Synthesis of collagen type I, type I trimer and type III by embryonic mouse dental epithelial and mesenchymal cells in vitro.

Epithelial and mesenchymal dental cells were grown in primary monolayer culture and the ability of both cell types to synthesize interstitial collagens was investigated. Pepsin-solubilized collagens were analyzed by CM-cellulose chromatography and both cell types were found to synthesize collagen type I, type III and type I trimer. The collagen phenotype of mesenchymal cells (type I: 82.4%, type III: 8.5%, type I trimer: 9.1%) was different from that of epithelial cells (type I: 71.8%, type III: 9.5%, type I trimer: 18.7%). The radioactivity incorporated into collagen molecules by mesenchymal cells was 34-times greater than the radioactivity incorporated by epithelial cells. This result agreed with previous observations obtained from tissue culture experiments (Lesot, H. and Ruch, J.V. (1979) Biol. Cell. 34, 23--37) which indicated a low synthesis of interstitial collagens by isolated dental epithelia when compared to isolated dental mesenchymes.

Production of monoclonal antibodies against mouse molar papilla cells.

To develop markers for the analysis of the molecular mechanisms of dental papilla cells differentiation, 10 monoclonal antibodies were produced against trypsin-isolated mouse molar dental papilla cells. These antibodies identify matrix components, cell membrane associated antigens and intracellular-constituents. Changes of the staining patterns were correlated with a typological hierarchy of dental papilla cells and with terminal differentiation of odontoblasts.

Polyamine depletion-mediated effects on murine odontogenesis are dependent on tooth developmental stage and culture conditions.

Polyamines are known to play a central role in processes such as growth and development. Virtually nothing is known about their importance in tooth development, an attractive and frequently used experimental model for studies of developmental processes. A polyamine-depleted state was created in tooth cells in an organ culture system. First lower molar germs from 16-and 17.5-day old mouse fetuses were used. alpha-difluoromethylornithine (DFMO) and methylglyoxal bis-(guanylhydrazone) (MGBG) were used to deplete the cells from their polyamine content. Polyamine interconversion and catabolism were prevented by aminoguanidine sulfate (AG). In day-16 germs cultured in serum-containing medium, DFMO reduced the frequency of cycling cells as shown by [3H]thymidine incorporation, and induced a delay of odontoblast differentiation of about 24 h. Under the same conditions, MGBG induced an arrest of histo-morphogenesis, correlated to a significant decrease in the rate of cell proliferation. Addition of polyamines prevented DFMO- and MGBG-induced delay of tooth differentiation. Interestingly, MGBG did not delay the terminal differentiation of odontoblasts and ameloblasts in cultured day-17.5 molars; in these, cells at the tip of the cusps are only a few hours before their withdrawal from the cell cycle. In serum-deprived medium, dental cytodifferentiations did not occur. Addition of putrescine or spermidine to serum-free media, however, allowed for tooth morphogenesis and cytodifferentiation. Tooth explants in a serum-deprived medium reacted to DFMO in a cytocidal fashion, whereas MGBG showed only a mild toxicity in some cell types. Addition of putrescine to DFMO-containing medium prevented its cytotoxic effect. Addition of spermidine to MGBG-containing medium not only prevented its mild toxicity but also allowed for predentin secretion by differentiated odontoblasts. The results are discussed with regard to the well-established developmental events of tooth germs cultured in vitro and with respect to present knowledge of polyamine metabolism and their involvement in cellular processes.

Odontoblast differentiation.

Odontoblasts are post-mitotic, neural crest-derived, cells which overtly differentiate according to tooth specific temporo-spatial patterns and secrete predentin-dentin components. Neither the timing nor the molecular mechanisms of their specification are known and the problem of their patterning in the developing jaws is far from being solved. On the other hand, some significative strides were made concerning the control of their terminal differentiation. Fibronectin interacting with a 165 kDa, non integrin, membrane protein intervenes in the cytoskeletal reorganization involved in odontoblast polarization and their terminal differentiation can be triggered in vitro by immobilized members of the TGF beta family. Histological aspects and the transcriptional phenotypes (transcripts of TGF beta s, BMPs, msxs, IGF1, fibronectin, osteonectin, bone sialoprotein genes) are very similar in vivo and in vitro. In vivo members of the TGF beta super family secreted by preameloblasts, trapped and activated by basement membrane associated components, might initiate odontoblast terminal differentiation.

Mouse odontogenesis in vitro: the cap-stage mesenchyme controls individual molar crown morphogenesis.

Day 14 ICR mouse first lower (M1) and upper molars (M1) as well as heterotopic recombinations of M1 epithelium/M1 mesenchyme and M1 epithelium/M1 mesenchyme were cultured for 6, 8 and 10 days on semi-solid medium. Computer-assisted 3D reconstructions were performed to follow the in vitro development of these explants. In vitro culture of cap-stage molars allowed for the emergence of unequivocal morphological features distinctive for M1 versus M1 including the cusp pattern, cusp inclination and tooth specific chronology for odontoblast and ameloblast terminal differentiations. Both M1 epithelium/M1 mesenchyme and M1 epithelium/M1 mesenchyme recombinations developed according to the known developmental fate of the mesenchyme. Our data demonstrate that the cap-stage dental ecto-mesenchyme not only directs tooth class specific morphogenesis, but also individual molar crown features. Furthermore, the mesenchyme apparently also controls the typical mirror symmetry of right and left handed teeth.

Contribution of 3-D computer-assisted reconstructions to the study of the initial steps of mouse odontogenesis.

The specific arrangement of mouse dentition in each dental quadrant (1-0-0-3) is supposed to result from the initiation of two independent dental laminae--one for the incisor and one for the three molars. In order to verify whether the adult mouse dental pattern really corresponds to the initial patterning, an analysis of development of the mouse antemolar part of the upper dental quadrant was performed in 10-13 day embryos using histological sections and computer-assisted 3-D reconstructions. Six primary dental laminae contributed to the formation of the upper incisor anlage, which is, therefore, a structure of multiple origin. In contrast to the lower diastema, where only a low epithelial band extended mesially from the first lower molar in 12-13 day embryos, in the upper diastema a dental lamina existed interconnecting transiently the incisor and molar anlagen and giving rise to 3 distinct epithelial rudiments. The rudiments exhibited growth retardation and regressed after reaching a maximum at the bud stage. Our results showed a discrepancy between the embryonic and adult dental patterns in the mouse upper jaw. The specific arrangement of the mouse dentition implied a reduction of the embryonic dental anlagen, which was achieved either by their integration into the one incisor primordium or regression in the prospective diastema. Odontogenesis in the mouse upper jaw provides a model of hypodontia of evolutionary origin, which can be employed in molecular studies of the control mechanisms of initiation, spatial organization and specific morphogenesis of teeth.

Effects of hepatocyte growth factor anti-sense oligodeoxynucleotides or met D/D genotype on mouse molar crown morphogenesis.

Hepatocyte growth factor (HGF) is considered to be one of the mediators of epithelio-mesenchymal interactions during early organogenesis and to be also involved in the development of murine molars. In the developing tooth, HGF is expressed in the cells of the dental papillae, and c-Met, its receptor, in the cells of dental epithelia. In order to study the functional role played by HGF in tooth development, we tested the effects of HGF translation arrest by anti-sense phosphorothioate oligodeoxynucleotides on E-14 molars cultured in vitro. We also analyzed the histo-morphogenesis and crown cytodifferentiation of transgenic met E-14 molars cultured in vitro. 3D reconstructions revealed perturbations of the cusp pattern. However, histo-morphogenesis and crown cytodifferentiation were normal at the histological level.

Multiple developmental origin of the upper incisor in mouse: histological and computer assisted 3-D-reconstruction studies.

Heads of 11-15-day-old mouse embryos were cut in frontal serial sections. Early development of the maxillary incisor was analyzed using series of thick (5 and 7 microns) and semi-thin (1 micron) frontal sections and computer assisted 3-D-reconstructions of the epithelial component. The enamel organ of the mouse maxillary incisor was found to be a complex structure of multiple origin, involving several epithelial anlagen--primary dental laminae--, which could hypothetically correspond to the 5 upper incisors of early mammals. The transitory existence of at once distinct and then fusing dental primordia could reflect heterochronic changes in ontogeny which might be related to phyletic trends.

Effects of retinoids on tooth morphogenesis and cytodifferentiations, in vitro.

The first embryonic lower mouse molar was used as a model system to investigate the effects of two retinoids, retinoic acid (RA) and a synthetic analogue, Ch55, on morphogenesis and cytodifferentiations in vitro. Exogenous retinoids were indispensable for morphogenesis of bud, cap and bell-stage molars in serum-free, chemically-defined, culture media. Transferrin and RA or transferrin and Ch55 acted synergistically in promoting morphogenesis from bud and cap-stage explants. Transferrin, per se, had no morphogenetic effect. Epithelial histogenesis, odontoblast functional differentiation and ameloblast polarization always occurred in RA-depleted explants. Comparison of the distributions of bromodeoxyuridine (BrdU) incorporation between explants cultured in the absence or presence of RA revealed that RA could modify the patterns of cell proliferation in the inner dental epithelium and dental mesenchyme. Inner dental epithelium cell proliferation is regulated by the dental mesenchyme through basement membrane-mediated interactions, and tooth morphogenesis is controlled by the dental mesenchyme. Laminin is a target molecule of retinoid action. Using a monospecific antibody, we immunolocalized laminin and/or structurally-related molecules sharing the laminin B chain in the embryonic dental mesenchyme and in the dental basement membrane and showed that RA could promote the synthesis or secretion of these molecules. Based on previous in situ hybridization data, it was speculated that CRABPs might regulate the effects of RA on embryonic dental cell proliferation. The fact that Ch55, a retinoid which does not bind to CRABPs, is 100 times more potent than RA in promoting tooth morphogenesis in vitro seems to rule out this hypothesis. On the other hand, the stage-specific inhibition of tooth morphogenesis by excess RA is consistent with the hypothesis that CRABPs might protect embryonic tissues against potentially teratogenic concentrations of free retinoids.

Regulated changes in chondroitin sulfation during embryogenesis: an immunohistochemical approach.

Chondroitin sulfate proteoglycans, which represent the main class of nonfibrous macromolecules found in the extracellular matrix of connective tissues, have been implicated in the control of a variety of cell activities during ontogenesis. The respective contributions of the chondroitin sulfate chains and of the protein moiety of the proteoglycan in morphogenesis and cytodifferentiation are not known. In this context, monoclonal antibodies identifying specific chondroitin sulfate chains are interesting new tools. A panel of well characterized monoclonal antibodies recognizing distinct epitopes present only in chondroitin sulfate chains was used in conjunction with immunohistochemical techniques for the purpose of identifying and mapping chondroitin sulfate isoforms during development in the mouse and rat fetus. Expression of chondroitin sulfate isoforms occurred in the tissues according to specific spatio-temporal patterns, suggesting that chondroitin sulfates differing in sulfation position and degree perform distinct functions in development.

Mouse molar morphogenesis revisited by three-dimensional reconstruction. III. Spatial distribution of mitoses and apoptoses up to bell-staged first lower molar teeth.

Computer-assisted 3D reconstructions were used to follow the development of the embryonic mouse first lower molar (M1). At ED 12.5, the thickening of the oral epithelium, which was thought to correspond to the molar dental lamina, regressed in its anterior part as a result of apoptosis. Only the posterior part later gave rise to molars. The transition to the cap stage entailed medial and lateral extensions of the dental epithelium. The growth and histo-morphogenesis of the enamel organ as well as cervical loop formation proceeded more rapidly in the anterior part of the M1 during the cap and early bell stages producing significant morphological differences along the antero-posterior axis. Apoptosis was temporarily intensive in the anterior part of the bud- and cap-shaped epithelium and thus pointed domains which do not participate in the formation of the final M1 enamel organ. In the well-formed cap, apoptoses displayed maximum concentration in the enamel knot (EK). No increase in the number of metaphases could be detected in the vicinity of the EK. Mitoses were distributed throughout the epithelial compartment until cap stage and then mainly concentrated in the inner dental epithelium at the early bell stage. At this later stage, either lateral views or thick virtual sections performed in the reconstruction demonstrated a clear cut distribution of mitoses and apoptoses in the enamel organ. At the early bell stage, mitoses in the mesenchyme demonstrated an increasing postero-anterior gradient.

The presence of rudimentary odontogenic structures in the mouse embryonic mandible requires reinterpretation of developmental control of first lower molar histomorphogenesis.

In the mouse embryonic maxilla, rudimentary tooth primordia have been identified, which can be mistaken for the first upper molar. In order to determine whether such a situation might exist in the lower jaw as well, tooth development was investigated in the mouse mandibular cheek region during ED 12.5-15.0. A combination of histology, morphometry and computer-aided 3D reconstructions demonstrated the existence of rudimentary dental structures, whose gradual appearance and regression was associated with the segmental progress of odontogenesis along the mesio-distal axis of the jaw: 1) At ED 12.5, the mesial segment (MS) was the most prominent part of the dental epithelial invagination. It included an asymmetrically budding dental lamina. The MS, although generally mistaken for the lower first molar (M1, primordium, regressed and did not finally participate in M1 cap formation. 2) At ED 13.5, a wide dental bud (called segment R2) appeared distally to the MS. Although the R2 segment transiently represented the predominant part of the dental epithelium at ED13.5, it participated only in the formation of the mesial end of the M1 cap. 3) The top of the R2 segment at ED13.5 was not the precursor of the enamel knot (EK), contrary to what has been assumed. 4) The central segment of the M1 cap as well as the EK developed later and distally to the R2 segment. 5) Time-space specific apoptosis correlated with the retardation in growth of the R2 segment as well as with strong regressive changes in the epithelium situated mesially to it. These highlight the need to reinterpret current molecular data on early M1 development in the mouse in order to correlate the expression of signalling molecules with specific morphogenetic events in the appropriate antemolar or molar segments of the embryonic mandible.

Regeneration of halved embryonic lower first mouse molars: correlation with the distribution pattern of non dividing IDE cells, the putative organizers of morphogenetic units, the cusps.

Recently we demonstrated that non-cycling, cap-stage, mouse molar inner dental epithelial (IDE) cells corresponding to the primary enamel knot (EK) area underwent a coordinated temporo-spatial patterning leading to their patchy irregular segregation at the tips of the forming cusps. These non-cycling cells were suggested to perhaps represent the organizers of the morphogenetic units (OMU), the cusps. The present study has analyzed the regenerative capacity of halved cap-stage first lower mouse molars through three dimensional (3D) reconstructions. Partial regeneration of the anterior half and possible complete regeneration of the posterior half were documented. Using BrdU (5-bromo-2'-deoxyuridine) labeling and 3D reconstructions of the IDE, we have correlated the patterns of cusp regeneration with the distribution of BrdU negative IDE cells. These data support a morphogenetic role for the non-cycling IDE cells.

Comparative analysis of TGF beta s, BMPs, IGF1, msxs, fibronectin, osteonectin and bone sialoprotein gene expression during normal and in vitro-induced odontoblast differentiation.

Immobilized TGF beta 1 and BMP2 are able to promote the differentiation of odontoblast-like cells in isolated mouse dental papillae cultured in vitro. These cells polarize and accumulate predentin-like matrix at their apical pole. Immobilized IGF1 mainly promoted polarization with disturbed matrix accumulation. In situ hybridization demonstrated that TGF beta 1 combined with heparin mirrored the physiological processes of odontoblast differentiation. Normal odontoblast and in vitro induced odontoblast-like cells expressed transcripts encoding for TGF beta 1 and 3, BMP2 and 4, bone sialoprotein and osteonectin whereas either ubiquitous expression or no expression could be detected for TGF beta 2, IGF1 or fibronectin mRNAs. Odontoblast-like cells obtained in the presence of IGF-1 combined with heparin did not express TGF beta 1 transcripts and expressed weakly TGF beta 3 transcripts. Our results suggest that in vivo an epithelial-derived member of the TGF beta family trapped by basement membrane-associated components interacts with competent preodontoblasts and promotes the polarization by triggering the transcription of growth factor gene(s) like TGF beta itself and/or selector gene(s) like msx2.

Aspects of cell proliferation kinetics of the inner dental epithelium during mouse molar and incisor morphogenesis: a reappraisal of the role of the enamel knot area.

First lower E-14 and E-16 mouse molars and E-13 lower incisors were cultured in vitro and either sequentially or continuously labelled with BrdU (5-bromo-2'-deoxyuridine). The behaviour of the non-cycling inner dental epithelial cells emerging from the enamel knot area of the molars was analysed by 3D (three dimensional) reconstructions of serial sections. These cells, as well as slow cycling cells underwent a coordinated temporo-spatial patterning leading to their patchy segregation at the tips of the forming cusps. In incisors (in vitro and in vivo), non-cycling cells were also present in the inner dental epithelium of the enamel knot area. However, these cells were not redistributed during incisor morphogenesis. These non-dividing inner dental epithelium cells of the enamel knot area which are either redistributed or not according to the tooth type specific morphogenesis might represent the organizers of morphogenetic units (OMU), the cusps.

Alterations in the incisor development in the Tabby mouse.

The X-linked tabby (Ta) syndrome in the mouse is homologous to the hypohidrotic ectodermal dysplasia (HED) in humans. As in humans with HED, Ta mice exhibit hypohidrosis, characteristic defects of hairs and tooth abnormalities. To analyze the effects of Ta mutation on lower incisor development, histology, morphometry and computer-aided 3D reconstructions were combined. We observed that Ta mutation had major consequences for incisor development leading to abnormal tooth size and shape, change in the balance between prospective crown- and root-analog tissues and retarded cytodifferentiations. The decrease in size of Ta incisor was observed at ED13.5 and mainly involved the width of the tooth bud. At ED14.5-15.5, the incisor appeared shorter and narrower in the Ta than in the wild type (WT). Growth alterations affected the diameter to a greater extent than the length of the Ta incisor. From ED14.5, changes in the shape interfered with the medio-lateral asymmetry and alterations in the posterior growth of the cervical loop led to a loss of the labio-lingual asymmetry until ED17.0. Although the enamel organ in Ta incisors was smaller than in the WT, a larger proportion of the dental papilla was covered by preameloblasts-ameloblasts. These changes apparently resulted from reduced development of the lingual part of the enamel organ and might be correlated with a possible heterogeneity in the development of the enamel organ, as demonstrated for upper incisors. Our observations suggest independent development of the labial and lingual parts of the cervical loop. Furthermore, it appeared that the consequences of Ta mutation could not be interpreted only as a delay in tooth development.

Morphogenesis of the lower incisor in the mouse from the bud to early bell stage.

The development of the lower incisor in the mouse was investigated from histological sections using computer-aided 3D reconstructions. At ED 13.0, the incisor was still at the bud stage. At ED 13.5, the initial cap was delimited by a short cervical loop, the development of which proceeded on the labial side, but was largely retarded on the medial side. This difference was maintained up to ED 15.0. From ED 16.0, the bell stage was achieved. Metaphases had a ubiquitous distribution both in the enamel organ and in the dental papilla from the bud to early bell stage. Apoptosis gradually increased in the mesenchyme posteriorly to the labial cervical loop from ED 13.5 to 14.0 and then disappeared; this apoptosis was not related to the posterior growth of the incisor. From ED 13.5, a high apoptotic activity was observed in the stalk. A focal area of apoptosis was observed at ED 13.5 in the enamel organ, approaching the epithelio-mesenchymal junction at the future tip of the incisor. There, the inner dental epithelium formed a bulbous protrusion towards dental papilla, reminiscent of the secondary enamel knot of mouse molars. This epithelial protrusion was still maintained at the bell stage. The enamel knot in the incisor demonstrated specific features, different from those characterizing the enamel knot in the molar: the concentric arrangement of epithelial cells was much less prominent and the occurrence of apoptosis was very transitory in the incisor at ED 13.5. The disappearance of the enamel knot despite a low apoptotic activity and the maintenance of the protrusion suggested a histological reorganization specific for rodent incisor.

Comparison of expression of the msx-1, msx-2, BMP-2 and BMP-4 genes in the mouse upper diastemal and molar tooth primordia.

The existence of transient putative tooth anlagen in the prospective mouse upper diastema region has been documented previously in morphological studies. By in situ hybridization we investigated the expression patterns of the msx-1, msx-2, BMP-2 and BMP-4 genes, supposed to regulate early tooth development, in day 10-14 mouse embryonic upper diastema and molar regions, using 49 series of frontal sections. On the basis of comparison of the temporo-spatial expression patterns in both diastemal and molar tooth primordia we conclude that each of the four genes was expressed at least for some period simultaneously and at a comparable developmental stage in the transient and persisting dental primordia. BMP-2 and BMP-4 expression was downregulated in the diastemal dental primordia during their regression starting at day 13. The temporo-spatial pattern of BMPs expression may be associated with the disappearance of diastemal rudiments. Contrary to the molar anlage, we did not detect msx-2 gene expression in the diastemal dental rudiments after the stage of epithelial thickening. The deficiency of the msx-2 gene products may play a role in the growth retardation of diastemal dental primordia resulting in their subsequent involution.

Reactionary dentinogenesis.

Reactionary dentinogenesis is the secretion of a tertiary dentine matrix by surviving odontoblast cells in response to an appropriate stimulus. Whilst this stimulus may be exogenous in nature, it may also be from endogenous tissue components released from the matrix during pathological processes. Implantation of isolated dentine extracellular matrix components in unexposed cavities of ferret teeth led to stimulation of underlying odontoblasts and a response of reactionary dentinogenesis. Affinity chromatography of the active components prior to implantation and assay for growth factors indicated that this material contained significant amounts of TGF-beta 1, a growth factor previously shown to influence odontoblast differentiation and secretory behavior. Reactionary dentinogenesis during dental caries probably results from solubilization of growth factors, TGF-beta in particular, from the dentine matrix which then are responsible for initiating the stimulatory effect on the odontoblasts. Compositional differences in tertiary dentine matrices beneath carious lesions in human teeth have also been shown indicating modulation of odontoblast secretion during reactionary and reparative dentinogenesis.

Apoptosis is involved in the disappearance of the diastemal dental primordia in mouse embryo.

Three transient dental primordia (D1, D2 and D3) exist in the upper diastema in mouse embryos and their regression is associated with the presence of cell death. In order to specify the type of cell death and its temporo-spatial distribution, staining with hematoxylin, supravital staining with Nile Blue, TUNEL method, electron microscopic analysis and computer assisted 3-D reconstructions were performed. These data demonstrated that apoptosis is involved in the disappearance of the diastemal dental rudiments. Apoptosis occurred first with prevalence in the buccal part of the epithelium of the diastemal dental primordia and extended later to the whole epithelium of the dental rudiments and the dental lamina interconnecting them with the incisor and molar epithelia. Cell death occurred only sporadically in the adjacent mesenchyme. The prospective upper diastema in mouse embryos may provide a model for studies of developmental determination of toothless areas in the jaw as well as a tool for analyses of regulatory mechanisms of programmed cell death in morphogenesis.