Cells Of Enamel Organ Research Articles

Ameloblatoma: An aggressive odontogenic neoplasm with altered vickers and gorlin criteria

Plexiform ameloblastoma is a rare aggressive odontogenic neoplasm comprising about 1% of all odontogenic lesions of the jaw arising from cells of enamel organ that has failed to differentiate to form the hard tissues of tooth. Clinically, Ameloblastoma presents as painless swelling of jaw occurring in 3 and 4 decade in the posterior region of the mandible with equal sex distribution. It shows multiple patterns including solid, unicystic, extra osseous and desmoplastic type. Due to its varied presentation and relative risk of recurrence, it possesses a great challenge to physician for early diagnosis. Vickers and Gorlin (V and G) criteria is an integral part of diagnosis of histological types of Ameloblastoma. This article aims to highlight the variations observed in V and G criteria in the histopathological features of mixed variety of Ameloblastoma in a middle aged man.

Structural and functional relations between the connective tissue and epithelium of enamel organ and their role during enamel maturation.

The morphological and possible functional interactions between the connective tissue and enamel organ cells were examined during the maturation phase of enamel formation, using immunohistochemical techniques. Decalcified mandibular sections(10µm) including incisors were used from Wistar rats ages 10-12weeks. Sections were incubated with one or two primary antibodies targeting cell cytoskeleton (vimentin, α-actin, α-tubulin), dendritic marker (OX6), gap junctions (cx-43), enzymes (nitric-oxide synthase (nos1) and cyclooxygenase (cox1)), and the ion transporters (Na+/H+ exchanger (NHE1) and Na+/Ca2+ exchanger (NCX)) for 24h, before incubation with the appropriate conjugated fluorescent secondary antibodies. Sections were examined by fluorescence microscopy. Haematoxylin-eosin slides were also employed. Cellular heterogeneity and morphological modulations were identified within enamel organ cells and connective tissue covering suggesting complex cellular interactions and indicating a new functional concept and possible complementary role during enamel maturation. Also, some ion transportation activity, and nos1 and cox1 signalling pathways have been identified, indicating intercellular communication between these regions. A hypothesis is suggested, to explain the morphological modulation of ameloblasts and papillary cells during enamel maturation which functions to increase the transporting membrane surface area to accomplish faster and bulker ion transportation to achieve controlled pH and to direct Ca2+ towards enamel.

Altered miRNA expression profiling in enamel organ of fluoride affected rat embryos

Evidence has shown that miRNAs could play a role in dental fluorosis, but there is no study has investigated the global expression miRNA profiles of fluoride-exposed enamel organ. In this study, we analysed the differentially expressed (DE) miRNAs between fluoride-treated and control enamel organ for the first time and found several candidate miRNAs and signaling pathways worthy of further research. Thirty Wistar rats were randomly distributed into three groups and exposed to drinking water with different fluoride contents for 10 weeks and during the gestation. The three groups were a control group (distilled water), medium fluoride group (75 mg/L NaF), and high fluoride group (150 mg/L NaF). On the embryonic day 19.5, the mandible was dissected for histological analysis, and the enamel organ of the mandibular first molar tooth germ was collected for miRNA sequencing (miRNA-seq) and quantitative real-time PCR analysis (qRT-PCR). Typical dental fluorosis was observed in the incisors of the prepregnant rats. In addition to the disorganized structure of enamel organ cells, 39 DE miRNAs were identified in the fluoride groups compared with the control group, and good agreement between the miRNA-seq data and qRT-PCR data was found. The functional annotation of the target genes of 39 DE miRNAs showed significant enrichment in metabolic process, cell differentiation, calcium signaling pathway, and mitogen-activated protein kinase(MAPK) signaling pathway terms. This study provides a theoretical reference for an extensive understanding of the mechanism of fluorosis and potential valuable miRNAs as therapeutic targets in fluorosis.

WITHDRAWN: Evaluation of expression of p63 and amelogenin in human tooth germ and ameloblastoma

BACKGROUND: The study was undertaken to evaluate the expression of p63 and amelogenin in human tooth germs and ameloblastomas by immunohistochemistry to find out whether the pattern of expression was related to differentiation of ameloblasts. MATERIALS AND METHODS: In this study fifteen human tooth germs of late bell stage, six human tooth germs of early bell stage and fifteen ameloblastoma samples were included and expression pattern of p63 and amelogenin were evaluated individually in all the samples. RESULTS: During the early bell stage, p63 expression was intense throughout the enamel organ. But, during the late bell stage, the number and intensity of p63 expression decreases in the cells of enamel organ. The peripheral cells in the ameloblastoma shows variable pattern of p63 expression. Expression of amelogenin was first evident in the presecretory ameloblasts at the cusp tip, followed by secretory ameloblasts and progresses cervically. But in ten late bell stage tooth germs, amelogenin expression was negative in the secretory ameloblasts and positive only at the secreting end i.e. tomes process. Intense staining was evident at the enamel matrix and in some dentinal tubules nearer to enamel matrix secretion. No amelogenin expression was evident in the dental papilla cells or odontoblasts throughout odontogenesis with the exception of dental follicle. Amelogenin expression was absent in eleven cases of ameloblastoma, but in four cases positive expression was evident at the peripheral cells. CONCLUSION: In the present study, the expression pattern of p63 and amelogenin in human tooth germ and ameloblastoma correlates with cytodifferentiation of ameloblasts.

Bivalency of Shh Modulates Cell Fate Decision of Dental Epithelium Stem Cells

Rodent incisor grows throughout whole life of the animal and owns every stage of dental epithelium development, thus it provides an ideal model to study the fate decision of dental epithelial stem cells (DESCs) as well as to understand the mechanisms of tooth regeneration. Bivalency, characterized as concurrence of H3K27me3 and H3K4me3, manages key developmental genes transcript in some organs, however no evidences answer whether it works on enamel organ development. Here using mouse incisor models, we first of time reveal the dynamic alterations of bivalency during DESCs differentiation, which is dominated via H3K27me3 rather than H3K4me3. Ezh2 is identified to catalyze this H3K27me3 change, then determining cell fate of DESCs. Ezh2 defecient DESCs show impaired stemness but activated amelogenic gene amelogenin (Amelx). Conversely, elevating Ezh2 maintains pluripotency and represses Amelx, which is rescued by deleting SET trimethylase activity domain in Ezh2. Mechanistically, Ezh2 is validated to catalyze H3K27me3 within promoter of Shh in a methytransferase activity dependent manner, thus determining Shh transcript. In vitro silencing Ezh2 results in activation of Shh expression, increasing the recruitment of GLI1 within Amelx promoter, however, Ezh2 overexpression represses such Shh signaling. In vivo bivalency related Shh activation occurs in pre-ameloblasts (PAB), which directly triggers Ptch1/Gli1 signaling to enhance the enrichment of Gli1 within promoter of Amelx, finally opens the door of differentiation. These results bring light to brand new insights on role of bivalency in enamel organ formation and cell fate choices of stem cell. Funding Sources: This work was supported by National Natural Science Foundation of China (NSFC grant 81470711&81771033 to LWZ), preeminent youth fund of Sichuan province (2016JQ0054 to LWZ), Sichuan Province Science and Technology Innovation Team Program (2015TD0011 to YL, 2017TD0016 to Y.Q). Conflicts of Interest: The authors declare no potential conflicts of interest with respect to the authorship and publication of this article. Ethics Approval Statement: All mouse experiments were conducted in accordance with protocols approved by ethical committees of the West China School of Stomatology, Sichuan University and State Key Laboratory of Oral Diseases.

Expression of desmosomal proteins and their implications during enamel organ morphogenesis

Background/Objective: The present article is between the fewest that describe the expression of desmosomal proteins in human tooth organ and to implicate desmosome and its components in tooth morphogenesis. Although present in all epithelia, desmosomes are particularly down regulated in undifferentiated, dedifferentiated and malignant cells. Methodology: In the present article, using immunohistochemical staining, we report the expression patterns of 6 desmosomal components; Desmoplakin, plakoglobin, desmoglein, plakophilin-1, plakophilin-2, and plakophilin-3. The dental, oral and ectodermal epithelia were investigated in 7 and 9 weeks' human embryos. Results: The expression of these components appeared to associate with cell differentiation, stage of development and epithelial type. The fundamental desmosomal proteins, desmoplakin, plakoglobin and desmoglein, produced almost identical profiles, suggesting that desmosomes are absent or under developed in negative and weak reacting areas respectively. Whilst the accessory component plakophilin-1 was always absent, plakophilin-2 and -3 were strongly labeled in the embryonic epidermis and moderately expressed in the oral epithelium. In the developing enamel organ, gradients of fundamental protein expressions were produced being strong towards the oral epithelium and weak/negative around the inner enamel epithelium. These gradients were also produced using antibodies to plakophilin-2 and -3 although the majority of enamel organ cells were unlabeled. Conclusion: Mirror image symmetry was foundwas found for the distribution patterns of these markers. Furthermore, the left-right asymmetry was found both for the maxillary and mandibular tooth germs, thus bringing further evidence implicating desmosome and its components in the mechanisms regulating tooth morphogenesis.

The Role of Na:K:2Cl Cotransporter 1 (NKCC1/SLC12A2) in Dental Epithelium during Enamel Formation in Mice.

Na+:K+:2Cl− cotransporters (NKCCs) belong to the SLC12A family of cation-coupled Cl− transporters. We investigated whether enamel-producing mouse ameloblasts express NKCCs. Transcripts for Nkcc1 were identified in the mouse dental epithelium by RT-qPCR and NKCC1 protein was immunolocalized in outer enamel epithelium and in the papillary layer but not the ameloblast layer. In incisors of Nkcc1-null mice late maturation ameloblasts were disorganized, shorter and the mineral density of the enamel was reduced by 10% compared to wild-type controls. Protein levels of gap junction protein connexin 43, Na+-dependent bicarbonate cotransporter e1 (NBCe1), and the Cl−-dependent bicarbonate exchangers SLC26A3 and SLC26A6 were upregulated in Nkcc1-null enamel organs while the level of NCKX4/SLC24A4, the major K+, Na+ dependent Ca2+ transporter in maturation ameloblasts, was slightly downregulated. Whole-cell voltage clamp studies on rat ameloblast-like HAT-7 cells indicated that bumetanide increased ion-channel activity conducting outward currents. Bumetanide also reduced cell volume of HAT-7 cells. We concluded that non-ameloblast dental epithelium expresses NKCC1 to regulate cell volume in enamel organ and provide ameloblasts with Na+, K+ and Cl− ions required for the transport of mineral- and bicarbonate-ions into enamel. Absence of functional Nkcc1 likely is compensated by other types of ion channels and ion transporters. The increased amount of Cx43 in enamel organ cells in Nkcc1-null mice suggests that these cells display a higher number of gap junctions to increase intercellular communication.

Integrative Temporo-Spatial, Mineralogic, Spectroscopic, and Proteomic Analysis of Postnatal Enamel Development in Teeth with Limited Growth.

Tooth amelogenesis is a complex process beginning with enamel organ cell differentiation and enamel matrix secretion, transitioning through changes in ameloblast polarity, cytoskeletal, and matrix organization, that affects crucial biomineralization events such as mineral nucleation, enamel crystal growth, and enamel prism organization. Here we have harvested the enamel organ including the pliable enamel matrix of postnatal first mandibular mouse molars during the first 8 days of tooth enamel development to conduct a step-wise cross-sectional analysis of the changes in the mineral and protein phase. Mineral phase diffraction pattern analysis using single-crystal, powder sample X-ray diffraction analysis indicated conversion of calcium phosphate precursors to partially fluoride substituted hydroxyapatite from postnatal day 4 (4 dpn) onwards. Attenuated total reflectance spectra (ATR) revealed a substantial elevation in phosphate and carbonate incorporation as well as structural reconfiguration between postnatal days 6 and 8. Nanoscale liquid chromatography coupled with tandem mass spectrometry (nanoLC-MS/MS) demonstrated highest protein counts for ECM/cell surface proteins, stress/heat shock proteins, and alkaline phosphatase on postnatal day 2, high counts for ameloblast cytoskeletal proteins such as tubulin β5, tropomyosin, β-actin, and vimentin on postnatal day 4, and elevated levels of cofilin-1, calmodulin, and peptidyl-prolyl cis-trans isomerase on day 6. Western blot analysis of hydrophobic enamel proteins illustrated continuously increasing amelogenin levels from 1 dpn until 8 dpn, while enamelin peaked on days 1 and 2 dpn, and ameloblastin on days 1–5 dpn. In summary, these data document the substantial changes in the enamel matrix protein and mineral phase that take place during postnatal mouse molar amelogenesis from a systems biological perspective, including (i) relatively high levels of matrix protein expression during the early secretory stage on postnatal day 2, (ii) conversion of calcium phosphates to apatite, peak protein folding and stress protein counts, and increased cytoskeletal protein levels such as actin and tubulin on day 4, as well as (iii) secondary structure changes, isomerase activity, highest amelogenin levels, and peak phosphate/carbonate incorporation between postnatal days 6 and 8. Together, this study provides a baseline for a comprehensive understanding of the mineralogic and proteomic events that contribute to the complexity of mammalian tooth enamel development.

CALCIFYING ODONTOGENIC CYST: CASE REPORT

The calcifying odontogenic cyst (COC) is an uncommon odontogenic lesion, presenting with histopathologic diversity and variable clinical behavior. Herein we report on a case of a male patient, 59 years old, referred for evaluation of an asymptomatic swelling, slow growth, soft consistency areas, in the anterior maxilla, identified 2 years ago. On radiographic examination, there was a radiolucent lesion, unilocular, and relatively well defined. A biopsy was performed and histopathologic examination revealed pathologic cavity presenting cystic lining, with inverted nuclear polarization of the basal layer and similar cells to the stellate reticulum of the enamel organ and numerous ghost cells. Under the diagnosis of COC, marsupialization was performed followed by enucleation of the lesion 2 months after. Six months after treatment, no tumor recurrence was seen. Enucleation is the appropriate treatment for most COC and, although it has low recurrence rates, it emphasizes the importance of close monitoring of patients.

Multiple Calcium Export Exchangers and Pumps Are a Prominent Feature of Enamel Organ Cells.

Calcium export is a key function for the enamel organ during all stages of amelogenesis. Expression of a number of ATPase calcium transporting, plasma membrane genes (ATP2B1-4/PMCA1-4), solute carrier SLC8A genes (sodium/calcium exchanger or NCX1-3), and SLC24A gene family members (sodium/potassium/calcium exchanger or NCKX1-6) have been investigated in the developing enamel organ in earlier studies. This paper reviews the calcium export pathways that have been described and adds novel insights to the spatiotemporal expression patterns of PMCA1, PMCA4, and NCKX3 during amelogenesis. New data are presented to show the mRNA expression profiles for the four Atp2b1-4 gene family members (PMCA1-4) in secretory-stage and maturation-stage rat enamel organs. These data are compared to expression profiles for all Slc8a and Slc24a gene family members. PMCA1, PMCA4, and NCKX3 immunolocalization data is also presented. Gene expression profiles quantitated by real time PCR show that: (1) PMCA1, 3, and 4, and NCKX3 are most highly expressed during secretory-stage amelogenesis; (2) NCX1 and 3, and NCKX6 are expressed during secretory and maturation stages; (3) NCKX4 is most highly expressed during maturation-stage amelogenesis; and (4) expression levels of PMCA2, NCX2, NCKX1, NCKX2, and NCKX5 are negligible throughout amelogenesis. In the enamel organ PMCA1 localizes to the basolateral membrane of both secretory and maturation ameloblasts; PMCA4 expression is seen in the basolateral membrane of secretory and maturation ameloblasts, and also cells of the stratum intermedium and papillary layer; while NCKX3 expression is limited to Tomes' processes, and the apical membrane of maturation-stage ameloblasts. These new findings are discussed in the perspective of data already present in the literature, and highlight the multiplicity of calcium export systems in the enamel organ needed to regulate biomineralization.

Store-operated Ca2+ Entry Modulates the Expression of Enamel Genes

Dental enamel formation is an intricate process tightly regulated by ameloblast cells. The correct spatiotemporal patterning of enamel matrix protein (EMP) expression is fundamental to orchestrate the formation of enamel crystals, which depend on a robust supply of Ca2+. In the extracellular milieu, Ca2+-EMP interactions occur at different levels. Despite its recognized role in enamel development, the molecular machinery involved in Ca2+ homeostasis in ameloblasts remains poorly understood. A common mechanism for Ca2+ influx is store-operated Ca2+ entry (SOCE). We evaluated the possibility that Ca2+ influx in enamel cells might be mediated by SOCE and the Ca2+ release-activated Ca2+ (CRAC) channel, the prototypical SOCE channel. Using ameloblast-like LS8 cells, we demonstrate that these cells express Ca2+-handling molecules and mediate Ca2+ influx through SOCE. As a rise in the cytosolic Ca2+ concentration is a versatile signal that can modulate gene expression, we assessed whether SOCE in enamel cells had any effect on the expression of EMPs. Our results demonstrate that stimulating LS8 cells or murine primary enamel organ cells with thapsigargin to activate SOCE leads to increased expression of Amelx, Ambn, Enam, Mmp20. This effect is reversed when cells are treated with a CRAC channel inhibitor. These data indicate that Ca2+ influx in LS8 cells and enamel organ cells is mediated by CRAC channels and that Ca2+ signals enhance the expression of EMPs. Ca2+ plays an important role not only in mineralizing dental enamel but also in regulating the expression of EMPs.

Expression of cytokeratins in enamel organ, junctional epithelium and epithelial cell rests of Malassez

After tooth formation is complete, it is suggested that continuity exists between the epithelial cell rests of Malassez (ERM), reduced enamel epithelium (REE) and subsequently the junctional epithelium. However, the junctional epithelium was reported to differ from REE and ERM. The developmental relationships between and among them remain controversial. Therefore, in the present study we examined the expression of cytokeratins in the three types of epithelia to investigate the epithelial phenotypes. The maxillae of Wistar rats, 1, 2, 3 and 7 wk of age, were used, and the expression of CK14, CK17, CK19, CK10/CK13 and AE1/AE3 was detected using immunoperoxidase techniques. There was negative staining for CK10/CK13 in all the epithelia. ERM stained strongly for AE1/AE3, CK14, CK17 and CK19. During the transformation of inner enamel epithelial (IEE) cells into reduced ameloblasts and subsequently into junctional epithelium, strong staining for CK14 was evident in IEE, REE and junctional epithelium, whereas the expression of AE1/AE3 and of CK19 were initially negative in IEE and then strong in REE and junctional epithelium, respectively. In particular, the expression of CK17 was strongly positive in ERM and REE, but was negative in IEE and junctional epithelium. ERM are of odontogenic origin and junctional epithelium has an epithelial phenotype different from REE and ERM. This is the first report to demonstrate that CK17 can be used as a marker to distinguish junctional epithelium from ERM.

Enamel maturation: a brief background with implications for some enamel dysplasias.

The maturation stage of enamel development begins once the final tissue thickness has been laid down. Maturation includes an initial transitional pre-stage during which morphology and function of the enamel organ cells change. When this is complete, maturation proper begins. Fully functional maturation stage cells are concerned with final proteolytic degradation and removal of secretory matrix components which are replaced by tissue fluid. Crystals, initiated during the secretory stage, then grow replacing the tissue fluid. Crystals grow in both width and thickness until crystals abut each other occupying most of the tissue volume i.e. full maturation. If this is not complete at eruption, a further post eruptive maturation can occur via mineral ions from the saliva. During maturation calcium and phosphate enter the tissue to facilitate crystal growth. Whether transport is entirely active or not is unclear. Ion transport is also not unidirectional and phosphate, for example, can diffuse out again especially during transition and early maturation. Fluoride and magnesium, selectively taken up at this stage can also diffuse both in an out of the tissue. Crystal growth can be compromised by excessive fluoride and by ingress of other exogenous molecules such as albumin and tetracycline. This may be exacerbated by the relatively long duration of this stage, 10 days or so in a rat incisor and up to several years in human teeth rendering this stage particularly vulnerable to ingress of foreign materials, incompletely mature enamel being the result.

NBCe1 (SLC4A4) a potential pH regulator in enamel organ cells during enamel development in the mouse.

During the formation of dental enamel, maturation-stage ameloblasts express ion-transporting transmembrane proteins. The SLC4 family of ion-transporters regulates intra- and extracellular pH in eukaryotic cells by cotransporting HCO3 (-) with Na(+). Mutation in SLC4A4 (coding for the sodium-bicarbonate cotransporter NBCe1) induces developmental defects in human and murine enamel. We have hypothesized that NBCe1 in dental epithelium is engaged in neutralizing protons released during crystal formation in the enamel space. We immunolocalized NBCe1 protein in wild-type dental epithelium and examined the effect of the NBCe1-null mutation on enamel formation in mice. Ameloblasts expressed gene transcripts for NBCe1 isoforms B/D/C/E. In wild-type mice, weak to moderate immunostaining for NBCe1 with antibodies that recognized isoforms A/B/D/E and isoform C was seen in ameloblasts at the secretory stage, with no or low staining in the early maturation stage but moderate to high staining in the late maturation stage. The papillary layer showed the opposite pattern being immunostained prominently at the early maturation stage but with gradually less staining at the mid- and late maturation stages. In NBCe1 (-/-) mice, the ameloblasts were disorganized, the enamel being thin and severely hypomineralized. Enamel organs of CFTR (-/-) and AE2a,b (-/-) mice (CFTR and AE2 are believed to be pH regulators in ameloblasts) contained higher levels of NBCe1 protein than wild-type mice. Thus, the expression of NBCe1 in ameloblasts and the papillary layer cell depends on the developmental stage and possibly responds to pH changes.

Gene-expression profile and localization of Na+/K+-ATPase in rat enamel organ cells

The sodium pump Na(+)/K(+)-ATPase, expressed in virtually all cells of higher organisms, is involved in establishing a resting membrane potential and in creating a sodium gradient to facilitate a number of membrane-associated transport activities. Na(+)/K(+)-ATPase is an oligomer of α, β, and γ subunits. Four unique genes encode each of the α and β subunits. In dental enamel cells, the spatiotemporal expression of Na(+)/K(+)-ATPase is poorly characterized. Using the rat incisor as a model, this study provides a comprehensive expression profile of all four α and all four β Na(+)/K(+)-ATPase subunits throughout all stages of amelogenesis. Real-time PCR, western blot analysis, and immunolocalization revealed that α1, β1, and β3 are expressed in the enamel organ and that all three are most highly expressed during late-maturation-stage amelogenesis. Expression of β3 was significantly higher than expression of β1, suggesting that the dominant Na(+)/K(+)-ATPase consists of an α1β3 dimer. Localization of α1, β1, and β3 subunits in ameloblasts was primarily to the cytoplasm and occasionally along the basolateral membranes. Weaker expression was also noted in papillary layer cells during early maturation. Our data support that Na(+)/K(+)-ATPase is functional in maturation-stage ameloblasts.

Reciprocal expressions between α-dystroglycan and integrin β1, perlecan receptors, in the murine enamel organ development

Signals of perlecan, an extracellular matrix molecule, which accumulates within the intercellular spaces of the stellate reticulum of the enamel organ, are mediated by at least two receptors, dystroglycan (DG) and integrin β1, in a case-dependent manner in various events in embryogenesis and pathogenesis. This study aims to understand the expression profiles of these two perlecan receptors at both protein and gene levels in murine enamel organ development. Before birth, α-DG was immunolocalized in stellate reticulum cells, in which perlecan was colocalized, while integrin β1 was mainly distributed in the peripheral enamel organ cells as well as the dental mesenchymal cells. On and after postnatal Day 1, the expression of α-DG was dramatically decreased in the stellate reticulum, while integrin β1 was enhanced around blood vessels within the enamel organ. Furthermore, biosyntheses of α-DG and integrin β1 by dental epithelial and pulp mesenchymal cells were confirmed in vitro by using immunofluorescence and reverse-transcriptase polymerase chain reaction. The results suggest that DG is a perlecan receptor that specifically functions in the stellate reticulum of the embryonic stage, but that dental epithelial and mesenchymal cells are maturated by capturing perlecan signals differentially through integrin β1.

Adaptor protein complex 2–mediated, clathrin-dependent endocytosis, and related gene activities, are a prominent feature during maturation stage amelogenesis

Molecular events defining enamel matrix removal during amelogenesis are poorly understood. Early reports have suggested that adaptor proteins (AP) participate in ameloblast-mediated endocytosis. Enamel formation involves the secretory and maturation stages, with an increase in resorptive function during the latter. Here, using real-time PCR, we show that the expression of clathrin and adaptor protein subunits are upregulated in maturation stage rodent enamel organ cells. AP complex 2 (AP-2) is the most upregulated of the four distinct adaptor protein complexes. Immunolocalization confirms the presence of AP-2 and clathrin in ameloblasts, with strongest reactivity at the apical pole. These data suggest that the resorptive functions of enamel cells involve AP-2 mediated, clathrin-dependent endocytosis, thus implying the likelihood of specific membrane-bound receptor(s) of enamel matrix protein debris. The mRNA expression of other endocytosis-related gene products is also upregulated during maturation including: lysosomal-associated membrane protein 1 (Lamp1); cluster of differentiation 63 and 68 (Cd63 and Cd68); ATPase, H(+) transporting, lysosomal V0 subunit D2 (Atp6v0d2); ATPase, H(+) transporting, lysosomal V1 subunit B2 (Atp6v1b2); chloride channel, voltage-sensitive 7 (Clcn7); and cathepsin K (Ctsk). Immunohistologic data confirms the expression of a number of these proteins in maturation stage ameloblasts. The enamel of Cd63-null mice was also examined. Despite increased mRNA and protein expression in the enamel organ during maturation, the enamel of Cd63-null mice appeared normal. This may suggest inherent functional redundancies between Cd63 and related gene products, such as Lamp1 and Cd68. Ameloblast-like LS8 cells treated with the enamel matrix protein complex Emdogain showed upregulation of AP-2 and clathrin subunits, further supporting the existence of a membrane-bound receptor-regulated pathway for the endocytosis of enamel matrix proteins. These data together define an endocytotic pathway likely used by ameloblasts to remove the enamel matrix during enamel maturation.

Unicuspid and bicuspid tooth crown formation in squamates

The molecular and developmental factors that regulate tooth morphogenesis in nonmammalian species, such as snakes and lizards, have received relatively little attention compared to mammals. Here we describe the development of unicuspid and bicuspid teeth in squamate species. The simple, cone-shaped tooth crown of the bearded dragon and ball python is established at cap stage and fixed in shape by the differentiation of cells and the secretion of dental matrices. Enamel production, as demonstrated by amelogenin expression, occurs relatively earlier in squamate teeth than in mouse molars. We suggest that the early differentiation in squamate unicuspid teeth at cap stage correlates with a more rudimentary tooth crown shape. The leopard gecko can form a bicuspid tooth crown despite the early onset of differentiation. Cusp formation in the gecko does not occur by the folding of the inner enamel epithelium, as in the mouse molar, but by the differential secretion of enamel. Ameloblasts forming the enamel epithelial bulge, a central swelling of cells in the inner enamel epithelium, secrete amelogenin at cap stage, but cease to do so by bell stage. Meanwhile, other ameloblasts in the inner enamel epithelium continue to secrete enamel, forming cusp tips on either side of the bulge. Bulge cells specifically express the gene Bmp2, which we suggest serves as a pro-differentiation signal for cells of the gecko enamel organ. In this regard, the enamel epithelial bulge of the gecko may be more functionally analogous to the secondary enamel knot of mammals than the primary enamel knot.

Morphogenetic roles of perlecan in the tooth enamel organ: An analysis of overexpression using transgenic mice

Perlecan, a heparan sulfate proteoglycan, is enriched in the intercellular space of the enamel organ. To understand the role of perlecan in tooth morphogenesis, we used a keratin 5 promoter to generate transgenic ( Tg) mice that over-express perlecan in epithelial cells, and examined their tooth germs at tissue and cellular levels. Immunohistochemistry showed that perlecan was more strongly expressed in the enamel organ cells of Tg mice than in wild-type mice. Histopathology showed wider intercellular spaces in the stellate reticulum of the Tg molars and loss of cellular polarity in the enamel organ, especially in its cervical region. Hertwig's epithelial root sheath (HERS) cells in Tg mice were irregularly aligned due to excessive deposits of perlecan along the inner, as well as on the outer sides of the HERS. Tg molars had dull-ended crowns and outward-curved tooth roots and their enamel was poorly crystallized, resulting in pronounced attrition of molar cusp areas. In Tg mice, expression of integrin β1 mRNA was remarkably higher at E18, while expression of bFGF, TGF-β1, DSPP and Shh was more elevated at P1. The overexpression of perlecan in the enamel organ resulted in irregular morphology of teeth, suggesting that the expression of perlecan regulates growth factor signaling in a stage-dependent manner during each step of the interaction between ameloblast-lineage cells and mesenchymal cells.

Chymotrypsin C (Caldecrin) Is Associated with Enamel Development

Two main proteases cleave enamel extracellular matrix proteins during amelogenesis. Matrix metalloprotease-20 (Mmp20) is the predominant enzyme expressed during the secretory stage, while kallikrein-related peptidase-4 (Klk4) is predominantly expressed during maturation. Mutations to both Mmp20 and Klk4 result in abnormal enamel phenotypes. During a recent whole-genome microarray analysis of rat incisor enamel organ cells derived from the secretory and maturation stages of amelogenesis, the serine protease chymotrypsin C (caldecrin, Ctrc) was identified as significantly up-regulated (> 11-fold) during enamel maturation. Prior reports indicate that Ctrc expression is pancreas-specific, albeit low levels were also noted in brain. We here report on the expression of Ctrc in the enamel organ. Quantitative PCR (qPCR) and Western blot analysis were used to confirm the expression of Ctrc in the developing enamel organ. The expression profile of Ctrc is similar to that of Klk4, increasing markedly during the maturation stage relative to the secretory stage, although levels of Ctrc mRNA are lower than for Klk4. The discovery of a new serine protease possibly involved in enamel development has important implications for our understanding of the factors that regulate enamel biomineralization.