Changes in the matrix proteins, fibronectin and collagen, during differentiation of mouse tooth germ.

The distribution of the matrix protein fibronectin was studied by indirect immunofluorescence in differentiating mouse molars from bud stage to the stage of dentin and enamel secretion, and compared to that of collagenous proteins procollagen type III and collagen type I. Fibronectin was seen in mesenchymal tissue, basement membranes, and predentin. The dental mesenchyme lost fibronectin staining when differentiating into odontoblasts. Fibronectin was not detected in mineralized dentin. Epithelial tissues were negative except for the stellate reticulum within the enamel organ. Particularly intense staining was seen at the epithelio-mesenchymal interface between the dental epithelium and mesenchyme. Fibronectin may here be involved in anchorage of the mesenchymal cells during their differentiation into odontoblasts. Procollagen type III was lost from the dental mesenchyme during odontoblast differentiation but reappeared with advancing vascularization of the dental papilla. Similarly, procollagen type III present in the dental basement membrane during the bud and cap stages disappeared from the cuspal area along with odontoblast differentiation. Weak staining was seen in predentin but not in mineralized dentin. The staining with anti-collagen type I antibodies was weak in dental mesenchyme but intense in predentin as well as in mineralized dentin.     

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.     

Epithelial-Directed Mesenchyme Differentiation in vitro

To assess the requirement for specific or possibly non-specific epithelial instructions for mesenchymal cell differentiation, we designed studies to evaluate and compare homotypic with heterotypic tissue recombinations across vertebrate species. These studies further tested the hypothesis that determined dental papilla mesenchyme requires epithelial-derived instructions to differentiate into functional odontoblast cells using a serumless, chemically-defined medium. Theiler stage 25 C57BL/6 or Swiss Webster cap stage mandibular first molar tooth organs or trypsin-dissociated, homotypic epithelial-mesenchymal tissue recombinants resulted in the differentiation of odontoblasts within 3 days. Epithelial differentiation into functional ameloblasts was observed within 7 days. Trypsin-dissociated and isolated mesenchyme did not differentiate into odontoblasts under these experimental conditions. Heterotypic recombinants between quail Hamburger-Hamilton stages 22–26 mandibular epithelium and Theiler stage 25 dental papilla mesenchyme routinely resulted in odontoblast differentiation within 3 days in vitro. Odontoblast differentiation and the production of dentine extracellular matrix continued throughout the 10 days in organ culture. Ultrastructural observations of the interface between quail and mouse tissues indicated the reconstitution of the basal lamina as well as the maintenance of an intact basal lamina during 10 days in vitro. Quail epithelial cells did not differentiate into ameloblasts and no enamel extracellular matrix was observed. These results show that quail mandibular epithelium can provide the required developmental instructions for odontoblast differentiation in the absence of serum or other exogenous humoral factors in a chemically-defined medium. They also suggest the importance of reciprocal epithelial-mesenchymal interactions during epidermal organogenesis.     

Basement Membrane Formation in Transfilter Tooth Culture and Its Relation to Odontoblast Differentiation

The mesenchymal cells of the developing tooth differentiate into odontoblasts as a result of an epithelio-mesenchymal interaction. Odontoblast differentiation was studied in vitro by cultivating dental mesenchyme and epithelium with interposed filters. Separation of the two components by enzyme treatment resulted in removal of the basement membrane. When the epithelium was grown alone, or transfilter from killed lens capsule, the basement membrane was not restored. Transfilter cultivation with dental mesenchyme resulted in basement membrane formation, but only if the filter pores allowed penetration of cytoplasmic processes. Hence, a close association between the epithelial and the mesenchymal cells seems to be a prerequisite for the restoration of the basement membrane. Differentiation of odontoblasts took place only in explants in which a basement membrane was formed. Differentiation did not occur when contact of the mesenchymal cells with the basement membrane was prevented by small pore size filters. Further experiments demonstrating an intact basement membrane suggested that membrane contacts between the epithelial and the mesenchymal cells are not needed for odontoblast differentiation. Hence, we suggest that differentiation of odontoblasts is triggered via contact of the mesenchymal cells with the basement membrane.     

Patterns of nerve growth factor (NGF), proNGF, and p75 NGF receptor expression in the rat incisor: comparison with expression in the molar

Abstract. Nerve growth factor (NGF), a target-derived neurotrophic substance, may have broader biological functions in various types of nons-neuronal differentiating cells. The effects of NGF are dependent on initial binding of NGF to specific cell-surface receptors (p75^NGFR and p140^prototrk) on responsive cells. The continously growing rat incisor offers an excellent model demonstrating defined territories of differentiation of specific cell populations. We used immunohistochemistry to determine sites of NGF. proNGF and p75^NGFR accumulation in the rat incisor, whereas NGF mRNA expression was visualized by in situ hybridization in the developing rat molar and incisor. Strictly similar patterns of NGF mRNA, proNGF and NGF expression were observed in differentiating cells responsible for the production of the main structural matrices of the tooth. Thus, proNGF-like and NGF-like immunoreactivity, as well as the NGF mRNA signal were observed in preameloblasts and young ameloblasts of the dental epithelium and in polarizing odontoblasts of the dental mesenchyme. In contrast, the distribution of p75^NGFR was correlated with differentiation events only in dental mesenchyme: polarizing odontoblasts expressed p75^NGFR whereas the molecule was absent in functional odontoblasts. In dental epithelium, the restricted expression of p75^NGFR in ameloblast precursor cells was correlated with proliferative phenomena. The patterns of proNGF, NGF and p75^NGFR expression in epithelium and mesenchyme implicate both an autocrine and paracrine mode of action of the NGF molecule in dental tissues. The findings reported here are important for understanding NGF action in specific dental cell populations and suggest that this molecule is involved in the cascade of events that directs tooth development.     

Laminin alpha2 is essential for odontoblast differentiation regulating dentin sialoprotein expression

Laminin alpha2 is subunit of laminin-2 (alpha2beta1gamma1), which is a major component of the muscle basement membrane. Although the laminin alpha2 chain is expressed in the early stage of dental mesenchyme development and localized in the tooth germ basement membrane, its expression pattern in the late stage of tooth germ development and molecular roles are not clearly understood. We analyzed the role of laminin alpha2 in tooth development by using targeted mice with a disrupted lama2 gene. Laminin alpha2 is expressed in dental mesenchymal cells, especially in odontoblasts and during the maturation stage of ameloblasts, but not in the pre-secretory or secretory stages of ameloblasts. Lama2 mutant mice have thin dentin and a widely opened dentinal tube, as compared with wild-type and heterozygote mice, which is similar to the phenotype of dentinogenesis imperfecta. During dentin formation, the expression of dentin sialoprotein, a marker of odontoblast differentiation, was found to be decreased in odontoblasts from mutant mice. Furthermore, in primary cultures of dental mesenchymal cells, dentin matrix protein, and dentin sialophosphoprotein, mRNA expression was increased in laminin-2 coated dishes but not in those coated with other matrices, fibronectin, or type I collagen. Our results suggest that laminin alpha2 is essential for odontoblast differentiation and regulates the expression of dentin matrix proteins.     

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.     

Sonic hedgehog regulates growth and morphogenesis of the tooth

During mammalian tooth development, the oral ectoderm and mesenchyme coordinate their growth and differentiation to give rise to organs with precise shapes, sizes and functions. The initial ingrowth of the dental epithelium and its associated dental mesenchyme gives rise to the tooth bud. Next, the epithelial component folds to give the tooth its shape. Coincident with this process, adjacent epithelial and mesenchymal cells differentiate into enamel-secreting ameloblasts and dentin-secreting odontoblasts, respectively. Growth, morphogenesis and differentiation of the epithelium and mesenchyme are coordinated by secreted signaling proteins. Sonic hedgehog (Shh) encodes a signaling peptide which is present in the oral epithelium prior to invagination and in the tooth epithelium throughout its development. We have addressed the role of Shh in the developing tooth in mouse by using a conditional allele to remove Shh activity shortly after ingrowth of the dental epithelium. Reduction and then loss of Shh function results in a cap stage tooth rudiment in which the morphology is severely disrupted. The overall size of the tooth is reduced and both the lingual epithelial invagination and the dental cord are absent. However, the enamel knot, a putative organizer of crown formation, is present and expresses Fgf4, Wnt10b, Bmp2 and Lef1, as in the wild type. At birth, the size and the shape of the teeth are severely affected and the polarity and organization of the ameloblast and odontoblast layers is disrupted. However, both dentin- and enamel-specific markers are expressed and a large amount of tooth-specific extracellular matrix is produced. This observation was confirmed by grafting studies in which tooth rudiments were cultured for several days under kidney capsules. Under these conditions, both enamel and dentin were deposited even though the enamel and dentin layers remained disorganized. These studies demonstrate that Shh regulates growth and determines the shape of the tooth. However, Shh signaling is not essential for differentiation of ameloblasts or odontoblasts.     

Epithelial-mesenchymal interactions in the differentiation of odontoblasts and adamantoblasts]

In the present study we report experiments which indicate that the adamantal epithelium (inner dental epithelium) plays a specific role in the initiation of differentiation and in the initial maintenance of odontoblasts. The preodontoblasts do not differentiate in the pulps cultures alone or in association of pulps with no specific epithelium. The early stages in the process of odontoblasts differentiation are labile and dependent upon their environment. The odontoblasts and the ameloblasts cannot differentiate in advance in the isochronic and heterochronic associations. A previous maturation of these cells or of their environment is indispensable.     

Expression of connexin 43 and ZO-1 in differentiating ameloblasts and odontoblasts from rat molar tooth germs

We studied the distribution of connexin (Cx) 43 and ZO-1 by confocal laser scanning microscopy at early stages of dentinogenesis and amelogenesis. Labeling for Cx43 was observed at early stages of differentiation in both the epithelial cells and differentiating odontoblasts. Immunolabeling was detected at the distal and medial regions of undifferentiated ameloblasts and between cells from stratum intermedium and stellate reticulum. Differentiating odontoblasts exhibited immunoreaction for this antibody at their distal end. Immunoreactivity for ZO-1 was observed at regions that correspond to the proximal and distal junctional complexes of differentiating ameloblasts. Staining for ZO-1 was observed at apical regions of odontoblasts with a punctate appearance. In more advanced stages, expression of Cx43 was more evident on ameloblasts, especially at the junctional complexes. Punctate immunolabeling for Cx43 was observed at the lateral sides of differentiating ameloblasts and between the other cells of the enamel organ. Immunoreaction for ZO-1 in ameloblasts was more evident than at the previous stage. It was also observed at the distal end of differentiated odontoblasts. The present study showed that differentiating ameloblasts and odontoblasts express Cx43 and ZO-1 as early as the start of the differentiation process. In addition, the expression of these junctional proteins increases as differentiation of cells continues.     

Immunohistochemical localization of LIM mineralization protein 1 during mouse molar development

LIM mineralization protein 1 (LMP-1) is an essential positive regulator of osteoblast differentiation, maturation and bone formation. Our previous investigations on the distribution of LMP-1 in mature human teeth indicated that LMP-1 might play a role in the odontoblast differentiation and dentin matrix mineralization. The aim of the present study was to use immunohistochemistry to determine the expression of LMP-1 during tooth development in mouse molars. In embryonic and postnatal Kunming mice, LMP-1 protein was expressed during molar development, but the expression levels and patterns differed at various developmental stages. At embryonic day 13.5 (E13.5), LMP-1 was found in the enamel organ. At E14.5, LMP-1 was detected in the entire enamel organ and in the underlying mesenchyme. At E16.5, LMP-1 was observed in the inner and outer enamel epithelium and the stratum intermedium. The expression also converged at the cusps in the dental papilla. At E18.5 and postnatal day 2.5 (P2.5), LMP-1 was restricted to the stratum intermedium, in differentiating dental papilla cells at cusps, while it disappeared in terminal differentiated ameloblasts and odontoblasts. At P13.5, no positive staining was detected in the odontoblasts or in the dental pulp cells. Therefore, LMP-1 showed spatiotemporal expression patterns during molar development and might participate in molar crown morphogenesis and odontoblast differentiation at late molar development.     

Membrane-cytoskeleton interactions: Inhibition of odontoblast differentiation by a monoclonal antibody directed against a membrane protein

It is known that high-molecular-weight (HMW) membrane proteins mediate interactions with constituents of the extracellular matrix and/or with cytoskeletal elements. To study participation of HMW membrane proteins in odontoblast or ameloblast differentiation, an immunological approach has been adopted. Antibodies directed against membrane proteins (Mr, 110-190) from mouse embryos have been produced by the hybridoma technique. Supernatants of hybridoma cultures were screened for their ability to stain dental tissues and also tested for their biological activities on dental cells in primary culture or on developing tooth germs in organ culture. An IgM monoclonal antibody, MC16A16, directed against a 165-kDa antigen present in plasma membrane preparations, reacted strongly with the dental epithelium and weakly with the mesenchyme. MC16A16 also reacted with the cell surface of nonpermeabilized cultured dental cells and could detach epithelial cells cultured on glass, but not mesenchymal cells which maintained vinculin-containing focal contacts. This antibody, which affected the organization of dental-cell microfilaments in primary culture, also inhibited the polarization of odontoblasts, but not that of ameloblasts.     

35S]autoradiographic study of sulfated GAG accumulation and turnover in embryonic mouse tooth germs

The accumulation of sulfated GAG in embryonic mouse molars before, during, and after terminal differentiation of odontoblasts was localized by [35S]autoradiography combined with the use of chondroitin ABC lyase. Much more sulfated GAG were accumulated in the dental papilla than in the dental epithelium. High incorporation of [35S]sulfate occurred at the epithelio-mesenchymal junction, which is the site of dental basement membrane and predentin. Before terminal differentiation of odontoblasts, the distribution of sulfated GAG was uniform at the basement membrane. After the onset of terminal differentiation of odontoblasts, much more sulfated GAG accumulated at the tip of principal cusps than at the apical (inferior) parts of cusps, and sulfated GAG were then found to be degraded more rapidly at the epithelio-mesenchymal junction than at other parts of the tooth germ. Thus regional variation in the rate of degradation of GAG exists in the tooth germs. Trypsin-isolated dental epithelia cultured in vitro synthesized a new basement membrane that could be labeled with [3H]glucosamine but not with 35SO4(-2). The epithelial-derived basal lamina contains little or no sulfatated GAG.     

Tooth development in a scincid lizard, Chalcides viridanus (Squamata), with particular attention to enamel formation

Comparative analysis of tooth development in the main vertebrate lineages is needed to determine the various evolutionary routes leading to current dentition in living vertebrates. We have used light, scanning and transmission electron microscopy to study tooth morphology and the main stages of tooth development in the scincid lizard, Chalcides viridanus, viz., from late embryos to 6-year-old specimens of a laboratory-bred colony, and from early initiation stages to complete differentiation and attachment, including resorption and enamel formation. In C. viridanus, all teeth of a jaw have a similar morphology but tooth shape, size and orientation change during ontogeny, with a constant number of tooth positions. Tooth morphology changes from a simple smooth cone in the late embryo to the typical adult aspect of two cusps and several ridges via successive tooth replacement at every position. First-generation teeth are initiated by interaction between the oral epithelium and subjacent mesenchyme. The dental lamina of these teeth directly branches from the basal layer of the oral epithelium. On replacement-tooth initiation, the dental lamina spreads from the enamel organ of the previous tooth. The epithelial cell population, at the dental lamina extremity and near the bone support surface, proliferates and differentiates into the enamel organ, the inner (IDE) and outer dental epithelium being separated by stellate reticulum. IDE differentiates into ameloblasts, which produce enamel matrix components. In the region facing differentiating IDE, mesenchymal cells differentiate into dental papilla and give rise to odontoblasts, which first deposit a layer of predentin matrix. The first elements of the enamel matrix are then synthesised by ameloblasts. Matrix mineralisation starts in the upper region of the tooth (dentin then enamel). Enamel maturation begins once the enamel matrix layer is complete. Concomitantly, dental matrices are deposited towards the base of the dentin cone. Maturation of the enamel matrix progresses from top to base; dentin mineralisation proceeds centripetally from the dentin–enamel junction towards the pulp cavity. Tooth attachment is pleurodont and tooth replacement occurs from the lingual side from which the dentin cone of the functional teeth is resorbed. Resorption starts from a deeper region in adults than in juveniles. Our results lead us to conclude that tooth morphogenesis and differentiation in this lizard are similar to those described for mammalian teeth. However, Tomes processes and enamel prisms are absent.     

Interaction between Fibronectin and β1 Integrin Is Essential for Tooth Development

The dental epithelium and extracellular matrix interact to ensure that cell growth and differentiation lead to the formation of teeth of appropriate size and quality. To determine the role of fibronectin in differentiation of the dental epithelium and tooth formation, we analyzed its expression in developing incisors. Fibronectin mRNA was expressed during the presecretory stage in developing dental epithelium, decreased in the secretory and early maturation stages, and then reappeared during the late maturation stage. The binding of dental epithelial cells derived from postnatal day-1 molars to a fibronectin-coated dish was inhibited by the RGD but not RAD peptide, and by a β1 integrin-neutralizing antibody, suggesting that fibronectin-β1 integrin interactions contribute to dental epithelial-cell binding. Because fibronectin and β1 integrin are highly expressed in the dental mesenchyme, it is difficult to determine precisely how their interactions influence dental epithelial differentiation in vivo. Therefore, we analyzed β1 integrin conditional knockout mice (Intβ1^lox-/lox-/K14-Cre) and found that they exhibited partial enamel hypoplasia, and delayed eruption of molars and differentiation of ameloblasts, but not of odontoblasts. Furthermore, a cyst-like structure was observed during late ameloblast maturation. Dental epithelial cells from knockout mice did not bind to fibronectin, and induction of ameloblastin expression in these cells by neurotrophic factor-4 was inhibited by treatment with RGD peptide or a fibronectin siRNA, suggesting that the epithelial interaction between fibronectin and β1 integrin is important for ameloblast differentiation and enamel formation.     

Follistatin regulates enamel patterning in mouse incisors by asymmetrically inhibiting BMP signaling and ameloblast differentiation

Rodent incisors are covered by enamel only on their labial side. This asymmetric distribution of enamel is instrumental to making the cutting edge sharp. Enamel matrix is secreted by ameloblasts derived from dental epithelium. Here we show that overexpression of follistatin in the dental epithelium inhibits ameloblast differentiation in transgenic mouse incisors, whereas in follistatin knockout mice, ameloblasts differentiate ectopically on the lingual enamel-free surface. Consistent with this, in wild-type mice, follistatin was continuously expressed in the lingual dental epithelium but downregulated in the labial epithelium. Experiments on cultured tooth explants indicated that follistatin inhibits the ameloblast-inducing activity of BMP4 from the underlying mesenchymal odontoblasts and that follistatin expression is induced by activin from the surrounding dental follicle. Hence, ameloblast differentiation is regulated by antagonistic actions of BMP4 and activin A from two mesenchymal cell layers flanking the dental epithelium, and asymmetrically expressed follistatin regulates the labial-lingual patterning of enamel formation.     

Changes in the distribution of tenascin during tooth development

Tenascin is an extracellular matrix molecule that was earlier shown to be enriched in embryonic mesenchyme surrounding the budding epithelium in various organs including the tooth. In the present study tenascin was localized by immunohistology throughout the course of tooth development in the mouse and rat using polyclonal antibodies against chick tenascin. The results indicate that tenascin is expressed by the lineage of dental mesenchymal cells throughout tooth ontogeny. The intensity of staining with tenascin antibodies in the dental papilla mesenchyme was temporarily reduced at cap stage when the tooth grows rapidly and undergoes extensive morphogenetic changes. During the bell stage of morphogenesis, the staining intensity increased and tenascin was accumulated in the dental pulp even after completion of crown development and eruption. Tenascin was present in the dental basement membrane at the time of odontoblast differentiation. The dental papilla cells ceased to express tenascin upon differentiation into odontoblasts and tenascin was completely absent from dentin. It can be speculated that the remarkable expression of tenascin in the dental mesenchymal cells as compared to other connective tissues is associated with their capacity to differentiate into hard-tissue-forming cells.     

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.