, 2-its expression in dental epithelium and mesenchyme during morphogenesis, 3-the membranous and endochondral bone and tooth phenotype of tricho-dento-osseous syndrome related to Dlx3 gene mutation and 4-recently emerging knowledge on Dlx family members in the bone field. Ameloblasts, odontoblasts, osteoblasts and chondrocytes were analyzed in vitro and in vivo. Dlx3 transcripts were detected by RT-PCR in established model systems (microdissected dental epithelium and mesenchyme; primary cultures of rat chondrocytes), as recently performed in osteoblasts in vitro. A human 414-bp Dlx3 probe was generated. A 4.5-kb human Dlx3 sense RNA was identified in maxillo-facial samples by Northern blotting. Immunolabeling and in situ hybridization were performed in mice from Theiler stage E 14.5 until birth. In teeth, although Dlx3 was still expressed in differentiated ameloblasts, it was down regulated during odontoblast polarization. During endochondral bone formation, Dlx3 protein was detected in chondrocytes and was most strongly expressed in the prehypertrophic cartilage zone and in differentiating and differentiated osteoblasts of metaphyseal periosteum. In vitro, real-time PCR studies supported this upregulation in prehypertrophic chondrocytes, closely correlated with Ihh variations. In membranous bone, Dlx3 was present in preosteoblasts, osteoblasts and osteoid-osteocytes. The present data on Dlx3 and recently published functional studies show that this transcription factor may be instrumental during growth in the control of matrix deposition and biomineralization in the entire skeleton.

Characteristic distribution of immunoreaction for estrogen receptor alpha in rat ameloblasts.

Estrogen has a diverse function, including cell proliferation and differentiation via estrogen receptors (ER), which have been reported to be the case in various tissues in addition to female reproductive organs. A recent immunocytochemical study has reported the expression of ERalpha, a subtype of ER, in rat odontoblasts, suggesting an involvement of estrogen in the differentiation of tooth-forming cells. However, there is no information on the ERalpha immunoexpression in ameloblasts. The present study was therefore undertaken to examine the localization of ERalpha immunoreaction in rat ameloblasts during amelogenesis. A computer-assisted quantitative analysis under a confocal laser scanning microscope was employed to demonstrate the stage-specific localization pattern of ERalpha immunoreaction. Immunohistochemistry of the rat enamel organ revealed ERalpha expression as nuclear localization in ameloblasts, stratum intermedium, stellate reticulum, and papillary layer, in addition to mature and immature odontoblasts. The ratio of immunopositive nuclei to total nuclei (immunopositive ratio) in ameloblasts was high at the apical loop region and gradually declined at the presecretory stage to zero at the secretory stage with statistically significant difference. The ERalpha immunolabeling pattern exhibited a periodic change at the maturation stage proper with constant higher labeling in ruffle-ended ameloblasts than in smooth-ended ameloblasts. The positive ratio was then followed by a statistically significant increase in immunolabeling thereafter. This stage-specific immunolabeling pattern during amelogenesis suggests a possible role of ERalpha in ameloblast proliferation and differentiation.

Immunohistochemical evidence for proteolipid protein and nestin expression in the late bell stage of developing rodent teeth.

In this study, the expression of proteolipid protein (PLP) and nestin is studied in the late bell stage of developing rodent teeth in neonatal rats. By using immunohistochemistry, it was shown that odontoblasts, ameloblasts and the stratum intermedium are positive for PLP in regions of active matrix deposition. Reactivity for nestin could be detected in the odontoblasts, stratum intermedium and in some of the apical processes of the ameloblasts. The fact that mutations in the PLP gene can cause disturbances in tooth form, number and eruption taken together with the presence of PLP reactivity in odontoblasts and ameloblasts of healthy animals, suggests a crucial role for PLP in developing teeth because of its structural supportive characteristics. These results also imply the possible use of PLP antibody as a new marker for, respectively, dentin and enamel-secreting odontoblasts and ameloblasts. PLP and nestin expression could point to a possible similarity in function between the oligodendrocyte and the odontoblast, both derived from the neural crest. To compare with the situation in human tissue, PLP and nestin expression were preliminarily tested on human dental pulp. The odontoblasts were positive for both PLP and nestin.

The expression pattern of hyaluronan synthase during human tooth development.

In previous studies, hyaluronan (HA) and its major cell surface receptor CD44 have been suggested to play an important role during tooth development. HA synthases (HASs) are the enzymes that polymerize hyaluronan. Data on the expression pattern of HASs during tooth development is lacking and the aim of the present study was to investigate the localisation of HAS by immunohistochemistry in human tooth germs from different developmental stages. The distribution pattern of HAS in the various tissues of the "bell stage" tooth primordia corresponded to that of hyaluronan in most locations: positive HAS immunoreactivity was observed in the dental lamina cells, inner- and outer-enamel epithelium. On the stellate reticulum cells, moderate HAS signal was observed, similar to the layers of the oral epithelium, where faint HAS immunoreactivity was detected. At the early phase of dental hard tissues mineralization, strong HAS immunoreactivity was detected in the odontoblasts and their processes, as well as in the secretory ameloblasts and their apical processes and also, the pulpal mesenchymal cells. The HAS signals observed in odontoblasts and ameloblasts gradually decreased with age. Our results demonstrate that hyaluronan synthesised locally by different dental cells and these results provide additional indirect support to the suggestion that HA may contribute both to the regulation of tooth morphogenesis and dental hard tissue formation.

The enamelin (tuftelin) gene.

This paper reviews the primary structure, characteristics and possible function of tuftelin/enamelin protein. It describes the distribution of tuftelin in the ameloblast cell and in the extracellular enamel matrix, employing high resolution protein-A gold immunocytochemistry. The chromosomal localization of the human tuftelin gene and its possible involvement in autosomally linked Amelogenesis Imperfecta, the most common hereditary disease of enamel, is also discussed.

Ameloblastin expression in rat incisors and human tooth germs.

We recently identified ameloblastin as an ameloblast-specific gene product from a rat incisor cDNA library (Krebsbach et al., J. Biol. Chem. 271: 4431-4435, 1996). Here we report the developmental pattern of expression of ameloblastin in rat incisors and human tooth germs as visualized by in situ hybridization and immunochemistry. Compared to the expression of amelogenin, the major ameloblast product, ameloblastin mRNA was more widely expressed in ameloblasts from the presecretory to the late maturation stage of development. Ameloblastin mRNA was first observed in the juxtanuclear cytoplasm or presecretory stage ameloblasts, gradually increased in the distal cytoplasm of secretory stage ameloblasts and was found throughout the cytoplasm of early to late maturation stage ameloblasts. The immunostaining of ameloblastin, using a monospecific antibody raised against a recombinant protein, showed intense reactivity in Tomes' processes of secretory stage ameloblasts and surrounding enamel. The immunoreaction was concentrated in the juxtanuclear cytoplasm of late maturation stage ameloblasts. High-resolution colloidal gold immunocytochemistry established the presence of ameloblastin antigenicity in the Golgi apparatus, secretory granules in Tomes' process and enamel. Human tooth germs in early to late bell stage also expressed ameloblastin mRNA and ameloblastin antigenicity in the ameloblasts. Western blot analysis of protein extracts from rat incisor tissues indicated that ameloblastin can be found in the enamel epithelial tissue and in mineralized enamel, as well as in the EDTA decalcification solution. These data indicate that ameloblastin is an ameloblast secretory product which is sequentially expressed from the presecretory to the late maturation stage in rat and human teeth. This unique developmental pattern suggests that ameloblastin may have a broader role in amelogenesis than amelogenin and tuftelin.

Amelogenins as potential buffers during secretory-stage amelogenesis.

Amelogenins are the most abundant protein species in forming dental enamel, taken to regulate crystal shape and crystal growth. Unprotonated amelogenins can bind protons, suggesting that amelogenins could regulate the pH in enamel in situ. We hypothesized that without amelogenins the enamel would acidify unless ameloblasts were buffered by alternative ways. To investigate this, we measured the mineral and chloride content in incisor enamel of amelogenin-knockout (AmelX(-/-)) mice and determined the pH of enamel by staining with methyl-red. Ameloblasts were immunostained for anion exchanger-2 (Ae2), a transmembrane pH regulator sensitive for acid that secretes bicarbonate in exchange for chloride. The enamel of AmelX(-/-) mice was 10-fold thinner, mineralized in the secretory stage 1.8-fold more than wild-type enamel and containing less chloride (suggesting more bicarbonate secretion). Enamel of AmelX(-/-) mice stained with methyl-red contained no acidic bands in the maturation stage as seen in wild-type enamel. Secretory ameloblasts of AmelX(-/-) mice, but not wild-type mice, were immunopositive for Ae2, and stained more intensely in the maturation stage compared with wild-type mice. Exposure of AmelX(-/-) mice to fluoride enhanced the mineral content in the secretory stage, lowered chloride, and intensified Ae2 immunostaining in the enamel organ in comparison with non-fluorotic mutant teeth. The results suggest that unprotonated amelogenins may regulate the pH of forming enamel in situ. Without amelogenins, Ae2 could compensate for the pH drop associated with crystal formation.

Immunolocalization of CD44 and the ezrin-radixin-moesin (ERM) family in the stratum intermedium and papillary layer of the mouse enamel organ.

We studied the immunohistochemical localization of CD44 and the ezrin-radixin-moesin (ERM) family of actin binding proteins in mouse enamel organ, using confocal laser scanning microscopy and transmission electron microscopy to clarify their role in cytoskeletal organization. At the differentiation stage of ameloblasts, immunoreactivity to CD44 was detected on the plasma membrane of the inner enamel epithelium, the stellate reticulum, the stratum intermedium, and the external enamel epithelium. In accordance with the differentiation of preameloblasts into secretory ameloblasts, immunoreactivity increased in the stratum intermedium cells. At the maturation stage, intense immunoreactivity was observed on the papillary layer cells. For the ERM family, the stratum intermedium and the papillary layer cells were stained with anti-ezrin and -radixin monoclonal antibodies but not with the anti-moesin antibody. Electron microscopic observations revealed that CD44, ezrin, and radixin were localized in the region at which preameloblasts came into contact with the stratum intermedium at the differentiation stage. At the secretory and maturation stages, they were concentrated in the microvilli of the stratum intermedium and the papillary layer cells. These findings suggest that the CD44-ezrin-radixin-actin filament system is involved in cell-cell interaction between preameloblasts and the stratum intermedium, and in the cytoskeletal organization of the cells in the stratum intermedium and the papillary layer.

Immunohistochemical similarities and differences between amelogenin and tuftelin gene products during tooth development.

Amelogenins and tuftelins are highly specialized proteins secreted into the developing enamel matrix during mammalian enamel formation. Both tuftelins and amelogenins have been associated with various functions during nucleation and maturation of the developing enamel matrix. In this study we conducted experiments to investigate whether tuftelins and portions of the amelogenin molecule were deposited and processed in spatially distinguished portions of the developing enamel matrix, using antibodies specific against tuftelin or amelogenins. The amelogenin antibodies were raised against recombinant and native amelogenins and also included an antibody against a polypeptide encoded by amelogenin exon 4. To compare spatial expression patterns of enamel protein epitopes, 3-day postnatal mouse molar tooth organs were processed for paraffin histology and cut into serial sections. Adjacent sections were exposed to antibodies against either tuftelin or various amelogenin epitopes. To investigate age-related changes of enamel protein expression, amelogenin and tuftelin antibodies were applied to tooth organs of developmental stages E19 and 1, 3, 5, 7, 9 and 11 postnatal days. Tuftelin was detected within the odontoblast processes during earlier stages of development (E19 and 1 day postnatal), whereas during later stages (3-11 days) it was recognized in a portion of the enamel layer adjacent to the dentine-enamel junction. In contrast, all four antibodies against amelogenins reacted with parts of the ameloblast cytoplasm and the entire enamel layer. Using immunohistochemistry, we were not able to detect any differences in the spatial distribution of the four amelogenin epitopes investigated. The spatial differences in the distribution of amelogenin and tuftelin as observed in this study may be interpreted as an indication of functional differences between both proteins during early enamel biomineralization.

Distribution of protein kinase C alpha and accumulation of extracellular Ca2+ during early dentin and enamel formation.

Activation of the protein kinase C (PKC)-related signal transduction system has been associated with phenotypic expression in a wide variety of cell types. In in vitro studies, it has often been activated by relatively small increases in the Ca2+ concentration ([Ca2+]) in the medium. The studies reported here explored the hypothesis that localized increases in the extracellular [Ca2+] and activation of the PKC-related pathway may be involved in early dentin and enamel formation. Whole-head, freeze-dried sections through the developing molars of 5-day-old rats were evaluated by methods that localized non-crystalline Ca2+. Immunohistochemical methods were adapted for use with the freeze-dried sections, and two monoclonal antibodies were used to localize PKC alpha in the formative cells of the developing teeth. Low concentrations of extracellular Ca2+ were observed in the early, unmineralized dentin in the area of ameloblast differentiation. Increased concentrations occurred at the point of initial dentin mineralization, immediately before the beginning of enamel matrix deposition. PKC alpha was localized in the differentiating odontoblasts, at the beginning of dentin matrix deposition. It was intensely localized in the distal borders of the pre-ameloblasts, and appeared to redistribute in the cells during ameloblast differentiation. These observations suggest that local increases in the extracellular [Ca2+] and the PKC signal transduction pathway may be involved in key inductions in the early stages of dentin and enamel formation.

Immunohistochemical localization of Galphaq, PLCbeta, Galphai1-2, PKA, and the endothelin B and extracellular Ca2+-sensing receptors during early amelogenesis.

Antibodies specific to Galphaq, PLCbeta, Galphai 1-2, and PKA were immunohistochemically (IHC) localized in the pre-ameloblasts up to initial dentin matrix deposition and continued in the distal ends of the pre-secretory ameloblasts to the beginning of enamel matrix secretion. It was hypothesized that the endothelin B receptor (ETBR) and/or the extracellular Ca2+-sensing receptor (CaR) would localize in the same locations as their known downstream signal transduction pathway (STP) effectors during events related to early amelogenesis. Localization was similar for the 4 signal transduction pathway elements and the CaR. The ETBR was not localized in any of the cells of the enamel organ. These findings indicate that the CaR and its related STPs are expressed in the pre-ameloblasts and pre-secretory ameloblasts in positions where they may be able to detect increases in extracellular Ca2+ concentrations observed in the pre-dentin matrix in a previous study. These observations are consistent with the hypothesis that increased levels of free Ca2+ in the pre-dentin matrix serve as a primary signal for modification of gene expression important to amelogenesis.

Immunohistochemical localization of connexin 43 in the developing tooth germ of rat.

Distribution of gap junction protein in maxillary tooth germs of 1-day-old rats was examined by immunohistochemistry, using an affinity-purified antibody specific to residues 360-376 of rat connexin (CX) 43. In 1-day-old rats, the maxillary second molar formed the shape of the cusp, but neither dentine nor enamel was formed between the cells of the dental papilla and the inner enamel epithelium. In the tooth germ, CX 43 was expressed in the cells of the stratum intermedium and the inner enamel epithelium. Labelling in the stratum intermedium was extensive and showed an increasing gradient from peripheral to cuspal regions. CX 43 detected in the inner enamel epithelium was at cell surfaces facing the interface between the dental papilla and the inner enamel epithelium. The cells of the dental papilla and the inner enamel epithelium began differentiation as odontoblasts and secretory ameloblasts respectively, in the cusps of the first molars, where predentine and dentine were formed but enamel matrix was not secreted. CX 43 was present in the stratum intermedium, inner enamel epithelium, preodontoblasts, odontoblasts and subodontoblasts. The incisors showed the most advanced stage of development, where the enamel matrix and calcified dentine were formed in the labial part of the teeth. The CX 43 epitope was seen in the stratum intermedium, inner enamel epithelium, preameloblasts, preodontoblasts, odontoblasts, and subodontoblasts. Immunolabelling was more extensive in the stratum intermedium and subodontoblasts than in preameloblasts, preodontoblasts, and odontoblasts. The immunolabelling in preameloblasts and predontoblasts was accumulated at cell surfaces facing the predentine.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunoblotting and cytochemical characterization of human enamel proteins.

Mature enamel proteins (tuft proteins) and fetal enamel proteins were extracted by an homogenizing buffer method, subjected to SDS-PAGE and immunoblotted with a polyclonal antibody raised against the mature enamel proteins. Both fetal and tuft proteins were recognized by this immunoblotting. With the same antibody, immunolocalization of the developing enamel proteins was done on semi-thin-sections of human fetal tissue at the secretory stage, using an immunoperoxidase technique. Specific labelling of the enamel protein matrix was observed. It is concluded that a polyclonal antibody against mature enamel proteins (anti-tuft) can recognize the developing protein matrix at the secretory stage. This suggests that a common antigenic determinant is maintained throughout the course of amelogenesis in human enamel.

The distribution pattern of the hyaluronan receptor CD44 during human tooth development.

The aim was to investigate the expression pattern of the major cell-surface hyaluronan receptor CD44, as there are no existing data on its presence or absence in human dental structures at different developmental stages. Immunohistochemical localization of CD44 was studied using a monoclonal antibody, H3, that specifically recognizes an epitope in the common backbone of all CD44 isoforms. The dental lamina displayed a strong CD44 signal; the external enamel epithelium was negative. In the coronal region of the tooth germ the presecretory ameloblasts showed an intense reaction whereas the less differentiated inner enamel epithelial cells showed no signal at the cervical loop where they meet the external enamel epithelium. In the stellate reticulum a moderate reaction was detected. The secretory ameloblasts and the stratum intermedium showed a strong cell-surface CD44 signal. A strong signal was also observed on the odontoblasts and their processes. In the pulp, close to the odontoblastic layer, weak labelling was seen in the walls of capillary vessels. The distribution of CD44 in the human tooth germ corresponds to that of hyaluronan in most locations, suggesting that during tooth development this transmembrane protein plays an important part in hyaluronan-mediated events.

Developmental pattern and subcellular localization of parvalbumin in the rat tooth germ.

The EF-hand calcium-binding protein parvalbumin has been extensively studied in nerve and muscle cells. Its possible role in biomineralization during tooth development was here investigated by determining its subcellular localization by immunogold cytochemistry. The developmental sequences of amelogenesis and dentinogenesis were studied in rat molars, and in continuously growing rat incisors. The findings confirm that parvalbumin is a nuclear and a cytosolic protein, not associated with any particular intracellular organelle. Epithelial and mesenchymal undifferentiated cells contained no specific parvalbumin immunolabelling. In differentiated ameloblasts, secretory-pole (Tomes' process) formation was associated with a proximal-distal gradient of parvalbumin labelling. But after the Tomes' process had formed, parvalbumin was evenly distributed throughout the cell. The parvalbumin contents of ruffle-ended and smooth-ended ameloblasts appeared to be very different. Differentiated odontoblasts were less heavily labelled than ameloblasts, and the label was restricted to the cell body during the whole of dentinogenesis. These data suggest that parvalbumin could contribute to membrane plasticity during differentiation, as shown during dendritic growth in the nervous cells. Moreover, as may occur in excitable cells, parvalbumin could buffer calcium specifically in the cells producing mineralized enamel and dentine during the later stages of tooth development.