High expression of 92-kDa type IV collagenase (gelatinase) in the osteoclast lineage during mouse development.
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Animals↗
Biomedical subjects
I Thesleff
Publications and source records attributed to I Thesleff.
High expression of 92-kD type IV collagenase (gelatinase B) in the osteoclast lineage during mouse development.
cDNA clones for murine 92 kD type IV collagenase (gelatinase B) were generated for the determination of its primary structure and for analysis of temporal and spatial expression in vivo. The mouse enzyme has 72% sequence identity with the human counterpart, the major difference being the presence of a 16-residue segment absent from the human enzyme. In situ hybridization analyses of embryonic and postnatal mouse tissues revealed intense signals in cells of the osteoclast cell lineage. Clear expression above background was not observed in macrophages, polymorphonuclear leukocytes, monocytes, or epithelial cells which have been shown to express the gene in vitro in cell cultures. Expression of the gene was first observed at early stage of cartilage and tooth development at E13, where signals were seen transiently in surrounding mesenchymal cells. At later developmental stages and postnatally strong expression was seen in large cells at the surface of bones. These cells were presumably osteoclasts as their location correlated with that of TRAP positive cells. Signals above background were not observed in a number of other tissues studied. The results represent the first demonstration of a highly osteoclast specific extracellular proteinase. The results suggest that during normal development of embryonic organs the 92-kD type IV collagenase does not have a major role in basement membrane degradation, but is rather mainly used for the turnover of bone matrix, possibly as a gelatinase required for the removal of denatured collagen fragments (gelatin) generated by interstitial collagenase.
Amino Acid Sequence↗
Degradation of the dental basement membrane during mouse tooth development in vitro.
During tooth development, the basement membrane is degraded at the late bell stage, but the developmental significance of this event is not known. Organ culture offers a method where developmental processes can be manipulated in controlled conditions. We cultured bell-stage tooth germs either in a chemically defined or a serum-containing medium and analyzed the degradation of the basement membrane by different methods. Type IV collagen was present throughout the dental basement membranes at the epithelial-mesenchymal interface at the onset of culture. After 10 days of culture, irrespective of the medium used, type IV collagen and laminin had disappeared from the cuspal areas but were present at the cervical loop. As was the case in vivo, the expression of 72 kDa type IV collagenase gene was intense in the differentiating preodontoblasts and in the odontoblasts during secretion of the first predentin matrix near the cuspal tips. Ultrastructural observations showed that the basal lamina had been removed in all cultured tooth organs. Also, the breakdown of the basement membrane occurred irrespective of the presence of mineral in the dentin matrix. Our observations suggest in contrast to earlier observations, that there are no major differences in basic events leading to dentino- and amelogenesis, when tooth organs are cultured in the presence or absence of serum.
Ameloblasts↗
Evidence for the role of the enamel knot as a control center in mammalian tooth cusp formation: non-dividing cells express growth stimulating Fgf-4 gene.
The main morphological features of the mammalian tooth crown are cusps, but the developmental mechanisms that cause the formation of cusps are unknown. Tooth cusp formation commences at cap-stage with the appearance of the enamel knot, which is a cluster of non-dividing epithelial cells. In this study, enamel knot was first seen in embryonic mice molar teeth at the onset of cap-stage. Later in tooth development, secondary enamel knot structures were observed at the cusp tips and their appearance corresponded to the formation of individual cusp morphology. Comparisons of the pattern of cell proliferation in embryonic mouse molars and the expression of fibroblast growth factor-4 (Fgf-4) gene revealed that expression of Fgf-4 mRNA is strictly localized to the non-dividing cells of the enamel knot. However, when FGF-4 protein was introduced onto isolated dental tissues in vitro, it stimulated the proliferation of both dental epithelial and mesenchymal cells. Based on these results, we suggest that the enamel knot may control tooth morphogenesis by concurrently stimulating cusp growth (via FGF-4 synthesis) and by directing folding of cusp slopes (by not proliferating itself).
Animals↗
Identification of BMP-4 as a signal mediating secondary induction between epithelial and mesenchymal tissues during early tooth development.
Growth factor-mediated signaling has been implicated in the regulation of epithelial-mesenchymal interactions during organogenesis. Bone morphogenetic protein 4 (BMP-4), a member of the transforming growth factor beta superfamily, is expressed in the presumptive dental epithelium at the initiation of tooth development. Subsequently, epithelial signaling leads to mesenchymal induction of BMP-4 expression. To address the role of this factor, BMP-4-releasing agarose beads were added to dental mesenchyme in culture. These beads induced a translucent mesenchymal zone similar to that induced by dental epithelium. Moreover, three transcription factors (Msx-1, Msx-2, and Egr-1) whose expression is governed by epithelial signaling were induced in response to BMP-4. In addition, BMP-4 induced its own mesenchymal expression. These findings support the hypothesis that BMP-4 mediates epithelial-mesenchymal interactions during early tooth development.
Animals↗
Initiation of secondary cartilage in the mandible of the Syrian hamster in the absence of muscle function.
The functioning lower jaw is a prerequisite of the ongoing secondary chondrogenesis in the mammalian mandibular condyle. Does the articular function also initiate secondary chondrogenesis in the mandible? The angular process of the fetal mammalian mandible possesses a large secondary cartilage without any apparent articular function. Past studies have shown that the anlage of the lower jaw of the mouse embryo grown in organ culture produces condylar and angular cartilages as in vivo. In order to clarify further the capacity of the mandibular anlage to initiate secondary chondrogenesis in a non-functional environment, mandibular arch explants taken from the prenatal hamster before any cartilage or bone formation was apparent were grown in organ culture. Both primary and secondary cartilage could be found in them within 9-10 days. The results thus indicate that initiation of the condylar and/or angular secondary cartilaginous development in the rodent mandible occurs in the absence of jaw-opening function, although the pertinent literature indicates that function maintains cartilaginous differentiation in the condyle.
Animals↗
Transient expression of type III collagen by odontoblasts: developmental changes in the distribution of pro-alpha 1(III) and pro-alpha 1(I) collagen mRNAs in dental tissues.
The expression of pro-alpha 1(III) and pro-alpha 1(I) collagen mRNAs in mouse and human dental tissues during tooth development and after its completion was analyzed by in situ hybridization, with use of [35S]-labeled RNA probes. The expression of pro-alpha 1(III) mRNA was also compared to that of the protein product, as localized by immunostaining with polyclonal antibodies to type III collagen and the N-terminal propeptide of type III procollagen. Contrary to many previous reports, our results suggest that odontoblasts express type III collagen. While pro-alpha 1(III) transcripts were less intensely expressed in odontoblasts than pro-alpha 1(I) transcripts, the amounts of both mRNAs increased in odontoblasts with progressing dentin formation, and decreased toward its completion. In contrast to pro-alpha 1(III) mRNA, pro-alpha 1(I) mRNA was still detectable in odontoblasts of fully developed teeth. Type III collagen immunoreactivity was observed in the early predentin, and again in predentin toward the completion of dentinogenesis, when mRNA was no longer detected. Also in the pulp, the protein product, unlike pro-alpha 1(III) mRNA, was relatively strongly expressed. Hence, these immunostaining patterns were inversely related to the expression of pro-alpha 1(III) mRNA, suggesting accumulation of the protein. The mesenchymal cells, when condensed in the region of the future mandibular bone, expressed pro-alpha 1(III) mRNA intensely, whereas osteoblasts expressed pro-alpha 1(I) but not pro-alpha 1(III) transcripts strongly. Cell type- and developmental stage-related differences in the expression of the two mRNAs suggest that type I/type III collagen ratio influences the structure of dental tissues.
Aging↗
Increased rate of salivary epidermal growth factor secretion in patients with juvenile periodontitis.
We compared salivary epidermal growth factor (EGF) concentrations in patients with juvenile periodontitis (JP) and periodontally healthy controls. In initial screening of 45 JP patients and a group of healthy controls, significantly higher salivary EGF concentrations were measured in the JP patients. Subsequently, 17 JP patients who had high EGF concentrations in some of their salivary samples were chosen, and a group of age- and sex-matched controls was selected. We then examined their EGF concentrations and EGF secretion rates under standardized conditions in stimulated and unstimulated saliva and studied the expression of EGF receptor (EGF-R) in their gingival tissues. The results showed that the mean EGF concentration (pmol/ml) was slightly higher in JP patients than in controls. However, the difference was statistically significant only in stimulated saliva and when calculated per milligram salivary protein. When EGF release was measured as the rate of EGF secretion (pg/min), significantly higher values were observed in JP patients than in controls both in unstimulated and stimulated saliva. Immunofluorescence microscopy (IF) of gingival samples from JP patients and their controls revealed no quantitative or qualitative differences in the expression of EGF-R. Our results demonstrate the complex nature of salivary EGF release. The elevated rate of salivary EGF secretion in JP patients may be associated with the pathogenetic mechanisms of juvenile periodontitis.
Adolescent↗
A chondroitin sulfate epitope in mammalian dental pulp and its developmental expression in mouse dental papilla.
The molecular specificity of the dental papilla of a bell-stage tooth was studied by production of dental-papilla-reactive monoclonal antibodies (Mabs). One of the Mabs, designated 7C5, recognized an epitope present in glycosaminoglycan. Several lines of evidence suggested that the 7C5-epitope consists of chondroitin 6-sulfate. The Mab did not react with mouse dental epithelium, but reacted uniformly with mesenchymal tissue in the mandibular process and accumulated in the dental sac and in the papilla of bell-stage tooth germs. The 7C5-staining was lost from the differentiating odontoblasts, while the staining in the molar tooth papilla was accumulated in the subodontoblastic layer. In the developing mouse incisor, the 7C5-epitope was restricted to the lingual-posterior area. The 7C5-epitope was also present in pulpal tissue and predentin of different types of teeth of various mammalian species, including man, sheep, swine, and rat. Collagenase pre-treatment of tissue sections abolished the bulk of the 7C5-reactivity in peridental mesenchyme during embryonic stages while leaving the staining of the dental papilla intact. In newborn and adult teeth, collagenase also impaired the reactivity in the pulp except for the subodontoblastic layer. This suggests the existence of different subpopulations of the 7C5-epitope containing proteoglycans in dental papilla and pulp. A high-molecular-weight proteoglycan, sensitive to chondroitinase ABC but not to heparinase or heparitinase, was immunoprecipitated by 7C5 from extracts of bell-stage mouse tooth germs. We suggest that the evolutionary conservation of chondroitin 6-sulfate in the dental pulp reflects its properties as non-terminally differentiated tissue and perhaps the retention of a potential to differentiate to odontoblasts.
Animals↗
Epithelial-mesenchymal interactions are required for msx 1 and msx 2 gene expression in the developing murine molar tooth.
Duplication of the msh-like homeobox gene of Drosophila may be related to the evolution of the vertebrate head. The murine homologues of this gene, msx 1 and msx 2 are expressed in the developing craniofacial complex including the branchial arches, especially in regions of epithelial-mesenchymal organogenesis including the developing tooth. By performing in vitro recombination experiments using homochronic dental and non-dental epithelial and mesenchymal tissues from E10 to E18 mouse embryos, we have found that the maintenance of homeobox gene expression in the tooth is dependent upon tissue interactions. In homotypic recombinants, dental-type tissue interactions occur, leading to expression of both genes in a manner similar to that seen during in vivo development. msx 1 is expressed exclusively in mesenchyme, both in the dental papilla and follicle. msx 2 is expressed in the dental epithelium and only in the mesenchyme of the dental papilla. In heterotypic recombinants, the dental epithelium is able to induce msx 1 expression in non-dental mesenchyme, this potential being lost at the bell stage. In these recombinants msx 2 was induced by presumptive dental epithelium prior to the bud stage but not thereafter. The expression of msx 1 and msx 2 in dental mesenchyme requires the presence of epithelium until the early bell stage. However, whereas non-dental, oral epithelium is capable of maintaining expression of msx 1 in dental mesenchyme throughout tooth development, induction of msx 2 was temporally restricted suggesting regulation by a specific epithelial-mesenchymal interaction related to the inductive events of tooth formation. msx 1 and msx 2, as putative transcription factors, may play a role in regulating the expression of other genes during tooth formation. We conclude that expression of msx 1 in jaw mesenchyme requires a non-specific epithelial signal, whereas msx 2 expression in either epithelium or mesenchyme requires reciprocal interactions between specialized dental cell populations.
Animals↗
Molecular cloning of murine 72-kDa type IV collagenase and its expression during mouse development.
We report the isolation of a cDNA clone providing the first and complete sequence of mouse 72-kDa type IV collagenase. The clone contains 2800 nucleotides with a 1986-nucleotide open reading frame coding for 662 amino acids. The amino acid sequence includes a 29-residue signal peptide, an 80-residue propeptide, and a 553-residue enzyme proper. The sequence identity between the mouse and human enzymes is 96% with all cysteine residues conserved. The carboxyl-terminal domain of the mouse enzyme contains two more residues than the human enzyme. Northern hybridization analysis revealed considerable expression of the enzyme gene in newborn mouse lung, heart, kidney, and psoas muscle tissues, whereas only weak or no signals were observed in liver, spleen, and brain. Expression of the gene was substantially reduced in the same tissues of 3-month-old mice. In situ hybridization analysis of 72-kDa type IV collagenase expression in 10-15-day-old mouse embryos showed that the gene was intensely expressed in mesenchymal cells. Brain and surface ectoderm were completely negative. The epithelial tissue component of developing organs was negative with the exception of salivary gland. Although the expression varied somewhat between different mesenchymal tissues, no temporal or spatial changes could be associated with the advancement of epithelial branching morphogenesis. These findings together with our previous data on the expression of 72-kDa type IV collagenase in human tumors indicate that this enzyme has some very specific roles both in the physiological and pathological degradation of extracellular matrix. Furthermore, it has become clear that the closely related 92-kDa type IV collagenase differs completely with respect to expression pattern as well as gene regulation. The mouse cDNA clones reported in this study may provide important tools unraveling the actual roles of these enzymes in vivo.
3T3 Cells↗
Epithelial-mesenchymal interactions in murine organogenesis.
Reciprocal, sequential interactions between embryonic epithelial and their mesenchymal stroma guide the cytodifferentiation and organization of both components. These morphogenetic interactions and their consequences are examined in two model systems in vitro: the mouse metanephric blastema and the tooth rudiment. Experimental approaches include dissection and recombination of the interacting tissues, localization of molecular changes by immunohistology and in situ hybridization. An early response of the mesenchyme is increased proliferation of cells in the vicinity of the epithelial inductor and their subsequent aggregation (condensation). In the kidney model disruption of this aggregation or prevention of assembly of the programmed cells results in impaired cytodifferentiation. If the cells are allowed to reaggregate, a phenotype is expressed not unlike that seen in normal in vivo conditions. Our present interest is focused on the early metabolic events associated with the condensation phenomenon. The cell surface proteoglycan syndecan and the matrix glycoprotein tenascin are expressed in the condensed mesenchyme and may mediate cell-matrix interactions. The expression patterns of certain growth factors suggest functions in signal transduction.
Animals↗
Coordinated induction of cell proliferation and syndecan expression in dental mesenchyme by epithelium: evidence for diffusible signals.
Epithelial-mesenchymal interactions induce the expression of syndecan, a cell surface proteoglycan, and tenascin, an extracellular matrix glycoprotein in the mesenchymal component of many organ rudiments including the tooth. Experimental recombination cultures of early dental epithelium and mesenchyme were analysed by double immunostaining to compare the distribution of syndecan, tenascin, and proliferating cells (BrdU incorporation) in the induced dental mesenchyme. After 5-9 hr in culture expression of syndecan and tenascin as well as an increase in BrdU incorporation were evident in the mesenchymal cells adjacent to the epithelium and the positive area enlarged with time. Syndecan and tenascin were colocalized only partially in some explants. The expression of syndecan and tenascin in the recombinants correlates with their stage-dependent expression pattern during early tooth development in vivo (Vainio and Thesleff, 1992). The area of increased cell proliferation in the mesenchyme correlated closely with syndecan expression. In none of the explants was increased BrdU incorporation observed in syndecan negative areas. Epithelium induced also condensation of the mesenchymal cells. Induction and spread of the syndecan-positive zone in the dental mesenchyme required close and continuous contact with the epithelium. The mechanism by which the induction of syndecan expression spreads in the mesenchyme was studied in rat-mouse interspecies recombination cultures, using syndecan antibodies that recognize mouse but not rat syndecan. The rat mesenchyme and epithelium were first cultured in contact for 24 hr. Then the epithelium was removed and freshly dissected, "uninduced" mouse mesenchyme was placed in contact with different aspects of the rat mesenchyme. The rat mesenchymal cells that had located next to the epithelial tissue stimulated syndecan expression in adjacent mouse mesenchyme. The induction potential was gradually lost toward the periphery of the rat mesenchyme. Based on these findings we suggest that diffusible signal molecules mediate the spread of syndecan induction in the mesenchyme and that syndecan plays a role in the regulation of cell proliferation.
Animals↗
Effect of surface processing on the attachment, orientation, and proliferation of human gingival fibroblasts on titanium.
The adhesion, orientation, and proliferation of human gingival fibroblasts was studied on electropolished (elpTi), etched (etchTi), and sandblasted (sblTi) titanium surfaces. The texture, chemical state, and composition of the titanium surfaces were analyzed using a surface tracing instrument and electron spectroscopy for chemical analysis. Considerable differences were evident in the surface texture and chemical composition of the differently treated titanium plates. Electropolishing produced the smoothest and cleanest surface. Human gingival fibroblasts attached, spread, and proliferated on all titanium surfaces. However, cells on elpTi exhibited an extremely flat morphology and seemed to form cellular bridges with adjacent cells, whereas the etchTi and sblTi surfaces harbored both round and flat cells with many long processes. Cells on elpTi appeared to grow in thick layers with no specific orientation, whereas on etchTi surfaces they were migrating along the parallel, irregular minor grooves caused by mechanical polishing, and on sblTi surfaces they seemed to grow in clusters. Stress-fiber type actin bundles and vinculin-containing focal adhesions were present in cells spreading on elpTi and etchTi surfaces but not in cells spreading on sblTi surfaces. Cell shape, orientation, and proliferation appear to depend on the texture of the titanium surface and probably also on the properties of the oxide layer and adjacent bulk material. Our findings suggest that smooth or finely grooved titanium surfaces could be optimal in implants adjacent to soft tissues as they support the attachment and growth of human gingival fibroblasts.
Actin Cytoskeleton↗
Association between the expression of murine 72 kDa type IV collagenase by odontoblasts and basement membrane degradation during mouse tooth development.
In situ hybridization was used to study the expression of the 72 kDa type IV collagenase gene and its association with morphogenesis and cell differentiation during advancing mouse tooth development. The epithelia were completely negative during all developmental stages. The dental mesenchyme was uniformly positive during the early stages of tooth morphogenesis, and no association of type IV collagenase with morphogenetic events was observed. However, at the bell stage the expression increased in differentiating preodontoblasts. Expression was intense in the odontoblasts during secretion of the first predentine matrix. The expression was, however, transient; it decreased around the time when mineralization of dentine started until it completely ceased. Transcripts for 72 kDa type IV collagenase also gradually disappeared from the dental pulp. The expression of 72 kDa type IV collagenase was also strong in the osteoblastic cell lineage. The preosteoblasts at the beginning of the formation of mandibular bone as well as the osteoblasts of the alveolar bone expressed more 72 kDa type IV collagenase than did other mesenchymal cells. The increased gene expression in the odontoblasts correlates with the disappearance of the dental basement membrane as shown by immunolabelling with antibodies against type IV collagen. The onset of increased expression in the odontoblasts preceded the disappearance of the basement membrane and at the time when type IV collagenase transcripts were lost from all odontoblasts the basement membrane was completely removed. It can be speculated that during early stages of tooth development the 72 kDa type IV collagenase acts as a gelatinase whereas during later stages, when odontoblasts and ameloblasts differentiate and the deposition of predentine and enamel matrix is initiated, the enzyme may act as a type IV collagenase and contribute to the degradation of the dental basement membrane.
Animals↗
Transient and recurrent expression of the Egr-1 gene in epithelial and mesenchymal cells during tooth morphogenesis suggests involvement in tissue interactions and in determination of cell fate.
We have analyzed the expression of early growth response gene (Egr-1) by mRNA in situ hybridization during mouse embryonic tooth development and in experimental recombinations of dental epithelium and mesenchyme. Egr-1 was transiently and recurrently expressed both in epithelial and mesenchymal cells starting from day 13 of gestation and up to 4 days after birth. The expression correlated with developmental transition points of dental mesenchymal and epithelial cells suggesting a role for Egr-1 in sequential determination and differentiation of cells. In recombination cultures of early dental epithelium and mesenchyme Egr-1 RNA was localized at the epithelial-mesenchymal interface in mesenchymal cells, and in two cases also in epithelial cells. These data indicate that Egr-1 expression may be regulated by epithelial-mesenchymal interactions when they are specific enough to initiate differentiation. We have also analyzed by in situ hybridization whether Wilms' tumour-1 gene (wt-1) is expressed in the developing tooth as it was proposed on the bases of in vitro studies that it may inhibit Egr-1 expression. No wt-1 expression was detected at any stage of tooth development showing that wt-1 is not obligatory for regulation of Egr-1 expression.
Animals↗
Sequential induction of syndecan, tenascin and cell proliferation associated with mesenchymal cell condensation during early tooth development.
The cell surface proteoglycan, syndecan, and the extracellular matrix glycoprotein, tenascin, are expressed in the mesenchyme during early development of many organs. We have studied the expression patterns of syndecan and tenascin during initiation of tooth development and in association with mesenchymal cell condensation and compared these with cell proliferation. Syndecan, tenascin and bromodeoxyuridine (BrdU) incorporation were localized by triple-labelling immunohistochemistry in serial sections of molar tooth germs of mouse embryos. Prior to formation of the epithelial tooth bud, syndecan accumulated in the mesenchymal cells which underlie the presumptive dental epithelium, but tenascin was not detected at this stage. Tenascin appeared during initiation of the epithelial down-growth at the lingual aspect of the tooth germ. During subsequent formation of the epithelial bud, at the late bud stage, syndecan and tenascin became exactly colocalized in the condensed mesenchyme which was clearly demarcated from other jaw mesenchyme. The expression of syndecan and tenascin was accompanied by rapid cell proliferation as indicated by marked BrdU incorporation. When development advanced to the cap stage, syndecan staining intensity in the dental papilla mesenchyme increased further whereas tenascin became reduced. In conclusion, the results demonstrate that the expression patterns of syndecan and tenascin overlap transiently during the period of mesenchymal cell condensation and that this is accompanied by cell proliferation. Syndecan and tenascin may play a role in growth control and in compartmentalization of the dental mesenchymal cells in the condensate.
Animals↗
Expression of type I collagen pro-alpha 2 chain mRNA in adult human permanent teeth as revealed by in situ hybridization.
The expression of the gene COL1A2, coding for the pro-alpha 2 chain of type I pro-collagen, was analyzed in fully developed human permanent teeth. The teeth were fixed with formalin, demineralized with EDTA for about ten weeks, and embedded in paraffin. Pro-alpha 2(I) mRNA was localized in the sections by in situ hybridization, with use of [35S)]-labeled single-stranded RNA probes. The amount of mRNA for pro-alpha 2(I) collagen chain, as indicated by the relative densities of silver grains and the grain counts per cell in autoradiography, was high in odontoblasts, whereas in pulpal fibroblasts it was low. High levels of pro-alpha 2(I)mRNA expression were also present in those odontoblasts which had elaborated new dentin matrix in response to dental caries. Expression in the periodontal ligament, including the cementoblast layer, was slightly stronger than that in odontoblasts. The intense expression of pro-alpha 2(I) mRNA in odontoblasts of adult teeth suggests that even after the completion of primary dentin formation, they continue to synthesize heterotrimeric type I collagen molecules. Cell type-specific differences in the expression of pro-alpha 2(I) mRNA imply that type I collagen probably plays a major role in the regulation of the structure and function of dental tissues. Finally, in situ hybridization enabled pro-alpha 2(I) collagen mRNA to be detected in tissue sections even after prolonged demineralization, and thus it proved to be a valuable technique for analysis of gene expression in adult dental tissues, as shown here for COL1A2.
Adult↗