Expression patterns of bone morphogenetic proteins (Bmps) in the developing mouse tooth suggest roles in morphogenesis and cell differentiation

Bone morphogenetic proteins (BMP) are secretory signal molecules which have a variety of regulatory functions during morphogenesis and cell differentiation. Teeth are typical examples of vertebrate organs in which development is controlled by sequential and reciprocal signaling between the epithelium and mesenchyme. In addition, tooth development is characterized by formation of mineralized tissues: the bone-like dentin and cementum as well as epithelially derived enamel. We have performed a comparative in situ hybridization analysis of the expression of six different Bmps (Bmp-2 to Bmp-7) starting from initiation of tooth development to completion of crown morphogenesis when dentine and enamel matrices are being deposited. Bmps-2, -4, and -7 were frequently codistributed and showed marked associations with epithelial-mesenchymal interactions. Their expression shifted between the epithelium and mesenchyme starting from the stage of tooth initiation. They were subsequently expressed in the enamel knot, the putative signaling center regulating tooth shape. Their expression domains prior to and during the differentiation of the dentine-forming odontoblasts and enamel-forming ameloblasts was in line with functions in regulation of cell differentiation and/or secretory activities of the cells. The expression of Bmp-3 was confined to mesenchymal cells, in particular to the dental follicle cells which give rise to the cementoblasts, forming the hard tissue covering the roots of teeth. Bmp-5 was expressed only in the epithelial ameloblasts. It was upregulated as the cells started to polarize and intense expression continued in the secretory ameloblasts. Bmp-6 was expressed only weakly in the dental mesenchyme during bud and cap stages. Our results are in line with regulatory functions of Bmps at all stages of tooth morphogenesis. Bmps-2, -4, and -7 are conceivably parts of signaling networks regulating tooth initiation and shape development. They as well as Bmp-5 may be involved in the induction and formation of dentine and enamel, and Bmp-3 in the development of cementum. The remarkable overlaps in the expression domains of different Bmp genes may implicate functional redundancy and/or formation of active heterodimers between different BMPs.     

Changes in pulmonary calcitonin gene-related peptide and protein gene product 9.5 innervation in rats infected with Mycoplasma pulmonis

Epithelial-mesenchymal interaction is a prerequisite for tooth morphogenesis. To study this interaction, inner enamel epithelium and dental papilla mesenchyme of molar tooth germs from a 16.5-day mouse embryo were dissociated enzymatically and cultured alone or after recombination. Characteristic matrix protein synthesized and secreted by recombined tooth germ was determined quantitatively by enzyme-linked immunosorbent assay. The protein was detected in the culture of recombined tooth germ but not of dissociated enamel epithelium alone. The amount of enamel protein increased until 8 days in culture. Morphological differentiation of the recombined epithelial rudiment into ameloblasts and enamel protein production were confirmed.     

Hepatocyte growth factor is involved in the morphogenesis of tooth germ in murine molars

The patterns of gene expression for hepatocyte growth factor (HGF) and its receptor, c-Met, were revealed in the tooth germ of rat mandibular molars using RT-PCR. In situ hybridization demonstrated that the HGF gene was expressed only in the cells of the dental papilla of the tooth germ in vivo. The characteristic temporospatial distribution of HGF and c-Met during germ development was revealed using immunohistochemical studies in vivo. In order to demonstrate the functional role played by HGF in tooth development, HGF translation arrest by antisense phosphorothioate oligodeoxynucleotide (ODN) was carried out in vitro. In the control experiment, explants of tooth germs from embryonic 14 day mice were cultured in a modification of Trowell's system under serum-free and chemically defined conditions for two weeks. Other explants were cultured with 15mer antisense or sense ODN targeted to the HGF mRNA. Both the control and the sense-treated explants showed normal histological structure, as observed in vivo. On the other hand, antisense-treated explants exhibited an abnormal structure in which the enamel organs were surrounded by a thin layer of dentin and dental papilla, appearing 'inside-out' compared to the control and sense-treated explants, although the cytodifferentiation of ameloblasts and odontoblasts was not inhibited. The explants treated with recombinant human HGF combined with antisense ODN showed normal development, indicating that exogenous HGF rescued the explants from the abnormal structure caused by antisense ODN. The findings of a BrdU incorporation experiment suggested that the imbalance between the proliferation activity of the inner enamel epithelium and that of the dental papilla caused by HGF translation arrest results in the abnormal structure of the tooth germ. These results indicate that HGF is involved in the morphogenesis of the murine molar.     

The epithelium-tooth interface--a basal lamina rich in laminin-5 and lacking other known laminin isoforms

The attachment of the marginal gingiva to the tooth surface is mediated by a thin nonkeratinized epithelium termed the junctional epithelium (JE). Ultrastructural studies have revealed that the attachment of the JE to the tooth surface occurs through hemidesmosomes (HD) and a basal lamina-like extracellular matrix termed the internal basal lamina (IBL). We have previously shown that neither type IV collagen nor prototypic laminin, two common components of basement membranes (BM), is present in the IBL between the epithelium and the tooth. In the present study, we show that laminin-5 is a major component of the IBL in both rodent and human tissues. By using in situ hybridization, we also show that the cells of the JE express the LAMC2 gene of laminin-5. In other parts of gingival epithelium, LAMC2 gene expression is less prominent. Our results indicate that the epithelium-tooth interface is a unique structure wherein epithelial cells are induced to secrete a basal lamina containing laminin-5 and no other presently known laminin isoform.     

Delta-notch signaling in odontogenesis: correlation with cytodifferentiation and evidence for feedback regulation

Recent data suggest that dental cells utilize the evolutonarily conserved Notch-mediated intercellular signaling pathway to regulate their fates. Here we report on the expression and regulation of Delta1, a transmembrane ligand of the Notch receptors, during mouse odontogenesis. Delta1 is weakly expressed in dental epithelium during tooth initiation and morphogenesis, but during cytodifferentiation, expression is upregulated in the epithelium-derived ameloblasts and the mesenchyme-derived odontoblasts. The expression pattern of Delta1 in ameloblasts and odontoblasts is complementary to Notch1, Notch2, and Notch3 expression in adjacent epithelial and mesenchymal cells. Notch1 and Notch2 are upregulated in explants of dental mesenchyme adjacent to implanted cells expressing Delta1, suggesting that feedback regulation by Delta-Notch signaling ensures the spatial segregation of Notch receptors and ligands. TGFbeta1 and BMPs induce Delta1 expression in dental mesenchyme explants at the stage at which Delta1 is upregulated in vivo, but not at earlier stages. In contrast to the Notch family receptors and their ligand Jagged1, expression of Delta1 in the tooth germ is not affected by epithelial-mesenchymal interactions, showing that the Notch receptors and their two ligands Jagged1 and Delta1 are subject to different regulations.     

Expression and localization of membrane type 1 matrix metalloproteinase in tooth tissues

Matrix metalloproteinases (MMPs) have been detected in forming dental enamel and are thought to play an important role during enamel biomineralization. Membrane-type 1 matrix metalloproteinase (MT1-MMP) is a membrane bound member of the MMP gene family that has previously been shown to be expressed by cells associated with bone and cartilage formation (osteoclasts, osteoblasts and chondrocytes). Thus, we asked if MT1-MMP was also expressed by the cells responsible for the formation of enamel and dentin. A porcine MT1-MMP cDNA composed of 3284 bp was isolated from an enamel organ-specific cDNA library. Multiple tissue Northern blot analysis revealed that the MT1-MMP message was expressed highly in the enamel organ and pulp organ when compared to the expression levels observed in other non-mineralizing tissues. Northern blot analysis of stage-specific enamel organs (early secretory, late secretory, or maturation stage) and their corresponding pulp organs revealed that MT1-MMP expression increased as the dentin matured. In the enamel organs, however, the MT1-MMP message level became reduced only during the late secretory stage. Immunohistochemical analysis showed that MT1-MMP was present on the surface of the cells (ameloblasts and odontoblasts) responsible for dentin and enamel formation. Thus, MT1-MMP is highly expressed in developing tooth tissues and may play a role in the biomineralization of enamel and dentin.     

Quantitative analysis of Na+ and Ca2+ current contributions on spike initiation in the newt olfactory receptor cell

The developmental localization patterns of collagen type IV alpha1-5 chains, laminin-1, laminin-5, and laminin alpha2 chain were analyzed in the embryonic mouse eye using isoform specific antibodies and immunofluorescence microscopy. Laminin-1 isoform and alpha1-2(IV) were ubiquitously expressed along the ocular surface basement membranes at a very early stage of eye development. Alpha3-5(IV) were first detected at later stages of development, and exhibited a variable distribution pattern along the ocular surface basement membrane. In contrast, expression of the laminin alpha2 chain was restricted to the conjunctival basement membrane, and was first detected during the same developmental period in which keratin K4-positive, differentiated conjunctival epithelial cells were observed. Although laminin-5 was uniformly expressed along the adult ocular surface basement membrane, during embryogenesis it was first incorporated into the conjunctival basement membrane structure. These data suggest that some of the laminin isoforms, including laminin alpha2 and laminin-5, may play a role in the formation of a conjunctival-type basement membrane. The temporal relationship between the localization of these molecules to the conjunctival basement membrane and the appearance of differentiated conjunctival epithelial cells suggests a role for external influence on the differentiation pathways of ocular surface epithelium.     

Extracellular proteolysis alters tooth development in transgenic mice expressing urokinase-type plasminogen activator in the enamel organ

By catalyzing plasmin formation, the urokinase-type plasminogen activator (uPA) can generate widespread extracellular proteolysis and thereby play an important role in physiological and pathological processes. Dysregulated expression of uPA during organogenesis may be a cause of developmental defects. Targeted epithelial expression of a uPA-encoding transgene under the control of the keratin type-5 promoter resulted in enzyme production by the enamel epithelium, which does not normally express uPA, and altered tooth development. The incisors of transgenic mice were fragile, chalky-white and, by scanning electron microscopy, their labial surface appeared granular. This phenotype was attributed to a defect in enamel formation during incisor development, resulting from structural and functional alterations of the ameloblasts that differentiate from the labial enamel epithelium. Immunofluorescence revealed that disorganization of the ameloblast layer was associated with a loss of laminin-5, an extracellular matrix molecule mediating epithelial anchorage. Amelogenin, a key protein in enamel formation, was markedly decreased at the enamel-dentin junction in transgenics, presumably because of an apparent alteration in the polarity of its secretion. In addition, increased levels of active transforming growth factor-beta could be demonstrated in mandibles of transgenic mice. Since the alterations detected could be attributed to uPA catalytic activity, this model provides evidence as to how dysregulated proteolysis, involving uPA or other extracellular proteases, may have developmental consequences such as those leading to enamel defects.     

Giant cell tumor (osteoclastoma) of the mandible: a diagnostic dilemma and a therapeutic challenge

We have recently identified a novel RNA sequence in ameloblasts, coding for amelin (Cerny et al., 1996). In the present paper, its expression has been compared with that of amelogenin in developing incisors and molars of rats, by means of in situ hybridization of paraffin sections. The RNAs for both amelin and amelogenin were highly expressed in secretory ameloblasts. The expression of RNA for amelogenin gradually decreased in the post-secretory ameloblasts. In contrast, the RNA expression for amelin remained high in post-secretory ameloblasts up to the stage of fusion between dental and oral epithelia at the time of tooth eruption. We suggest that amelin might be involved in the mineralization of enamel or in the attachment of ameloblasts to the enamel surface. The whole-mount in situ hybridization procedure is described for the first time in dental research. It proved to be a useful method and confirmed the results of the conventional in situ hybridization.     

Secular trend in body height since the Neolithic period

Rat mesangial cells express two unique isoforms of laminin which can be modulated by culture medium composition. To define further the nature of laminin expressed by cultured rat mesangial cells, synthesis of individual laminin chains, as well as their trimeric association, was examined. Based on data from Northern analysis of mRNA expression, immunoblots, immunofluorescence staining and radioimmunoprecipitation of biosynthetically labeled proteins, mesangial cells express laminin beta1, beta2, and gamma1 chains. Mesangial cells do not express laminin alpha1 or alpha2. MC produce a unique alpha chain, designated alpha'm. These laminin chains assemble into two major isoforms. One contains alpha'mbeta1gamma1, co-precipitates with entactin and is assembled into the fibrillar extracellular matrix. The second isoform contains alpha'mbeta2 and a presumed gamma chain that migrates in gel slightly ahead of gamma1. The beta2-containing isoform is concentrated in punctate sites on the cell surface. In addition, mesangial cells display different phenotypes when plated on laminin-1 (alpha1beta1gamma1), as compared to purified beta2. An LRE-containing peptide of laminin beta2 serves as an attachment site for mesangial cells and is sufficient to induce the phenotype observed with intact beta2. These data suggest that laminin isoform expression plays an important role in mesangial cell phenotype and function.     

Alpha 6 beta 4 integrin and newly deposited laminin-1 and laminin-5 form the adhesion mechanism of gastric carcinoma. Continuous expression of laminins but not that of collagen VII is preserved in invasive parts of the carcinomas: implications for acquisition of the invading phenotype

We studied the expression and distribution of different laminin chains, the alpha 6 beta 4 integrin and type VII collagen, i.e., components of the epithelial adhesion complex, in gastric carcinomas and in suggested preneoplastic stages of this malignancy. Intestinal-type gastric carcinomas showed strong reactivity for laminin alpha 1, alpha 3, beta 1, and beta 3 chains, the components of laminin-1 and -5, at the interface between malignant cells and tumor stroma. The reactivities were continuous throughout the carcinomas, even in structures invading through the smooth muscle layers of the gastric wall. The expression of different laminin chains was accompanied by strong polarized reactivity for the alpha 6 beta 4 integrin, which is a receptor for both laminin-1 and laminin-5. Collagen type VII was only occasionally present at sites showing reactivity for laminin-5 and was totally absent from the cell islands invading through the gastric wall. Intestinalized gastric epithelium showed a similar expression pattern of laminins and the alpha 6 beta 4 integrin as the gastric carcinomas. Our results suggest that gastric carcinomas use the alpha 6 beta 4 integrin and newly deposited laminin-1 and -5, accompanied by the disappearance of type VII collagen, as their mechanism of adhesion during the invasion through surrounding tissues. Unlike in previous studies, the reactivity for the laminin-5 protein was not restricted to the invading cells but surrounded the malignant glandular structures throughout the tumor. Our results also show that both intestinal-type gastric carcinoma, and intestinal metaplasia mimic the gastric surface epithelium in the expression pattern of laminins and the beta 4 integrin subunit. This supports previous studies proposing a pathogenetic sequence from intestinal metaplasia to gastric carcinoma.     

Adenomatous polyposis coli protein is expressed in alternate stages of the ameloblast life cycle

Mutations of the adenomatous polyposis coli gene protein (APC) are associated with familial polyposis and also sporadic colon adenomas, both preconditions to cancer formation. Some familial polyposis patients also develop Gardner's syndrome, a condition characterized by supernumerary teeth, mandibular osteomas, and other maladies. We investigated participation of APC in normal tooth development. Using a monoclonal antibody to study APC expression in the forming rat incisor, we found no APC staining in differentiating ameloblasts, then strong staining in secreting ameloblasts and stratum intermedium cells, followed by cells in the transition stage which did not stain. Intense APC staining resumes in maturation-stage ameloblasts and proximal papillary cells. APC staining disappears again in reduced ameloblasts at the conclusion of amelogenesis. APC staining was not seen in any other odontogenic cells. We report a unique system in which APC expression is upregulated and downregulated twice during the normal life cycle of ameloblasts. APC, therefore, is important in the normal maturation of both colonic epithelium and odontogenic epithelium. At this point, we cannot rule out any of the known functions of APC, which include: modulation of cell adhesion by binding to catenin, regulation of beta-catenin as a differentiative signaling molecule, and promotion of microtubule assembly. In this respect, the rat incisor enamel organ provides a unique tissue for studying the regulation and functions of APC.     

Breast cancer associated mucin: a review

Rabbit polyclonal antibody against mouse EHS laminin was used to investigate the distribution and composition of laminin in the rat first molar tooth germ. Immunohistochemical analysis showed that laminin is expressed in the inner and outer epithelia of the enamel organ and in small blood vessels in the dental papilla and strellate reticulum. Immunoblots revealed that tooth germ laminin differs from EHS laminin. Tooth germ laminin contains beta chains, while the alpha 1 chain is substituted by a 300-kDa chain. Two-dimensional electrophoresis analysis of tooth germ extract showed that beta chains appeared as four spots with approximate pI values of 6.6, 7.5, 7.8 and 8.5. These results indicate that more than-one type of laminin isoform is present in the first molar tooth germ. Additionally, we have shown that despite the early degradation of tooth germ basement membrane, the laminin molecule is still intact at the time of birth.     

Developmental expression of laminin beta 2 in rat retina. Further support for a role in rod morphogenesis

PURPOSE: The authors previously hypothesized that laminin beta 2 (S-laminin) plays a role in directing photoreceptor development. The aim of this study was to examine the temporal and spatial expression pattern of beta 2 laminins in rat retina to test this hypothesis. METHODS: Retinas from Sprague-Dawley rats were harvested on embryonic days (E) 14, 16, and 21, as well as on postnatal days (P) 2, 5, and 10. Cryostat sections were probed with antibodies directed against beta 2 laminin, laminin-1 (alpha 1-beta 1-gamma 1), and von Willebrand factor. RESULTS: At the onset of rod photoreceptor birth (E14), laminin beta 2 surrounds the cells of the retinal pigmented epithelium (RPE) and is present on the apical surface of the retinal neuroepithelium. At E16, laminin beta 2 persists on the apical surface of the neuroepithelium and the subjacent apical surface of the RPE. At birth, laminin beta 2 fills the matrix between the juxtaposed surfaces of the RPE and neuroepithelium; moreover, laminin beta 2 immunoreactivity penetrates the neural retina. Throughout postnatal development, laminin beta 2 immunoreactivity surrounds maturing inner and outer segments. Laminin beta 2 also is found in association with blood vessels in the neural retina itself, as well as with choroidal blood vessels; in both places, it is co-localized with an endothelial marker, von Willebrand factor, and laminin-1. CONCLUSIONS: The spatial and temporal expression of laminin beta 2 is consistent with its hypothesized role in rod development. Laminin beta 2 is in a unique position to interact with mitotically active cells (in early retinal development), uncommitted progenitors (in late embryonic development), developing rods (in early postnatal development), and mature outer segments (throughout adulthood). Together with our earlier functional data, these data support our hypothesis that this molecule is an important component of the interphotoreceptor matrix.     

Parathyroid hormone-related protein is required for tooth eruption

Parathyroid hormone (PTH)-related protein (PTHrP)-knockout mice die at birth with a chondrodystrophic phenotype characterized by premature chondrocyte differentiation and accelerated bone formation, whereas overexpression of PTHrP in the chondrocytes of transgenic mice produces a delay in chondrocyte maturation and endochondral ossification. Replacement of PTHrP expression in the chondrocytes of PTHrP-knockout mice using a procollagen II-driven transgene results in the correction of the lethal skeletal abnormalities and generates animals that are effectively PTHrP-null in all sites other than cartilage. These rescued PTHrP-knockout mice survive to at least 6 months of age but are small in stature and display a number of developmental defects, including cranial chondrodystrophy and a failure of tooth eruption. Teeth appear to develop normally but become trapped by the surrounding bone and undergo progressive impaction. Localization of PTHrP mRNA during normal tooth development by in situ hybridization reveals increasing levels of expression in the enamel epithelium before the formation of the eruption pathway. The type I PTH/PTHrP receptor is expressed in both the adjacent dental mesenchyme and in the alveolar bone. The replacement of PTHrP expression in the enamel epithelium with a keratin 14-driven transgene corrects the defect in bone resorption and restores the normal program of tooth eruption. PTHrP therefore represents an essential signal in the formation of the eruption pathway.     

Corrective gene transfer of keratinocytes from patients with junctional epidermolysis bullosa restores assembly of hemidesmosomes in reconstructed epithelia

Herlitz junctional epidermolysis bullosa (H-JEB) provides a promising model for somatic gene therapy of heritable mechano-bullous disorders. This genodermatosis is caused by the lack of laminin-5 that results in absence of hemidesmosomes (HD) and defective adhesion of squamous epithelia. To establish whether re-expression of laminin-5 can restore assembly of the dermal-epidermal attachment structures lacking in the H-JEB skin, we corrected the genetic mutation hindering expression of the beta 3 chain of laminin-5 in human H-JEB keratinocytes by transfer of a laminin beta 3 transgene. The transduced keratinocytes synthesized a recombinant beta 3 polypeptide that assembled with the endogenous laminin alpha 3 and gamma 2 chains into a biologically active laminin-5 that was secreted, processed and deposited into the extracellular matrix. Re-expression of laminin-5 induced cell spreading, nucleation of hemidesmosomal-like structures and enhanced adhesion to the culture substrate. Organotypic cultures performed with the transduced keratinocytes, reconstituted epidermis closely adhering to the mesenchyme and presenting mature hemidesmosomes, bridging the cytoplasmic intermediate filaments of the basal cells to the anchoring filaments of the basement membrane. Our results provide the first evidence of phenotypic reversion of JEB keratinocytes by somatic gene therapy and demonstrate that genetic treatment of the mild forms of skin blistering diseases and other inherited extracellular matrix pathologies is a realistic goal.