Biomedical subjects
H Lesot
Publications and source records attributed to H Lesot.
Distribution of transforming growth factor beta1-binding proteins and low-affinity receptors during odontoblast differentiation in the mouse.
Transforming growth factor-beta1 (TGF-beta1) was immunolocalized within differentiated odontoblasts and ameloblasts while LAP-beta1 was detected at the apicol pole of odonotoblasts and ameloblasts and in predentine. Anti-LAP-beta1 antibodies also stained the epithelial-mesenchymal junction (EMJ). Decorin was immunolocalized in young functional odonotoblasts and in both predentine and dentine. Biglycan was similarly distributed but absent from dentine. Immunostaining with anti-latent TGF-beta1 binding protein-1 (LTBP-1) showed fibrillar structures located at the EMJ and between predontoblasts and odontoblasts; at older states staining was restricted to the dental papilla and sac. Thus differentiated odonotoblasts express TGF-beta1 and in a more restricted manner decorin, biglycan and LAP-beta1; it can be assumed that TGF-beta1 is able to interact with the three molecules present in predentine. Earlier, LTBP-1 and LAP-beta1, both present at the EMJ, may contribute to odontoblast differentiation.
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.
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.
Mouse molar morphogenesis revisited by three dimensional reconstruction. I. Analysis of initial stages of the first upper molar development revealed two transient buds.
Early stages of tooth development in the maxillary cheek region in the mouse were investigated by combined analysis of histological sections, computer assisted 3D reconstructions and morphometry. In ED 12.5 embryos, 3D reconstructions revealed an accessory epithelial bud (R1) and a large bud (R2), which appeared as a single bud-shaped epithelium in frontal sections. This developmentally most advanced dental epithelium in the mouse embryonic maxilla until ED 13.5, generally considered as the bud of the first molar, regressed during later development. Meanwhile the bud and cap of the first upper molar originated more posteriorly, from ED 13.5. The regression of R1 and R2 was associated with epithelial apoptosis. Apoptotic cells and bodies were apparent on sections in the R1 epithelium from ED 12.5. The R2 epithelium maintained the large bud-shaped appearance on sections, representing the largest part of the dental epithelium in the maxillary cheek region until ED 14.0; apoptoses were detected there as late as from ED 13.5. During regression, the R2 rudiment was transformed into the medial and lateral epithelial ridges, posteriorly in continuity with the arising cap of the first molar. The reduced R1 epithelium seemed to contribute to the medial ridge. These results should be taken into consideration in the interpretation of early odontogenesis in the upper jaw in the mouse. The interesting problem of the identification of tooth homology of the rudiments should be elucidated by further comparative morphological and paleontological investigations.
Mouse molar morphogenesis revisited by three-dimensional reconstruction. II. Spatial distribution of mitoses and apoptosis in cap to bell staged first and second upper molar teeth.
Tooth morphogenesis is a complex multifactorial process in which differential mitotic activities and cell death play important roles. Upper first (m1) and second (m2) molars from mouse embryos were investigated from early cap to bell stage. m2 differed from m1 by delayed origin of the enamel grooves delimiting the protrusion of the cap bottom towards the dental papilla, and retardation of the enamel knot formation. The width of the m2 enamel organ was conspicuously smaller during cap formation and length remained smaller throughout the period of observation. Formation of the cap depression was comparable in m1 and m2, however margins delimiting the enamel organ cavity arose in m1 and m2 as mirror images. Attempts were made to correlate changes in the distribution of apoptotic cells and bodies and/or mitoses with morphogenesis. These cellular activities were recorded from histological sections and represented in space using computer-assisted three-dimensional reconstructions. Mitoses in the epithelial compartment were associated with the development of the cervical loop. In the mesenchyme of m1 at early bell stage, a postero-anterior increasing gradient of mitoses was observed which might be correlated with the anterior growth of the molar. Cells in the enamel knot demonstrated a high level of apoptosis, retarded in m2, but absolutely no division. Apoptotic processes were also involved in the anterior delimitation of the m1 epithelium. Apoptosis might correspond to the programmed destruction of cells whose function had to be suppressed or whose potential activity had to be avoided.
Cultured incisors display major modifications in basal lamina deposition without further effect on odontoblast differentiation.
Matrix-mediated epithelio-mesenchymal interactions play a crucial role in the control of dental cytodifferentiations. Ultrastructural observation of the epithelio-mesenchymal junction in cultured embryonic mouse molars showed discrete zones with duplicated or multilayered basal laminae. The use of synthetic peptides demonstrated that the process was RGD*-independent, did not involve the YIGSR* sequence present on laminin and could occur spontaneously. Cultured incisors showed a similar but much more dramatic multiplication of the basal laminae. Furthermore, the deposition of multilayered basal laminae was specific for the labial aspect of the tooth and could be detected after 6 h of culture. Despite these alterations, preodontoblasts differentiated and gradients of differentiation were maintained, suggesting that among basement membrane constituents, the basal lamina itself does not play a critical role. More important is the inner dental epithelium which may still control odontoblast differentiation by means of diffusible molecules able to reach surface receptors expressed by preodontoblasts or matrix receptors underlying the basal lamina. Gradients of odontoblast differentiation could result from a progressive acquisition of competence by preodontoblasts.
Comparison of the effects of growth hormone, insulin-like growth factor-I and fetal calf serum on mouse molar odontogenesis in vitro.
The effects of growth hormone, its mediator insulin-like growth factor-I (IGF-I), and fetal calf serum on odontogenesis were compared to those of serum-free medium. Explanted, 16-day, fetal mouse first molar tooth germs in early bell stage were grown on semisolid, serum-free medium supplemented with ascorbic and retinoic acids. Recombinant human growth hormone at 50 or 100 ng/ml, IGF-I at 100 or 200 ng/ml, or fatal calf serum at 20% concentration were added to the media. Volumetric changes in serial sections of six tooth germs per treatment over 3 days of treatment (4, 5, 6 days in vitro) were compared by digitized morphometry. Mitotic indices were also compared and the cell densities of the dental papillae recorded. Qualitative ratings of differentiation were ascribed to each tooth germ by light microscopy. Differences in volume, mitotic activity and cell densities were found. The growth hormone-treated tooth germs were not larger than the serum-free ones but had increased mitotic indices and higher cell densities in the dental papillae. IGF-I-treated tooth germs had larger volumes than with all other treatments, e.g. germs treated with 200 ng/ml of IGF-I, after 6 days in culture, were significantly larger than with all other treatments (p<0.01-<0.001). Whilst IGF-I-treated germs displayed the greatest extent of differentiation, growth hormone-treated germs also showed advanced differentiation compared to those on serum-free medium. These results suggest that growth hormone and IGF-I are involved in odontogenesis of murine teeth in vitro by affecting mitotic activity, tissue volume and cell differentiation. In conjunction with previous immunohistochemical studies that show expression of growth hormone receptor and IGF-I in developing teeth, these results provide evidence that both growth hormones and its mediator play a part in odontogenesis.
Immunocytochemical localization of fibronectin and a 165-kDa membrane protein in the odontoblast layer under initial carious lesions in man.
The possible role of fibronectin in dental tissue repair was investigated by comparing its distribution and that of the 165-kDa fibronectin-binding membrane protein (165 kDa-FnBP) in odontoblasts underlying carious and sound dentine. By immunoperoxidase and light microscopy, fibronectin was localized in the dentine underlying the carious lesion, mainly on the surface of the tubule walls, whereas it could not be detected in neighbouring sound zones. The antibody to the 165 kDa-FnBP strongly reacted with the membrane of odontoblasts underlying the lesion, although those facing sound dentine did not express this antigen. Ultrastructurally the 165 kDa-FnBP was localized in the cell membrane at the apical portion of odontoblasts, including the process membrane, beneath the initial lesion; fibronectin was detected in the dentinal area close to the process, and also in contact with its external surface. By a high-resolution immunogold procedure, the proteins were colocalized at the external surface of odontoblast processes. These data suggest that fibronectin present in human carious dentine could modulate the behaviour of underlying odontoblasts by means of newly expressed 165 kDa-FnBP.
Induction of odontoblast-like cell differentiation in dog dental pulps after in vivo implantation of dentine matrix components.
The effects of dentine extracellular matrix components on dental mesenchymal cells were studied by light and transmission electron microscopy after their implantation at central sites of mechanically exposed pulps in dog molar teeth. The implants were Millipore filters that had been soaked with solutions containing 30 or 300 micrograms/ml of an EDTA-soluble fraction of rabbit incisor dentine. Control filters were soaked with dog albumin or phosphate buffered saline. Columnar, polarized cells were consistently seen after 8 days in close proximity to the filters coated with both concentrations of dentine matrix components. Characteristic features of these polarized cells included widened cisternae of the rough endoplasmic reticulum, a rich microfilamentous network in the long cytoplasmic extensions invading the filter pores and numerous cytoplasmic bodies. These cells also showed evidence of functional as well as cytological differentiation. Polarized processing of secretory granules could be observed after 8 days' implantation, and also the presence of matrix vesicles and deposition of a fine, collagenous matrix into the filters apically to the distal end of the cytoplasmic processes. After 24 days' implantation, secretion of a tubular matrix could be consistently seen in association with the odontoblast-like cells. No changes in cell organization or matrix synthesis were seen after implantation of control filters. These studies demonstrate that bioactive components present in the EDTA-soluble dentine matrix fraction are able to directly induce cell polarization and apical secretion of tubular matrix when implanted in contact with dental pulp cells at sites remote from the odontoblast layer.
Influence of substrate nature and immobilization of implanted dentin matrix components during induction of reparative dentinogenesis.
The biological effects of isolated soluble dentin extracellular matrix components on the induction of reparative dentinogenesis in exposed cavities in ferret canine teeth have been shown to be blocked by immobilizing the extracellular matrix components on nitrocellulose or Millipore membranes during implantation. This contrasts with the picture of induction of odontoblast-like cell differentiation and reparative dentin deposition on existing insoluble dentin matrix of the exposure walls when the extracellular matrix components are implanted in lyophilized form. These data indicate the importance of an existing insoluble dentin matrix in providing a substrate to potentiate the growth factor-like activity of soluble isolated dentin extracellular matrix components in the induction of reparative dentinogenesis.
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.
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.
Odontoblast stimulation in ferrets by dentine matrix components.
The possible effects of isolated dentine matrix components on odontoblast secretory activity were investigated in vivo by implantation of lyophilized fractions of these components into cavities prepared in ferret canine teeth. After implantations as short as 14 days there was significant deposition of reactionary dentine by the odontoblasts beneath the cavity and this response increased in a non-linear manner with time of implantation. In contrast, control cavities lacking the dentine matrix components showed no evidence of reactionary dentine deposition. Examination of teeth at early periods of implantation (2 and 5 days) indicated that odontoblast death had not occurred as a result of the operative procedures and that the response was one of stimulation of existing odontoblasts rather than that of induction of a new generation of odontoblast-like cells. The mechanisms of odontoblast stimulation by the dentine matrix components remain to be elucidated, but could be mediated by growth factors within the dentine matrix preparations.
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.
Absence of interaction between the 165-kDa fibronectin-binding protein involved in mouse odontoblast differentiation and vinculin.
Previous data suggested that matrix could control the organization of microfilaments in differentiating odontoblasts and that this process involved a complex of fibronectin-165-kDa membrane protein-vinculin. The use of two different gel systems and microsequence analysis demonstrated that two distinct 165-kDa proteins interact, one with fibronectin and the other with vinculin.
Interaction of vinculin with the clathrin heavy chain.
To further document the interaction of vinculin with the clathrin heavy chain (CHC) which was observed by using gel overlay, co-sedimentation experiments were performed and attempts were made to localize the domains involved on both molecules. The binding properties of proteolytic fragments of vinculin were investigated after cleavage with V8 protease. Neither the isolated globular domain, nor the C-terminal rod domain were able to interact with the CHC. Either the interaction involved the portion of vinculin which links these two domains, or the region of vinculin mediating the interaction was present on one of the two major fragments, but the cleavage itself resulted in conformational changes which abolished the binding. The first hypothesis could be ruled out using alpha-chymotrypsin generated fragments of vinculin, suggesting that the native conformation of vinculin might play an important role. Proteolytic cleavage of CHC with trypsin demonstrated that the interaction with vinculin is mediated by the proximal or distal segment of the CHC. Presence of clathrin light chain (CLC) associated with the CHC did not affect its interaction with vinculin. Vinculin did not interact with the CLC.
The carboxy-terminal extension of the collagen binding domain of fibronectin mediates interaction with a 165 kDa membrane protein involved in odontoblast differentiation.
Terminal differentiation of the odontoblast is characterized by an elongation and a polarization of the cell. The change in the cell shape and the reorganization of the cytoplasm involve the microfilament system. An immunological approach has previously implicated a transmembrane interaction between fibronectin and vinculin in the control of odontoblast differentiation. A 165 kDa protein localized on the cell-surface of odontoblasts mediated this interaction. In order to define the nature of the interaction of the 165 kDa protein with fibronectin, peptides were prepared by proteolytic cleavage of fibronectin with alpha-chymotrypsin. The results indicate that the 165 kDa protein interacted with a 62 kDa peptide located towards the amino-terminal extremity of fibronectin, but not with a 47 kDa related fragment. Both these 62 kDa and 47 kDa peptides included the collagen-binding domain and were retarded on a heparin-Ultrogel column. Microsequences demonstrated that the 62 kDa and 47 kDa fragments had the same amino-terminal extremity and that the larger fragment was extended in the carboxy-terminal direction. This carboxy-terminal extension of the collagen binding domain of fibronectin is implicated in the interaction of this molecule with the 165 kDa protein. On the other hand, odontoblasts differentiated normally when tooth germs were cultured in the presence of GRGDS synthetic peptide, suggesting that RGD-dependent integrins were not involved in odontoblast differentiation. Staining of dental mesenchymal cells in primary culture and of differentiated odontoblasts in situ with antibodies directed against the beta 1-subunit of integrins confirmed previous observations and showed that although beta 1 integrins are involved in the attachment of cultured dental cells, they are not implicated in the process of odontoblast differentiation.