Expression of clock proteins in developing tooth

Morphological and functional changes during ameloblast and odontoblast differentiation suggest that enamel and dentin formation is under circadian control. Circadian rhythms are endogenous self-sustained oscillations with periods of 24h that control diverse physiological and metabolic processes. Mammalian clock genes play a key role in synchronizing circadian functions in many organs. However, close to nothing is known on clock genes expression during tooth development. In this work, we investigated the expression of four clock genes during tooth development. Our results showed that circadian clock genes Bmal1, clock, per1, and per2 mRNAs were detected in teeth by RT-PCR. Immunohistochemistry showed that clock protein expression was first detected in teeth at the bell stage (E17), being expressed in EOE and dental papilla cells. At post-natal day four (PN4), all four clock proteins continued to be expressed in teeth but with different intensities, being strongly expressed within the nucleus of ameloblasts and odontoblasts and down-regulated in dental pulp cells. Interestingly, at PN21 incisor, expression of clock proteins was down-regulated in odontoblasts of the crown-analogue side but expression was persisting in root-analogue side odontoblasts. In contrast, both crown and root odontoblasts were strongly stained for all four clock proteins in first molars at PN21. Within the periodontal ligament (PDL) space, epithelial rests of Malassez (ERM) showed the strongest expression among other PDL cells. Our data suggests that clock genes might be involved in the regulation of ameloblast and odontoblast functions, such as enamel and dentin protein secretion and matrix 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.     

Functional role of Rho-kinase in ameloblast differentiation

During tooth development, inner enamel epithelial (IEE) cells differentiate into enamel-secreting ameloblasts, a polarized and elongated cellular population. The molecular underpinnings of this morphogenesis and cytodifferentiation, however, are not well understood. Here, we show that Rho-associated coiled-coil-containing protein kinase (ROCK) regulates ameloblast differentiation and enamel formation. In mouse incisor organ cultures, inhibition of ROCK, hindered IEE cell elongation and disrupted polarization of differentiated ameloblasts. Expression of enamel matrix proteins, such as amelogenin and ameloblastin, and formation of the terminal band structure of actin and E-cadherin were also perturbed. Cultures of dental epithelial cells revealed that ROCK regulates cell morphology and cell adhesion through localization of actin bundles, E-cadherin, and β-catenin to cell membranes. Moreover, inhibition of ROCK promoted cell proliferation. Small interfering RNA specific for ROCK1 and ROCK2 demonstrated that the ROCK isoforms performed complementary functions in the regulation of actin organization and E-cadherin-mediated cell-cell adhesion. Thus, our results have uncovered a novel role for ROCK in amelogenesis.     

Stratum intermedium lineage diverges from ameloblast lineage via Notch signaling

The stratum intermedium develops as flattened cell layers on the proximal side of the ameloblast layer during tooth development. However, little information is available regarding the origin and the role. In this study, we indicate that some stratum intermedium cells originate from the inner enamel epithelium (IEE) in rat incisor organ cultures using DiI as a tracer. Immunohistochemical and in situ hybridization studies showed that the stratum intermedium cells express the Notch1 protein and Hes1 mRNAs, while the IEE and ameloblasts express the Jagged1. Further, we examined the role of Notch signaling using the dental epithelial cell line HAT-7. Recombinant Jagged1 protein enhanced the appearance of stratum intermedium cells in HAT-7 cultures and neutralization with an anti-Jagged1 antibody inhibited these effects. Additionally, overexpression of the Notch1 internal domain increased the number of stratum intermedium cells. We hypothesize that the stratum intermedium lineage differentiates from the ameloblast lineage via Notch signaling.     

Localization of epidermal growth factor receptors in cells of the enamel organ of the rat incisor.

Epidermal growth factor (EGF) is a peptide shown to effect precocious incisor tooth eruption in rat pups. Binding sites for EGF were visualized in the continuously erupting adult rat incisor by light and electron microscope radioautography after in vivo injection of 125I-EGF. These binding sites represented EGF receptors because of (i) competition between 125I-EGF binding at 2 min after injection and a coinjected excess of unlabeled EGF; (ii) the receptor-mediated endocytosis of 125I-EGF at 15 and 30 min after injection; and (iii) the demonstration of EGF receptor kinase activation in vivo. The stem and the mitotic cells in the epithelial odontogenic organ at the growing end of the tooth develop into two nondividing layers of the enamel organ: (i) ameloblasts which secrete enamel and are subsequently involved in the enamel maturation process, and (ii) papillary layer cells situated between the blood supply and the ameloblasts. Although few EGF receptors were present at the mitotic end, receptor density was highest at the mature end of the enamel organ. High levels of 125I-EGF binding were found on papillary layer cells and ruffle-ended, but not smooth-ended, ameloblasts. This implies a cyclical exteriorization and internalization of receptors during modulations between the two cell types. These data suggest that the EGF receptor mediates a major function of the enamel organ in the formation of enamel.     

Inner enamel epithelia synthesize and secrete enamel proteins during mouse molar occlusal "enamel-free area" development

Mesenchyme-derived instructions for odontogenic epithelial differentiation into ameloblasts and the production of enamel matrix has been well established. However, it is not known how position-specific differences within the enamel organ of rodent molar tooth organs regulate the enamel-forming vs. the enamel free areas in the developing cusp. Light microscopy, transmission electron microscopy, and immunocytochemistry using a rabbit anti-mouse amelogenin antibody, were used to map the position-specific patterns within the enamel organ. In the enamel-forming area, ameloblasts were associated with stratum intermedium. In the enamel-free area, another cell type was interposed between inner enamel epithelia (IEE) and stratum intermedium. IEE in the enamel-free area did not have Tomes' processes and secreted enamel matrix not only toward dentin but also between IEE cells. IEE became confluent with stellate reticulum; at this position stratum intermedium cells were no longer detected. The thickness and orientation of dentin matrix collagen fibers in the enamel-free area were different from the fibers in the enamel-forming area. These results suggest that the patterns of epithelial cell-cell and cell-matrix associations during position-specific enamel organ epithelial differentiation may regulate ameloblast matrix synthesis and/or the matrix secretion pathway.     

A novel role of periostin in postnatal tooth formation and mineralization

Periostin plays multiple functions during development. Our previous work showed a critical role of this disulfide-linked cell adhesion protein in maintenance of periodontium integrity in response to occlusal load. In this study, we attempted to address whether this mechanical response molecule played a direct role in postnatal tooth development. Our key findings are 1) periostin is expressed in preodontoblasts, and odontoblasts; and the periostin-null incisor displayed a massive increase in dentin formation after mastication; 2) periostin is also expressed in the ameloblast cells, and an enamel defect is identified in both the adult-null incisor and molar; 3) deletion of periostin leads to changes in expression profiles of many non-collagenous protein such as DSPP, DMP1, BSP, and OPN in incisor dentin; 4) the removal of a biting force leads to reduction of mineralization, which is partially prevented in periostin-null mice; and 6) both in vitro and in vivo data revealed a direct regulation of periostin by TGF-β1 in dentin formation. In conclusion, periostin plays a novel direct role in controlling postnatal tooth formation, which is required for the integrity of both enamel and dentin.     

Immunolocalization of enamelysin (matrix metalloproteinase-20) in the forming rat incisor.

In the rat model, we used the continuously growing incisor to study the expression pattern of matrix metalloproteinase-20 (MMP-20) during the formation of mineralized dental tissues. Casein zymography analysis of extracts of the forming part of the incisor revealed lysis bands corresponding to both the latent form at 57 kD and the active 46- and 41-kD forms, whereas omission of proteinase inhibitors during protein extraction resulted in a single band at 21 kD. A higher molecular weight form of 78 kD was also stained with MMP-20 and TIMP-2 antibodies in Western blotting, and was therefore believed to correspond to an MMP-20/TIMP-2 complex. Immunohistochemical and immunogold electron microscopic results demonstrated strong MMP-20 staining in the forming outer enamel, which diminished near the dentino-enamel junction, but dentin and predentin were unstained. A strong concentration of MMP-20 was seen in the stratum intermedium (SI), particularly at the earlier stages of enamel development. Our results confirm the presence of MMP-20 protein in ameloblasts and odontoblasts of rat incisor and show it to be localized in the same sites of the forming enamel as amelogenin. Their expression is transient in odontoblasts but persists in ameloblasts, and in both cases the expression of amelogenin preceded that of MMP-20 suggesting a developmentally controlled regulation.     

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.     

Multiple functions for NGF receptor in developing, aging and injured rat teeth are suggested by epithelial, mesenchymal and neural immunoreactivity

We have used immunocytochemistry to analyse expression of nerve growth factor receptor (NGFR) in developing, aging and injured molar teeth of rats. The patterns of NGFR immunoreactivity (IR) in developing epithelia and mesenchyme matched the location of NGFR mRNA assayed by in situ hybridization with a complementary S35-labeled RNA probe. The following categories of NGFR expression were found. (1) There was NGFR-IR in the dental lamina epithelium and in adjacent mesenchyme during early stages of third molar formation. (2) NGFR-IR nerve fibers were posterior and close to the bud epithelium. (3) During crown morphogenesis NGFR expression was prominent in internal enamel epithelium and preodontoblasts; it faded as preameloblasts elongated and as odontoblasts began to make predentin matrix; and it was weak or absent from outer enamel epithelium, the cervical loop, and differentiated ameloblasts and odontoblasts. (4) When NGFR-IR nerve fibers entered the molars late in the bell stage, they innervated the most mature peripheral pulp and dentin in an asymmetric pattern which correlated more with asymmetric enamel synthesis than with mesenchymal NGFR-IR distribution. (5) The mesenchymal pulp cells continued to have intense NGFR expression in adult teeth, especially near coronal tubular dentin. (6) The pulpal NGFR-IR decreased in very old rats or subjacent to reparative dentin (naturally occurring or experimentally induced). (7) During root formation, the preodontoblasts had NGFR-IR but most root mesenchymal cells and Hertwig's epithelial root sheath did not. This work suggests that there are important epithelial and mesenchymal targets of NGF regulation during molar morphogenesis that differ for crown and root development and that do not correlate with neural development. The continuing expression of NGFR-IR by pulpal mesenchymal cells in adult rats was most intense near coronal odontoblasts making tubular dentin; and it was lost during aging, or subjacent to sites of dentin injury that caused a phenotypic change in the odontoblast layer.     

Ca-ATPase and ALPase activities at the initial calcification sites of dentin and enamel in the rat incisor

Summary Enzymatic activities of calcium-magnesium dependent adenosine triphosphatase (Ca-ATPase) and nonspecific alkaline phosphatase (ALPase) were localized at the initial calcification sites of dentin and enamel of rat incisor teeth using electron-microscopic cytochemistry.Ca-ATPase was localized in the Golgi cisternae, cytoplasmic vesicles and along the outer surface of the presecretory and secretory ameloblasts, whereas it was totally absent from the odontoblasts in the pulp. Inversely, ALPase reaction was localized along the outer surface of the odontoblasts, but almost completely absent from the ameloblasts.Diffuse extracellular reactions of both enzymes were distributed throughout the unmineralized fibrous matrix of mantle dentin in which a large number of matrix vesicles were scattered. Both Ca-ATPase and ALPase reactions, which appeared in the matrix vesicles in the process of formation of mantle dentin, became most conspicuous at the site of initial dentin calcification. At this stage, an intense Ca-ATPase reaction also appeared along some of the collagen fibrils adjacent to the reactive matrix vesicles. No ALPase reaction was localized along these Ca-ATPase reactive collagen fibrils.Our observations suggest strongly that Ca-ATPase in the matrix vesicles originates from the inner enamel epithelium and/or preameloblasts whereas ALPase originates from the odontoblasts in the pulp. The importance of the coexistence of both enzymes for the control of initial calcification of dental hard tissues is suggested.     

Ameloblast apoptosis and IGF-1 receptor expression in the continuously erupting rat incisor model

Enamel-producing cells (ameloblasts) pass through several phenotypic and functional stages during enamel formation. In the transition between secretory and maturation stages, about one quarter of the ameloblasts suddenly undergo apoptosis. We have studied this phenomenon using the continuously erupting rat incisor model. A special feature of this model is that all stages of ameloblast differentiation are presented within a single longitudinal section of the developing tooth. This permits investigation of the temporal sequence of gene and growth factor receptor expression during ameloblast differentiation and apoptosis. We describe the light and electron microscopic morphology of ameloblast apoptosis and the pattern of insulin-like growth factor-1 receptor expression by ameloblasts in the continuously erupting rat incisor model. In the developing rat incisor, ameloblast apoptosis is associated with downregulated expression of the insulin-like growth factor-1 receptor. These data are consistent with the hypothesis that ameloblasts are hard wired for apoptosis and that insulin-like growth factor-1 receptor expression is required to block the default apoptotic pathway. Possible mechanisms of insulin-like growth factor-1 inhibition of ameloblast apoptosis are presented. The rat incisor model may be useful in studies of physiological apoptosis as it presents apoptosis in a predictable pattern in adult tissues.     

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.     

Movement of entire cell populations during renewal of the rat incisor as shown by radoioautography after labeling with 3H-thymidine. The concept of a continuously differentiating cross-sectional segment. (With an appendix on the development of the periodontal ligament).

Renewal of the rat incisor was studied in three dimensions by employing a serial cross-sectioning technique to locate the boundary between labeled and unlabeled cells in the enamel organ and odontoblast layer at various times after a single injection of 3H-thymidine. This boundary, or leading edge of the front of labeling, was graphically illustrated through point-plotting reconstruction of the labial surface of the incisor. At one hour after the injection of 3H-thymidine the front of labeled ameloblasts was located within the presecretory zone related to early predentin secretion. This front formed a "C"-shaped curve stretching across the labial surface of the tooth from the lateral to the mesial cemento-enamel junction. The "C" was open anteriorly and the lateral arm extended almost twice as far incisally as the mesial arm. The edge of the front of labeled odontoblasts was positioned apical to and parallel with this "C"-shaped curve. The morphological appearance of all cells along each respective front was found to be similar. As the fronts of labeled ameloblasts and labeled odontoblasts moved forward with the erupting incisor, the cells along these fronts differentiated simultaneously and subsequently formed enamel and dentin. Throughout this movement the distance between fixed points along the leading edge of the front of labeled ameloblasts, and its positional relationship to the front of labeled odontoblasts, did not change appreciably. This indicated that cells of the tooth were being carried incisally at a uniform speed. It was concluded that renewal in the rat incisor consists of the generation by the bulbous part of the odontogenic organ of epithelial "U"-shaped cross-sectional segments which enclose a core of pulp. As this segment is transported towards the gingival margin, cellular differentiation and subsequent formation of hard tissue is seen to begin at the central labial side of the segment and to progress in a mesial and lateral direction towards the lingual side. In the process, the limits of the enamel organ at the mesial and lateral cemento-enamel junctions are established and the entire circumference of the segment is eventually enclosed by a rim of dentin.     

Temporo-spatial distribution of matrix and microfilament components during odontoblast and ameloblast differentiation

Summary Several extracellular matrix components (procollagen type III, fibronectin, collagen type IV, laminin and nidogen) and microfilament constituents (actin, α-actinin and vinculin) were localized by indirect immunofluorescence microscopy in frozen sections of embryonic mouse molars. Nidogen was present at the epithelio-mesenchymal junction during polarization and initial steps of functional differentiation of odontoblasts. Nidogen disappeared at a stage where direct contacts between preameloblasts and predentin were required to allow the initiation of ameloblast polarization. Our observations concerning the distribution of procollagen type III and fibronectin during odontoblast differentiation add to current knowledge. Procollagen type III and fibronectin surrounding preodontoblasts accumulated at the apical part of polarizing and functional odontoblasts secreting “initial” predentin. Procollagen type III, but not fibronectin, disappeared in front of functional odontoblasts synthesizing “late” predentin and dentin. Fibronectin, present in “initial” predentin, was no longer detected in “late” predentin and dentin but was found between odontoblasts secreting “late” predentin and dentin. Actin, α-actinin and vinculin were concentrated in the peripheral cytoplasm of preameloblasts and accumulated at the apical and basal poles of functional ameloblasts. During differentiation of odontoblasts, the three proteins accumulated at the apical pole of these cells. Time and space correlations between matrix and microfilament modifications during odontoblast and ameloblast differentiation are documented. The possibility is discussed that there is transmembranous control of the cytoskeletal activities of odontoblasts and ameloblasts by the extracellular matrix.     

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.     

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.     

Electron probe analysis of maturation ameloblasts of the rat incisor and calf molar

Rapidly frozen upper incisor teeth of rats and molar teeth of calves were freeze fractured, freeze dried and dry dissected in preparation for energy dispersive x-ray emission microanalysis in the scanning electron microscope. Successive zones of ameloblasts adjacent to maturing rat incisor enamel were examined, beginning with cells adjacent to the least mature enamel and progressing to cells over increasingly more mature enamel. Pronounced Kalpha1,2 x-ray peaks were obtained for P, S, Cl, K and Fe but not for Ca. Ca levels were also very low compared with P, S, Cl and K in calf molar maturation ameloblasts, whereas they were high in the distal poles of the secretory odontoblasts in the same specimens. The findings indicate that both intra- and extracellular Ca levels are extremely low in maturation ameloblasts. It is concluded that Ca is neither stored nor concentrated in large amounts by the maturation ameloblasts prior to its entry into the enamel. The suggestion is made that the maturation ameloblasts might regulate entry of calcium into enamel by serving as a selective barrier.