Tooth formation and the 28,000-dalton vitamin D-dependent calcium-binding protein: an immunocytochemical study

Antiserum to the 28-kilodalton vitamin D-dependent calcium-binding protein (CaBP) was used to localize CaBP in histologic sections of the continuously erupting incisor in mandibles obtained from normal rats. With the peroxidase--anti-peroxidase technique, no CaBP was detected in undifferentiated ameloblasts or in those which had become columnar and were facing pulp. Calcium-binding protein was first noted in the cytoplasm of random ameloblasts facing dentin in the presecretion zone. As the ameloblasts became more mature in the zone of enamel secretion, CaBP was uniformly present in their cytoplasm. Ameloblasts with Tome's processes clearly contained CaBP in these processes as well as in the cell-body cytoplasm. Near the later developmental stages of the zone of enamel secretion, some of the adjacent underlying cells of the stratum intermedium also contained CaBP in their cytoplasm. In some stratum intermedium cells and papillary cells, CaBP extended into the zone of enamel maturation, but not to the end of that zone. Cytoplasmic CaBP continued to be present in ameloblasts as they progressed through the zone of enamel maturation to the final, shortened cells at the gingival margin of the erupting incisor. No CaBP was detected in odontoblasts, pulpal cells, the stellate reticulum, or the outer dental epithelium.     

Urokinase-type Plasminogen Activator mRNA is Expressed in Normal Developing Teeth and Leads to Abnormal Incisor Enamel in αMUPA Transgenic Mice

The urokinase-type plasminogen activator (uPA) is a secreted, inducible serine protease implicated in extracellular proteolysis and tissue remodeling. Here we detected uPA mRNA through in situ hybridization in developing molar and incisor teeth of normal mice at multiple sites of the cap and bell developmental stages. The mRNA was confined to epithelial cells, however, was undetectable in ameloblasts or their progenitor preameloblasts and the inner enamel epithelium. Furthermore, mice of five lines of previously described αMUPA transgenic mice, carrying a transgene consisting of the uPA cDNA linked downstream from the αA-crystallin promoter, overexpressed uPA mRNA in the same epithelial sites. In addition, αMUPA mice showed remarkably high levels of uPA mRNA in ameloblasts, however, exclusively in two specific sites late in incisor development. First, at the late secretory stage, but only on sides of the ameloblast layer. Second, in a limited zone of ameloblasts near the incisal end, coinciding with a striking morphological change of the ameloblast layer and the enamel matrix. In adult αMUPA mice, the incisor teeth displayed discoloration and tip fragility, and reduction of the outer enamel as determined by scanning electron microscopy. These results suggest that balanced uPA activity could play a role in normal tooth development. The αMUPA tooth phenotype demonstrates a remarkable sensitivity to excessive extracellular proteolysis at the incisor maturation stage of amelogenesis.     

Rat forming incisor requires a rigorous ECM remodeling modulated by MMP/RECK balance

Reversion-inducing-cysteine-rich protein with Kazal motifs (RECK) is a single membrane-anchored MMP-regulator and regulates matrix metalloproteinases (MMP) 2, 9 and 14. In turn, MMPs are endopeptidases that play a pivotal role in remodeling ECM. In this work, we decided to evaluate expression pattern of RECK in growing rat incisor during, specifically focusing out amelogenesis process. Based on different kinds of ameloblasts, our results showed that RECK expression was conducted by secretory and post-secretory ameloblasts. At the secretory phase, RECK was localized in the infra-nuclear region of the ameloblast, outer epithelium, near blood vessels, and in the stellate reticulum. From the transition to the maturation phases, RECK was strongly expressed by non-epithelial immuno-competent cells (macrophages and/or dendritic-like cells) in the papillary layer. From the transition to the maturation stage, RECK expression was increased. RECK mRNA was amplified by RT-PCR from whole enamel organ. Here, we verified the presence of RECK mRNA during all stages of amelogenesis. These events were governed by ameloblasts and by non-epithelial cells residents in the enamel organ. Concluding, we found differential expression of MMPs-2, -9 and RECK in the different phases of amelogenesis, suggesting that the tissue remodeling is rigorously controlled during dental mineralization.     

Ameloblast differentiation in the human developing tooth: Effects of extracellular matrices

Tooth enamel is formed by epithelially-derived cells called ameloblasts, while the pulp dentin complex is formed by the dental mesenchyme. These tissues differentiate with reciprocal signaling interactions to form a mature tooth. In this study we have characterized ameloblast differentiation in human developing incisors, and have further investigated the role of extracellular matrix proteins on ameloblast differentiation. Histological and immunohistochemical analyses showed that in the human tooth, the basement membrane separating the early developing dental epithelium and mesenchyme was lost shortly before dentin deposition was initiated, prior to enamel matrix secretion. Presecretary ameloblasts elongated as they came into contact with the dentin matrix, and then shortened to become secretory ameloblasts. In situ hybridization showed that the presecretory stage of odontoblasts started to express type I collagen mRNA, and also briefly expressed amelogenin mRNA. This was followed by upregulation of amelogenin mRNA expression in secretory ameloblasts. In vitro, amelogenin expression was upregulated in ameloblast lineage cells cultured in Matrigel, and was further up-regulated when these cells/Matrigel were co-cultured with dental pulp cells. Co-culture also up-regulated type I collagen expression by the dental pulp cells. Type I collagen coated culture dishes promoted a more elongated ameloblast lineage cell morphology and enhanced cell adhesion via integrin α2β1. Taken together, these results suggest that the basement membrane proteins and signals from underlying mesenchymal cells coordinate to initiate differentiation of preameloblasts and regulate type I collagen expression by odontoblasts. Type I collagen in the dentin matrix then anchors the presecretary ameloblasts as they further differentiate to secretory cells. These studies show the critical roles of the extracellular matrix proteins in ameloblast differentiation.     

Light and electron microscopical immunohistochemical localization of large proteoglycans in human tooth germs at the bell stage

Summary The immunohistochemical localization of large hyaluronate-binding proteoglycans has been studied in human tooth germs at the bell stage using a monoclonal antibody, 5D5, which is derived from bovine sclera and specifically recognizes the core protein of large proteoglycans, such as versican, neurocan and brevican, but not that of aggrecan. In the early bell stage before predentine secretion, when the enamel organs consisted of the inner and outer enamel epithelia, stratum intermedium and stellate reticulum, the enamel organs were not stained by 5D5, but the dental papillae and follicles stained strongly. Concomitant with the secretion of predentine, dentine and subsequent enamel matrix, strong 5D5 immunostaining distributed over the entire cell surfaces of secretory ameloblasts was observed. The forming enamel matrix showed strong staining. While most of the inner and outer enamel epithelia and stratum intermedium lacked staining, the cervical loop region and stellate reticulum showed weak staining. Although the forming dentine and odontoblasts appeared to lack 5D5 affinity, the predentine, dental papilla and dental follicle demonstrated moderate to strong reactivity. At the ultrastructural level, specific immunoreaction by immunogold particle deposition was clearly detected over the basal lamina of presecretory ameloblasts, secretion granules of secretory ameloblasts and the forming enamel matrix. These results indicate that a marked increase in the large proteoglycan associated with secretory ameloblasts may correlate with cell differentiation and enamel matrix biosynthesis. This revised version was published online in November 2006 with corrections to the Cover Date.     

Spatial and temporal expression of histone demethylase,Kdm2a,during murine molar development

The histone demethylase, lysine (K)-specific demethylase 2A (Kdm2a), is highly conserved and expressed ubiquitously. Kdm2a can regulate cell proliferation and osteo/dentinogenic, adipogenic and chondrogenic differentiation of mesenchymal stem cells (MSCs) derived from dental tissue. We used quantitative real-time RT-PCR analysis and immunohistochemistry to detect Kdm2a expression during development of the murine molar at embryonic days E12, E14, E16 and E17 and postnatal days P3 and P14. Immunohistochemistry results showed no positive staining of Kdm2a at E12. At E14, Kdm2a was expressed weakly in the inner enamel epithelium, stellate reticulum cells and dental sac. At E16, Kdm2a was expressed mainly in the inner and outer enamel epithelium, stratum intermedium and dental sac, but weaker staining was found in cervical loop and dental papilla cells adjacent to the basement membrane. At E17, the strongest Kdm2a staining was detected in the ameloblasts and stronger Kdm2a staining also was detected in the stratum intermedium, outer enamel epithelium and dental papilla cells compared to the expression at E16. Postnatally, we found that Kdm2a was localized in secretory and mature ameloblasts and odontoblasts, and dentin was unstained. Real-time RT-PCR showed that Kdm2a mRNA levels in murine germ cells increased from E12 to E14 and from E14 to E16; no significant change occurred at E16, E17 or P3, then the levels decreased at P14 compared to P3. Kdm2a expression may be closely related to cell proliferation, to ameloblast and odontoblast differentiation and to the secretion of extracellular enamel and dentin during murine tooth development.     

Establishment of primary cultures for mouse ameloblasts as a model of their lifetime

To understand how the properties of ameloblasts are spatiotemporally regulated during amelogenesis, two primary cultures of ameloblasts in different stages of differentiation were established from mouse enamel epithelium. Mouse primary ameloblasts (MPAs) prepared from immature enamel epithelium (MPA-I) could proliferate, whereas those from mature enamel epithelium (MPA-M) could not. MPA-M but not MPA-I caused apoptosis during culture. The mRNA expression of amelogenin, a marker of immature ameloblasts, was down-regulated, and that of enamel matrix serine proteiase-1, a marker of mature ameloblasts, was induced in MPA-I during culture. Using green fluorescence protein as a reporter, a visualized reporter system was established to analyze the promoter activity of the amelogenin gene. The region between -1102bp and -261bp was required for the reporter expression in MPA-I. These results suggest that MPAs are valuable in vitro models for investigation of ameloblast biology, and that the visualized system is useful for promoter analysis in MPAs.     

Dynamic assembly of tight junction-associated proteins ZO-1, ZO-2, ZO-3 and occludin during mouse tooth development

Tight junctions might play a role during tissue morphogenesis and cell differentiation. In order to address these questions, we have studied the distribution pattern of the tight junction-associated proteins ZO-1, ZO-2, ZO-3 and occludin in the developing mouse tooth as a model. A specific temporal and spatial distribution of tight junction-associated proteins during tooth development was observed. ZO-1 appeared discontinuously in the cell membrane of enamel organ and dental mesenchyme cells. However, endothelial cells of the dental mesenchyme capillaries displayed a continuous fluorescence at the cell membrane. Inner dental epithelium first showed an evident signal for ZO-1 at the basal pole of the cells at bud/cap stage, but ZO-1 was accumulated at the basal and apical pole of preameloblast/ameloblasts at late bell stage. Surprisingly, in the incisor ZO-1 decreased as the inner dental epithelium differentiated, and was re-expressed in secretory and mature ameloblasts. On the contrary, ZO-2 was confined to continuous cell-cell contacts of the enamel organ in both molars and incisors. The lateral cell membrane of inner dental epithelial cells was specifically ZO-2 labeled. However, ZO-3 was expressed in oral epithelium whereas dental embryo tissues were negative. In addition, occludin was hardly detected in dental tissues at the early stage of tooth development, but was distributed continuously at the cell membrane of endothelial cells of ED19.5 dental mesenchyme. In incisors, occludin was detected at the cell membrane of the secretory pole of ameloblasts. The occurrence and relation during tooth development of tight junction proteins ZO-1, ZO-2 and occludin, but not ZO-3, suggests a combinatory assembly in tooth morphogenesis and cell differentiation.     

Pleiotrophin expression during odontogenesis

Pleiotrophin (PTN) is an extracellular matrix-associated growth factor and chemokine expressed in mesodermal and ectodermal cells. It plays an important role in osteoblast recruitment and differentiation. There is limited information currently available about PTN expression during odontoblast differentiation and tooth formation, and thus the authors aimed to establish the spatiotemporal expression pattern of PTN during mouse odontogenesis. Immortalized mouse dental pulp (MD10-D3, MD10-A11) and odontoblast-like (M06-G3) and ameloblast-like (EOE-3M) cell lines were grown and samples prepared for immunocytochemistry, Western blot, and conventional and quantitative PCR analysis. Effects of BMP2, BMP4, and BMP7 treatment on PTN expression in odontoblast-like M06-G3 cells were tested by quantitative PCR. Finally, immunohistochemistry of sectioned mice mandibles and maxillaries at developmental stages E16, E18, P1, P6, P10, and P28 was performed. The experiments showed that PTN, at both the mRNA and protein level, was expressed in all tested epithelial and mesenchymal dental cell lines and that the level of PTN mRNA was influenced differentially by the bone morphogenetic proteins. The authors observed initial expression of PTN in the inner enamel epithelium with prolonged expression in the ameloblasts and odontoblasts throughout their stages of maturation and strong expression in the terminally differentiated and enamel matrix-secreting ameloblasts and odontoblasts of the adult mouse incisors and molars.     

Localization of actin during differentiation of the ameloblast, its related epithelial cells and odontoblasts in the rat incisor using NBD-phallacidin

Using NBD-phallacidin, which specifically binds to F-actin, we investigated changes in the localization of actin during the differentiation of ameloblasts, related epithelial cells and odontoblasts in rat incisors. In cryosections treated with NBD-phallacidin, intense fluorescence was observed in undifferentiated epithelial cells in the apical loop and at the proximal extremity of undifferentiated inner enamel epithelial cells. During differentiation, the distal extremity began to exhibit strong fluorescence. In cross-sections of secretory ameloblasts, the fluorescence took the form of polygons of uniform intensity at the proximal end, and of rectangles of non-uniform intensity at the distal end. At the distal end, the fluorescence was more intense at right angles to the long axis of the incisor. At the distal end, this pattern was established just before the appearance of the enamel layer. These patterns were maintained during the secretory stage of ameloblasts. The location, pattern and time of appearance of these sites were identical to those of the terminal webs in ameloblasts. NBD-phallacidin weakly labelled the peripheral cytoplasm of the cell body of ameloblasts, and also labelled Tomes' process. The cells forming the stratum intermedium were mainly labelled at their periphery (i.e. forming larger polygons), while the overlying epithelial cells exhibited labelling throughout their cytoplasm. Except for the terminal webs, the cell bodies of odontoblasts were weakly labelled throughout the period of differentiation. Young odontoblasts secreting predentin were first labelled on the terminal web, with the fluorescence becoming gradually more intense as the thickness of the dentin increased.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression and localization of laminin 5, laminin 10, type IV collagen,and amelotin in adult murine gingiva

The biochemical composition of the internal and external basal laminae in the junctional epithelium differs significantly, and the precise cellular origin of their respective molecules remains to be determined. In the present study, the expression and localization of three basement membrane-specific molecules—laminin 5 (γ2 chain), type IV collagen (α1 chain), and laminin 10 (α5 chain)—and one tooth-specific molecule, amelotin, was analyzed in adult murine gingiva by using in situ hybridization and immunohistochemistry. The results showed that the outermost cells in junctional epithelium facing the tooth enamel strongly expressed laminin 5 mRNA, supporting the immunohistochemical staining data. This suggests that laminin 5 is actively synthesized in junctional epithelial cells and that the products are incorporated into the internal basal lamina to maintain firm epithelial adhesion to the tooth enamel throughout life. Conversely, no amelotin mRNA signals were detected in the junctional epithelial cells, suggesting that the molecules localized on the internal basal lamina are mainly derived from maturation-stage ameloblasts. Weak and sporadic expression of type IV collagen in addition to laminin 10 in the gingiva indicates that these molecules undergo turnover less frequently in adult animals.     

Itm2a Expression in the Developing Mouse First Lower Molar,and the Subcellular Localization of Itm2a in Mouse Dental Epithelial Cells

Itm2a is a type II transmembrane protein with a BRICHOS domain. We investigated the temporospatial mRNA and protein expression patterns of Itm2a in the developing lower first molar, and examined the subcellular localization of Itm2a in murine dental epithelial (mDE6) cells. From the initiation to the bud stage, the in situ and protein signals of Itm2a were not detected in either the dental epithelial or mesenchymal cells surrounding the tooth bud. However, at the bell stage, these signals of Itm2a were primarily observed in the inner enamel epithelium of the enamel organ. After the initiation of the matrix formation, strong signals were detected in ameloblasts and odontoblasts. Itm2a showed a punctate pattern in the cytoplasm of the mDE6 cells. The perinuclear-localized Itm2a displayed a frequent overlap with the Golgi apparatus marker, GM130. A tiny amount of Itm2a was colocalized with lysosomes and endoplasmic reticulum. Minimal or no overlap between the Itm2a-EGFP signals with the other organelle markers for endoplasmic reticulum, lysosome and mitochondria used in this study noted in the cytoplasm. These findings suggest that Itm2a may play a role in cell differentiation during odontogenesis, rather than during the initiation of tooth germ formation, and may be related to the targeting of proteins associated with enamel and dentin matrices in the secretory pathway.     

In situ expression of 15 kDa interferon alpha responsive gene in the developing tooth germ of the mouse lower first molar

We previously performed cDNA subtraction between the mouse mandibles at embryonic day 10.5 (E10.5) and E12.0 to make a profile of the regulator genes for odontogenesis. Fifteen kDa interferon alpha responsive gene (Ifrg15) is one of several highly-expressed genes in the E12.0 mandible. The current study examined the precise expression patterns of Ifrg15 mRNA in the mouse mandibular first molar by in situ hybridization to evaluate the possible functional roles of this gene in odontogenesis. Ifrg15 mRNA was expressed in the epithelial and mesenchymal tissues of the mandible at E10.5 and E12.0. The Ifrg15 in situ signal was detected in the epithelial bud and the surrounding mesenchyme at E14.0, and was present in the enamel organ including the primary enamel knot, and in the underlying mesenchyme at E15.0. The in situ signal was restricted in the inner and outer enamel epithelia and the stratum intermedium at E16.0. The signal of Ifrg15 mRNA was further restricted to the inner enamel epithelium and the adjacent stratum intermedium at E17.0 and E18.0. Consequently, the expression of Ifrg15 mRNA was localized in the ameloblasts and odontoblasts at postnatal days 1.0 to 3.0. However, the in situ signal was markedly weaker than at the embryonic period. The expression of Ifrg15 mRNA was coincidently observed in various craniofacial organs as well as in the tooth germ. These results suggest that Ifrg15 is closely related to odontogenesis, especially the differentiation of the ameloblasts and odontoblasts, and to the morphogenesis of the craniofacial organs.     

Expression and localization of Nell-1 during murine molar development

Nel-like molecule-1 (Nell-1) is a recently discovered secreted protein that plays an important role in osteoblast differentiation, bone formation, and bone regeneration. However, its expression and distribution during tooth development are largely unknown. The aim of this study was to investigate the expression patterns of Nell-1 during murine molar development by immunohistochemistry. Nell-1 protein was expressed during molar development in embryonic and postnatal Kunming mice, but its expression levels and patterns at various developmental stages differed. At embryonic day 13.5 (E13.5) and E14.5, Nell-1 was found in both the entire enamel organ and the underlying mesenchyme. At E16.5, it was detected in the inner and outer enamel epithelia, stratum intermedium, secondary enamel knot, and dental papilla. At E18.5, Nell-1 was expressed in the differentiating ameloblasts, differentiating odontoblasts, and stratum intermedium. Positive staining was also found in the outer enamel epithelium. At postnatal day 2.5 (P2.5), P5, and P7, Nell-1 appeared in the secretory and mature ameloblasts and odontoblasts (odontoblastic bodies and processes) as well as immature enamel. Hertwig’s epithelial root sheath also stained positively at P7. At P13.5, positive staining was restricted to the reduced dental epithelium and odontoblasts, whereas Nell-1 disappeared in the mature enamel. During tooth eruption, Nell-1 was observed only in the odontoblastic bodies, odontoblastic processes, and endothelial cells of blood vessels. The spatiotemporal expression patterns of Nell-1 during murine tooth development suggest that it might play an important role in ameloblast and odontoblast differentiation, secretion and mineralization of the extracellular enamel matrix, molar crown morphogenesis, as well as root formation.     

Immunocytochemical and immunochemical study of enamelins, using antibodies against porcine 89-kDa enamelin and its N-terminal synthetic peptide, in porcine tooth germs

Enamelins comprise an important family of the enamel matrix proteins. Porcine tooth germs were investigated immunochemically and immunocytochemically using two antibodies: a polyclonal antibody raised against the porcine 89-kDa enamelin (89 E) and an affinity purified anti-peptide antibody against the porcine enamelin amino-terminus (EN). Immunochemical analysis of layers of immature enamel from the matrix formation stage detected immunopositive protein bands ranging from 10 kDa to 155 kDa in the outer layer enamel sample irrespective of the antibodies used. In contrast, the middle and inner enamel layer mainly contained lower molecular weight enamelins. In immunocytochemical analyses of the differentiation stage, 89 E stained enamel matrix islands around mineralized collagen fibrils of dentin, while EN stained both enamel matrix islands and stippled material. At the matrix formation stage, both antibodies intensely stained enamel prisms located in the outer layer. In the inner layer, 89 E moderately stained enamel matrix homogeneously, while EN primarily stained the prism sheath. The intense immunoreaction over the surface layer of enamel matrix at the matrix formation stage, following staining with 89 E and EN, disappeared by the end of the transition stage and the early maturation stage, respectively. The Golgi apparatus and secretory granules in the ameloblasts from the late differentiation stage to the transition stage were immunostained by both antibodies. These results suggest that expression of enamelin continues from late differentiation to the transition stage and the cleavage of N-terminal region of enamelin occurs soon after secretion. Some enamelin degradation products, which apparently have no affinity for hydroxyapatite crystals, concentrate in the prism sheaths during enamel maturation.