Parathyroid hormone-related peptide (1–34) promotes tooth eruption and inhibits osteogenesis of dental follicle cells during tooth development

Dental follicle cells (DFCs) activate and recruit osteoclasts for tooth development and tooth eruption, whereas DFCs themselves differentiate into osteoblasts to form alveolar bone surrounding tooth roots through the interaction with Hertwig's epithelial root sheath (HERS). Also during tooth development, parathyroid hormone-related peptide (PTHrP) is expressed surrounding the tooth germ. Thus, we aimed to investigate the effect of PTHrP (1–34) on bone resorption and osteogenesis of DFCs in vitro and in vivo. In vitro studies demonstrated that DFCs cocultured with HERS cells expressed higher levels of BSP and OPN than the DFCs control group, whereas cocultured DFCs treated with PTHrP (1–34) had lower expressions of ALP, RUNX2, BSP, and OPN than the cocultured DFCs control group. Moreover, we found PTHrP (1–34) inhibited osteogenesis of cocultured DFCs by inactivating the Wnt/β-catenin pathway. PTHrP (1–34) also increased the expression of RANKL/OPG ratio in DFCs. Consistently, in vivo study found that PTHrP (1–34) accelerated tooth eruption and inhibited alveolar bone formation. Therefore, these results suggest that PTHrP (1–34) accelerates tooth eruption and inhibits osteogenesis of DFCs by inactivating Wnt/β-catenin pathway.     

Fate of the Molar Dental Lamina in the Monophyodont Mouse

The successional dental lamina (SDL) plays an essential role in the development of replacement teeth in diphyodont and polyphyodont animals. A morphologically similar structure, the rudimental successional dental lamina (RSDL), has been described in monophyodont (only one tooth generation) lizards on the lingual side of the developing functional tooth. This rudimentary lamina regresses, which has been proposed to play a role in preventing the formation of future generations of teeth. A similar rudimentary lingual structure has been reported associated with the first molar in the monophyodont mouse, and we show that this structure is common to all murine molars. Intriguingly, a lingual lamina is also observed on the non-replacing molars of other diphyodont mammals (pig and hedgehog), initially appearing very similar to the successional dental lamina on the replacing teeth. We have analyzed the morphological as well as ultrastructural changes that occur during the development and loss of this molar lamina in the mouse, from its initiation at late embryonic stages to its disappearance at postnatal stages. We show that loss appears to be driven by a reduction in cell proliferation, down-regulation of the progenitor marker Sox2, with only a small number of cells undergoing programmed cell death. The lingual lamina was associated with the dental stalk, a short epithelial connection between the tooth germ and the oral epithelium. The dental stalk remained in contact with the oral epithelium throughout tooth development up to eruption when connective tissue and numerous capillaries progressively invaded the dental stalk. The buccal side of the dental stalk underwent keratinisation and became part of the gingival epithelium, while most of the lingual cells underwent programmed cell death and the tissue directly above the erupting tooth was shed into the oral cavity.     

Cellular expression of neurotrophin mRNAs during tooth development

. Target-derived neurotrophins support and sustain peripheral sensory neurons during development. In addition, it has been suggested that these growth factors could have developmental functions in non-neuronal tissues. To further elucidate the possible roles of neurotrophins in tooth morphogenesis and innervation, we have used in-situ hybridization to determine the specific sites of neurotrophin gene activity in pre- and postnatal rat jaws from E16 to P7. All four neurotrophins were expressed during tooth development with specific temporospatial patterns. Nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) mRNAs were mainly detected in the dental papilla/pulp in postnatal animals, and the pattern of expression correlated with the onset of dental innervation. In contrast, neurotrophin 3 (NT3) and neurotrophin 4 (NT4) mRNA expression patterns were predominantly epithelial and were strongest during early developmental stages when teeth are not yet innervated. Dental papilla NGF-mRNA expression was first seen in both epithelium and mesenchyme and later shifted to the odontoblast layer and the subodontoblast zone. BDNF-mRNA labeling was present in low levels in the early dental organ, but increased in the pulp and in the odontoblast cell layer of the developing teeth at later developmental stages. Both NT3 and NT4 mRNA were observed in the prenatal oral epithelium and the inner dental epithelium. NT3-mRNA labeling was seen mainly in the cervical loop region, fissure system depressions and cuspal tops, while NT4 mRNA was more evenly distributed in the dental epithelium. At P7, NT3-mRNA labeling was below detection level and NT4 mRNA expression was lower than at prior stages. Complementary to reports on the presence of low-affinity neurotrophin receptor (LANR), trkB and trkC mRNA in the developing teeth, our results suggest that neurotrophins may have multiple functions during tooth morphogenesis. Neurotrophins might participate in epithelial-mesenchymal interactions in early tooth morphogenetic events such as proliferation and differentiation of epithelial and mesenchymal cells. In addition, based on mRNA localization in postnatal animals, we also suggest that NGF and BDNF (beside glial cell line-derived neurotrophic factor) might participate in establishing and maintaining the innervation of the teeth, thus acting as classical neurotrophic factors.     

Expression pattern of E‐cadherin during development of the first tooth in zebrafish (Danio rerio)

Although the importance of cell adhesion in morphogenesis is already known for quite some time, there are remarkably few studies on the distribution and function of adhesion molecules in tooth development. We have chosen the zebrafish to study the role of specific cell adhesion molecules in the development and renewal of teeth. Zebrafish lack an oral dentition but have pharyngeal teeth which are renewed throughout life. Here we focus on the expression of E (epithelial)‐cadherin during the development of the first tooth to develop in the dentition, ‘initiator tooth’ 4V¹. E‐cadherin is expressed exclusively in the pharyngeal epithelium and in the enamel organ throughout all stages of development of this first‐generation tooth. Further studies are needed to compare this expression pattern with protein distribution, both in this and other first‐generation teeth as well as in replacement teeth.     

Cell surface proteoglycan expression correlates with epithelial-mesenchymal interaction during tooth morphogenesis

Tooth morphogenesis and differentiation of the dental cells are guided by interactions between epithelial and mesenchymal tissues. Because the extracellular matrix is involved in these interactions, the expression of matrix receptors located at the cell surface may change during this developmental sequence. We have examined the distribution of an epithelial cell surface proteoglycan antigen, known to behave as a receptor for interstitial matrix, during tooth morphogenesis. Intense staining was seen around the cells of the embryonic oral epithelium as well as the dental epithelium at the early bud stage. With development, expression was greatly reduced in the enamel organ. Differentiation of these cells into ameloblasts was associated with the loss of expression, while the epithelial cells remaining in the stratum intermedium and stellate reticulum regained intense staining. The PG antigen was weakly expressed in the loose neural crest-derived jaw mesenchyme but it became strongly reactive in the condensed dental papilla mesenchyme when extensive morphogenetic movements took place. With development, the PG antigen disappeared from the advanced dental papilla mesenchyme but persisted in the dental sac mesenchyme, which gives rise to periodontal tissues. The PG antigen was not expressed by odontoblasts. Hence, the expression of the PG antigen changes during the epithelial-mesenchymal interactions of tooth development and is lost during terminal cell differentiation. The expression follows morphogenetic rather than histologic boundaries. The acquisition and loss of expression in epithelial and mesenchymal tissues during tooth development suggest that this proteoglycan has specific functions in the epithelial-mesenchymal interactions that guide morphogenesis.     

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.     

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.     

Gene deployment for tooth replacement in the rainbow trout (Oncorhynchus mykiss): a developmental model for evolution of the osteichthyan dentition

Repeated tooth initiation occurs often in nonmammalian vertebrates (polyphyodontism), recurrently linked with tooth shedding and in a definite order of succession. Regulation of this process has not been genetically defined and it is unclear if the mechanisms for constant generation of replacement teeth (secondary dentition) are similar to those used to generate the primary dentition. We have therefore examined the expression pattern of a sub-set of genes, implicated in tooth initiation in mouse, in relation to replacement tooth production in an osteichthyan fish (Oncorhynchus mykiss). Two epithelial genes pitx2, shh and one mesenchymal bmp4 were analyzed at selected stages of development for O. mykiss. pitx2 expression is upregulated in the basal outer dental epithelium (ODE) of the predecessor tooth and before cell enlargement, on the postero-lingual side only. This coincides with the site for replacement tooth production identifying a region responsible for further tooth generation. This corresponds with the expression of pitx2 at focal spots in the basal oral epithelium during initial (first generation) tooth formation but is now sub-epithelial in position and associated with the dental epithelium of each predecessor tooth. Co-incidental expression of bmp4 and aggregation of the mesenchymal cells identifies the epithelial-mesenchymal interactions and marks initiation of the dental papilla. These together suggest a role in tooth site regulation by pitx2 together with bmp4. Conversely, the expression of shh is confined to the inner dental epithelium during the initiation of the first teeth and is lacking from the ODE in the predecessor teeth, at sites identified as those for replacement tooth initiation. Importantly, these genes expressed during replacement tooth initiation can be used as markers for the sites of "set-aside cells," the committed odontogenic cells both epithelial and mesenchymal, which together can give rise to further generations of teeth. This information may show how initial pattern formation is translated into secondary tooth replacement patterns, as a general mechanism for patterning the vertebrate dentition. Replacement of the marginal sets of teeth serves as a basis for discussion of the evolutionary significance, as these dentate bones (dentary, premaxilla, maxilla) form the restricted arcades of oral teeth in many crown-group gnathostomes, including members of the tetrapod stem group.     

Constitutively active PTH/PTHrP receptor in odontoblasts alters odontoblast and ameloblast function and maturation

Parathyroid hormone (PTH)-related protein (PTH-rP) is an important autocrine/paracrine attenuator of programmed cell differentiation whose expression is restricted to the epithelial layer in tooth development. The PTH/PTHrP receptor (PPR) mRNA in contrast is detected in the dental papilla, suggesting that PTHrP and the PPR may modulate epithelial-mesenchymal interactions. To explore the possible interactions, we studied the previously described transgenic mice in which a constitutively active PPR is targeted to osteoblastic cells. These transgenic mice have a vivid postnatal bone and tooth phenotype, with normal tooth eruption but abnormal, widened crowns. Transgene mRNA expression was first detected at birth in the dental papilla and, at 1 week postnatally, in odontoblasts. There was no transgene expression in ameloblasts or in other epithelial structures. Prenatally, transgenic molars and incisors revealed no remarkable change. By the age of 1 week, the dental papilla was widened, with disorganization of the odontoblastic layer and decreased dentin matrix. In addition, the number of cusps was abnormally increased, the ameloblastic layer disorganized, and enamel matrix decreased. Odontoblastic and, surprisingly, ameloblastic cytodifferentiation was impaired, as shown by in situ hybridization and electron microscopy. Interestingly, ameloblastic expression of Sonic Hedgehog, a major determinant of ameloblastic cytodifferentiation, was dramatically altered in the transgenic molars. These data suggest that odontoblastic activation of the PPR may play an important role in terminal odontoblastic and, indirectly, ameloblastic cytodifferentiation, and describe a useful model to study how this novel action of the PPR may modulate mesenchymal/epithelial interactions at later stages of tooth morphogenesis and development.     

Localization and quantitation of 125I-epidermal growth factor binding in mouse embryonic tooth and other embryonic tissues at different developmental stages

We have shown earlier that epidermal growth factor (EGF) inhibits morphogenesis and cell differentiation in mouse embryonic teeth in organ culture. This inhibition depends on the stage of tooth development so that only teeth at early developmental stages respond to EGF (A-M. Partanen, P. Ekblom, and I. Thesleff (1985) Dev. Biol. 111, 84-94). We have now studied the quantity and pattern of EGF binding in teeth at various stages of development by incubating the dissected tooth germs with 125I-labeled EGF. Although the quantity of 125I-EGF binding per microgram DNA stays at the same level, localization of 125I-EGF binding by autoradiography reveals that the distribution of binding sites changes dramatically. In bud stage the epithelial tooth bud that is intruding into the underlying mesenchyme has binding sites for EGF, but the condensation of dental mesenchymal cells around the bud does not bind EGF. At the cap stage of development the dental mesenchyme binds EGF, but the dental epithelium shows no binding. This indicates that the dental mesenchyme is the primary target tissue for the inhibitory effect of EGF on tooth morphogenesis during early cap stage. During advanced morphogenesis the binding sites of EGF disappear also from the dental papilla mesenchyme, but the dental follicle which consists of condensed mesenchymal cells surrounding the tooth germ, binds EGF abundantly. We have also studied EGF binding during the development of other embryonic organs, kidney, salivary gland, lung, and skin, which are all formed by mesenchymal and epithelial components. The patterns of EGF binding in various tissues suggest that EGF may have a role in the organogenesis of epitheliomesenchymal organs as a stimulator of epithelial proliferation during initial epithelial bud formation and branching morphogenesis. The results of this study indicate that EGF stimulates or maintains proliferation of undifferentiated cells during embryonic development and that the expression of EGF receptors in different organs is not related to the age of the embryo, but is specific to the developmental stage of each organ.     

Parathyroid hormone-related protein maintains mammary epithelial fate and triggers nipple skin differentiation during embryonic breast development

Prior reports have demonstrated that both parathyroid hormone-related protein (PTHrP) and the type I PTH/PTHrP receptor are necessary for the proper development of the embryonic mammary gland in mice. Using a combination of loss-of-function and gain-of-function models, we now report that PTHrP regulates a series of cell fate decisions that are central to the survival and morphogenesis of the mammary epithelium and the formation of the nipple. PTHrP is made in the epithelial cells of the mammary bud and, during embryonic mammary development, it interacts with the surrounding mesenchymal cells to induce the formation of the dense mammary mesenchyme. In response, these mammary-specific mesenchymal cells support the maintenance of mammary epithelial cell fate, trigger epithelial morphogenesis and induce the overlying epidermis to form the nipple. In the absence of PTHrP signaling, the mammary epithelial cells revert to an epidermal fate, no mammary ducts are formed and the nipple does not form. In the presence of diffuse epidermal PTHrP signaling, the ventral dermis is transformed into mammary mesenchyme and the entire ventral epidermis becomes nipple skin. These alterations in cell fate require that PTHrP be expressed during development and they require the presence of the PTH/PTHrP receptor. Finally, PTHrP signaling regulates the epidermal and mesenchymal expression of LEF1 and (&bgr;)-catenin, suggesting that these changes in cell fate involve an interaction between the PTHrP and Wnt signaling pathways.     

The human homologue of the Aspergillus nuclear migration gene nudC is preferentially expressed in dividing cells and ciliated epithelia

An early event in apoptosis is exposure of phosphatidylserine, an aminophospholipid normally present in the inner leaflet of the plasma membranes, at the outer leaflet of the plasma membrane facing the extracellular space. Annexin V (Anx-V) is a 35-kDa protein with high affinity for phosphatidylserine, which can be applied to detect apoptosis. We injected biotin-labelled Anx-V intravenously in adult mice and examined the tissue distribution of Anx-V-labelled cells in dental and periodontal tissues using ABC-peroxidase histochemistry. In the continuously erupting incisors, strong and frequent immunostaining was observed in transitional stage and late maturation stage ameloblasts with less frequent staining in preameloblasts. Frequency of staining in odontoblasts and pulp cells was low but increased slightly at older stages of dentinogenesis. Labelling was also seen in phagocytic or phagocytic-like cells in the enamel organ and pulp. A positive staining was furthermore found in fibroblasts of the periodontal ligament in continuously erupting incisors and in fully erupted molar teeth. Staining intensity and the number of positive cells were enhanced by antigen retrieval using high-pressure cooking. We conclude that Anx-V-biotin labels dental cells in early stages of cell death and indirectly cells that have ingested labelled apoptotic cells during the course of the experiment. The data confirm that during amelogenesis most cell death occurs in transitional stage and late maturation stage ameloblasts. Thus, labelling with Anx-V is a useful marker for studying cell death and the dynamics of clearance of apoptotic cells during tooth development.     

体外培养的人牙胚上皮细胞的成釉分化

人表皮干细胞可以作为牙齿再生中上皮源性的种子细胞,但是其成釉分化的效率低下. 本研究分离培养了人牙胚上皮细胞,利用E13.5的小鼠牙间充质与其重组,构建重组牙胚,对其成釉分化的潜能和机制进行研究. 研究结果发现,体外培养的P1代人牙胚上皮细胞成釉率高达50%. 随着传代次数的增加,成釉率明显下降. 通过对牙上皮发育分化相关基因的表达检测和分析表明,重组牙胚成牙分化能力和成釉潜能的下降与牙上皮发育相关基因的表达状态密切相关. 特别是FGF8表达水平的下调以及PITX2不同亚型在人牙胚细胞中表达量的不均衡,可能是导致人牙胚细胞成釉潜能下降并丧失的主要原因. 本研究结果为理解牙齿再生过程中上皮源性的种子细胞的成釉机制提供了新的实验数据,对进一步提高表皮干细胞在牙齿再生过程中的成釉率有指导意义.     

Nuclear PTHrP targeting regulates PTHrP secretion and enhances LoVo cell growth and survival

Parathyroid hormone-related protein (PTHrP) is expressed by human colon cancer tissue and cell lines; expression correlates with colon carcinoma severity. PTHrP is synthesized as a prepro isoform and contains two targeting sequences — a signal sequence and a nuclear localization signal (NLS). The signal peptide (SP) directs PTHrP to the secretory pathway, where it exerts autocrine/paracrine effects. The NLS directs PTHrP to the nucleus/nucleolus, where it exerts intracrine effects. In this study, we used the human colon cancer cell line LoVo as a model system to study the effects of autocrine/paracrine and intracrine PTHrP action on cell growth and survival, hallmarks of malignant tumor cells. We report that PTHrP increases cell growth and survival, protects cells from serum-starvation-induced apoptosis, and promotes anchorage-independent cell growth via an intracrine pathway. Conversely, autocrine/paracrine PTHrP action decreases cell growth and survival. We also show an inverse relationship between secreted and nuclear PTHrP levels, in that cells overexpressing NLS-deleted PTHrP secrete higher PTHrP levels than those overexpressing the wild-type isoform. Conversely, SP deletion results in higher nuclear PTHrP levels. These observations provide evidence of a link between intracrine PTHrP action and cell growth and survival. Targeting PTHrP production in colon cancer may thus prove therapeutically beneficial.     

Expression patterns of the homeobox gene, Hox-8, in the mouse embryo suggest a role in specifying tooth initiation and shape.

We have studied the expression patterns of the newly isolated homeobox gene, Hox-8 by in situ hybridisation to sections of the developing heads of mouse embryos between E9 and E17.5, and compared them to Hox-7 expression patterns in adjacent sections. This paper concentrates on the interesting expression patterns of Hox-8 during initiation and development of the molar and incisor teeth. Hox-8 expression domains are present in the neural crest-derived mesenchyme beneath sites of future tooth formation, in a proximo-distal gradient. Tooth development is initiated in the oral epithelium which subsequently thickens in discrete sites and invaginates to form the dental lamina. Hox-8 expression in mouse oral epithelium is first evident at the sites of the dental placodes, suggesting a role in the specification of tooth position. Subsequently, in molar teeth, this patch of Hox-8 expressing epithelium becomes incorporated within the buccal aspect of the invaginating dental lamina to form part of the external enamel epithelium of the cap stage tooth germ. This locus of Hox-8 expression becomes continuous with new sites of Hox-8 expression in the enamel navel, septum, knot and internal enamel epithelium. The transitory enamel knot, septum and navel were postulated, long ago, to be involved in specifying tooth shape, causing the inflection of the first buccal cusp, but this theory has been largely ignored. Interestingly, in the conical incisor teeth, the enamel navel, septum and knot are absent, and Hox-8 has a symmetrical expression pattern. Our demonstration of the precise expression patterns of Hox-8 in the early dental placodes and their subsequent association with the enamel knot, septum and navel provide the first molecular clues to the basis of patterning in the dentition and the association of tooth position with tooth shape: an association all the more intriguing in view of the evolutionary robustness of the patterning mechanism, and the known role of homeobox genes in Drosophila pattern formation. At the bell stage of tooth development, Hox-8 expression switches tissue layers, being absent from the differentiating epithelial ameloblasts and turned on in the differentiating mesenchymal odontoblasts. Hox-7 is expressed in the mesenchyme of the dental papilla and follicle at all stages. This reciprocity of expression suggests an interactive role between Hox-7, Hox-8 and other genes in regulating epithelial mesenchymal interactions during dental differentiation.(ABSTRACT TRUNCATED AT 400 WORDS)