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.     

Immunocytochemical and biochemical detection of EMMPRIN in the rat tooth germ: differentiation-dependent co-expression with MMPs and co-localization with caveolin-1 in membrane rafts of dental epithelial cells

In tooth development matrix metalloproteinases (MMPs) are under the control of several regulatory mechanisms including the upregulation of expression by inducers and downregulation by inhibitors. The aim of the present study was to monitor the occurrence and distribution pattern of the extracellular matrix metalloproteinase inducer (EMMPRIN), the metalloproteinases MMP-2 and MT1-MMP and caveolin-1 during the cap and bell stage of rat molar tooth germs by means of immunocytochemistry. Strong EMMPRIN immunoreactivity was detected on the cell membranes of ameloblasts and cells of the stratum intermedium in the bell stage of the enamel organ. Differentiating odontoblasts exhibited intense EMMPRIN immunoreactivity, especially at their distal ends. Caveolin-1 immunoreactivity was evident in cells of the internal enamel epithelium and in ameloblasts. Double immunofluorescence studies revealed a focal co-localization between caveolin-1 and EMMPRIN in ameloblastic cells. Finally, western blotting experiments demonstrated the expression of EMMPRIN and caveolin-1 in dental epithelial cells (HAT-7 cells). A substantial part of EMMPRIN was detected in the detergent-insoluble caveolin-1-containing low-density raft membrane fraction of HAT-7 cells suggesting a partial localization within lipid rafts. The differentiation-dependent co-expression of MMPs with EMMPRIN in the enamel organ and in odontoblasts indicates that EMMPRIN takes part in the induction of proteolytic enzymes in the rat tooth germ. The localization of EMMPRIN in membrane rafts provides a basis for further investigations on the role of caveolin-1 in EMMPRIN-mediated signal transduction cascades in ameloblasts.     

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.     

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

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

Accelerated dentinogenesis by inhibiting the mitochondrial fission factor,dynamin related protein 1

Undifferentiated odontogenic epithelium and dental papilla cells differentiate into ameloblasts and odontoblasts, respectively, both of which are essential for tooth development. These differentiation processes involve dramatic functional and morphological changes of the cells. For these changes to occur, activation of mitochondrial functions, including ATP production, is extremely important. In addition, these changes are closely related to mitochondrial fission and fusion, known as mitochondrial dynamics. However, few studies have focused on the role of mitochondrial dynamics in tooth development. The purpose of this study was to clarify this role. We used mouse tooth germ organ cultures and a mouse dental papilla cell line with the ability to differentiate into odontoblasts, in combination with knockdown of the mitochondrial fission factor, dynamin related protein (DRP)1. In organ cultures of the mouse first molar, tooth germ developed to the early bell stage. The amount of dentin formed under DRP1 inhibition was significantly larger than that of the control. In experiments using a mouse dental papilla cell line, differentiation into odontoblasts was enhanced by inhibiting DRP1. This was associated with increased mitochondrial elongation and ATP production compared to the control. These results suggest that DRP1 inhibition accelerates dentin formation through mitochondrial elongation and activation. This raises the possibility that DRP1 might be a therapeutic target for developmental disorders of teeth.     

Expression of Set-α during Morphogenesis of Mouse Lower First Molar

The detailed in situ expression pattern of the Set-α gene has been studied. Previously we showed that Set-α is a differentially expressed gene in the embryonic mouse mandible at day 10.5 (E10.5) gestational age. Cells expressing Set-α were widely distributed in both the epithelial and underlying ectomesenchymal cells at E10.5. At E12, they were slightly aggregated in an area where tooth germ of the lower first molar is estimated to be formed. At E13.5, Set-α was strongly expressed in the tooth germ. At the cap stage, Set-α was expressed in the enamel organ and dental papilla. At the bell stage, Set-α was distinctly expressed in the inner enamel epithelial and dental papilla cells facing the inner enamel epithelial layer, which were intended to differentiate into ameloblasts and odontoblasts, respectively. Interestingly, Set-α was also expressed in several embryonic craniofacial tissues derived from the ectoderm. This study is the first report that Set-α is distinctly expressed in the developing tooth germ, and suggests that Set-α plays an important role in both the initiation and the growth of the tooth germ, as well as in the differentiation of ameloblasts and odontoblasts.     

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.     

Notch2 protein distribution in human teeth under normal and pathological conditions

Notch signaling is essential for the appropriate differentiation of many cell types during development and, furthermore, is implicated in a variety of human diseases. Previous studies have shown that although the Notch1, -2, and -3 receptors are expressed in developing and injured rodent teeth, Notch2 expression was predominant after a lesion. To pursue the role of the Notch pathway in tooth development and disease, we have analyzed the expression of the Notch2 protein in embryonic and adult wounded human teeth. During the earlier stages of tooth development, the Notch2 protein was expressed in the epithelium, but was absent from proliferating cells of the inner enamel epithelium. At more advanced stages, Notch2 was expressed in the enamel-producing ameloblasts, while it was absent in mesenchyme-derived odontoblasts that synthesize the dentin matrix. Although Notch2 was not expressed in the pulp of adult intact teeth, it was reexpressed during dentin repair processes in odontoblasts and subodontoblastic cells. Transforming growth factor beta-1, which stimulates odontoblast differentiation and hard tissue formation after dental injury, downregulated Notch2 expression in cultured human dental slices, in vitro. These observations are consistent with the notion that Notch signaling is an important element in dental physiological and pathogenic conditions.     

PHEX neutralizing agent inhibits dentin formation in mouse tooth germ

The mutation of phosphate-regulating gene with homologies to endopeptidases on the X-chromosome (PHEX) can lead to human X-linked hypophosphatemic rickets which displays hypo-mineralization in bone and dentin. To study its possible roles in teeth, PHEX antibody was injected into pregnant mice on E15 to explore its roles on the formation of enamel and dentin. Mallory trichrome staining results showed that arrangements of ameloblasts and odontoblasts were irregular after PHEX antibody treatment. Differentiation of odontoblasts and the formation of dentin were inhibited. Spatiotemporal distribution of PHEX protein was observed in various stages of tooth germ. Immunohistochemical results showed positive PHEX signals appeared in the inner enamel epithelium on E16 and became stronger on E18. Ameloblasts and odontoblasts showed much higher PHEX expression on P1 and P3. Expression of PHEX in odontoblasts decreased accordingly. However, enamel formation was only slightly affected. The findings proved that a decrease in PHEX expression could suppress dentin formation.     

Dentin sialoprotein: biosynthesis and developmental appearance in rat tooth germs in comparison with amelogenins,osteocalcin and colagen type-I

A non-collagenous protein, extracted from rat incisor dentin, is a dentin sialoprotein (DSP). We examined immunohistochemically the developmental appearance and tissue distribution of DSP in 1 to 3-day-old rat molar and incisor tooth germs. The earliest staining for DSP was observed in newly differentiated odontoblasts. In more advanced stages, immunostaining for DSP gradually increased in pre-dentin, odontoblasts and dentin, and appeared in many cells of the dental papilla. In early stages of development before the breakdown of the dental basement membrane, pre-ameloblasts were also positive for DSP. This staining disappeared from the ameloblast cell body soon after deposition of the first layer of mineralized dentin. Radiolabelling of tooth matrix proteins with ¹⁴C-serine in vitro followed by immunoprecipitation and fluorography confirmed that DSP was synthesized by tooth-forming cells. The immunolocalization for DSP was different from that of either collagen type-I, osteocalcin or the amelogenins. Whereas collagen type-I and osteocalcin were restricted to the mesenchymal dental tissues, the amelogenins were detectable in both epithelial and mesenchymal dental cells and tissues at the epithelio-mesenchymal interface at early stages of development, prior to the onset of dentin mineralization. We conclude that DSP is expressed in and secreted by odontoblasts and some dental papilla cells from early stages of dentinogenesis onwards, i.e. later than type-I collagen, but before deposition of the first layer of mineralized dentin. In pre-mineralizing stages, some of the matrix proteins may be endocytosed from the pre-dentin by both cell types involved in the epithelio-mesenchymal interaction.     

Cell pellets from dental papillae can reexhibit dental morphogenesis and dentinogenesis

We isolated dental papilla mesenchymal cells (DPMCs) from different rat incisor germs at the late bell stage and incubated them as cell pellets in polypropylene tubes. In vitro pellet culture of DPMCs presented several crucial characteristics of odontoblasts, as indicated by accelerated mineralization, positive immunostaining for dentin sialophosphoprotein and dentin matrix protein 1, and expression of dentin sialophosphoprotein mRNA. The allotransplantation of these pellets into renal capsules was also performed. Despite the absence of dental epithelial components, dissociated DPMCs with a complete loss of positional information rapidly underwent dentinogenesis and morphogenesis, and formed a cusp-like dentin-pulp complex containing distinctive odontoblasts, predentin, dentin, and dentinal tubules. These results imply that DPMCs at the late bell stage can reexhibit the dental morphogenesis and dentinogenesis by themselves, and epithelial-mesenchymal interactions at this stage may not be indispensable. Furthermore, different DPMC populations from the similar stage may keep the same developmental pattern.     

FAM20C Plays an Essential Role in the Formation of Murine Teeth

FAM20C is highly expressed in bone and tooth. Previously, we showed that Fam20C conditional knock-out (KO) mice manifest hypophosphatemic rickets, which highlights the crucial roles of this molecule in promoting bone formation and mediating phosphate homeostasis. In this study, we characterized the dentin, enamel, and cementum of Sox2-Cre-mediated Fam20C KO mice. The KO mice exhibited small malformed teeth, severe enamel defects, very thin dentin, less cementum than normal, and overall hypomineralization in the dental mineralized tissues. In situ hybridization and immunohistochemistry analyses revealed remarkable down-regulation of dentin matrix protein 1 (DMP1) and dentin sialophosphoprotein in odontoblasts, along with a sharply reduced expression of ameloblastin and amelotin in ameloblasts. Collectively, these data indicate that FAM20C is essential to the differentiation and mineralization of dental tissues through the regulation of molecules critical to the differentiation of tooth-formative cells.     

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.     

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.     

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

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

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.     

Fate map of the dental mesenchyme: dynamic development of the dental papilla and follicle

At the bud stage of tooth development the neural crest derived mesenchyme condenses around the dental epithelium. As the tooth germ develops and proceeds to the cap stage, the epithelial cervical loops grow and appear to wrap around the condensed mesenchyme, enclosing the cells of the forming dental papilla. We have fate mapped the dental mesenchyme, using in vitro tissue culture combined with vital cell labelling and tissue grafting, and show that the dental mesenchyme is a much more dynamic population then previously suggested. At the bud stage the mesenchymal cells adjacent to the tip of the bud form both the dental papilla and dental follicle. At the early cap stage a small population of highly proliferative mesenchymal cells in close proximity to the inner dental epithelium and primary enamel knot provide the major contribution to the dental papilla. These cells are located between the cervical loops, within a region we have called the body of the enamel organ, and proliferate in concert with the epithelium to create the dental papilla. The condensed dental mesenchymal cells that are not located between the body of the enamel organ, and therefore are at a distance from the primary enamel knot, contribute to the dental follicle, and also the apical part of the papilla, where the roots will ultimately develop. Some cells in the presumptive dental papilla at the cap stage contribute to the follicle at the bell stage, indicating that the dental papilla and dental follicle are still not defined populations at this stage. These lineage-tracing experiments highlight the difficulty of targeting the papilla and presumptive odontoblasts at early stages of tooth development. We show that at the cap stage, cells destined to form the follicle are still competent to form dental papilla specific cell types, such as odontoblasts, and produce dentin, if placed in contact with the inner dental epithelium. Cell fate of the dental mesenchyme at this stage is therefore determined by the epithelium.