Light microscopic detection of sugar residues in rabbit embryo teeth with lectin-horseradish peroxidase conjugates

We investigated the binding of five HRP-conjugated lectins to rabbit tooth germs at the cap and late bell stages of development. The results revealed some changes in the glycosylation patterns of the glycoconjugates. Sugar residues, such as α-D-mannose, methyl-D-glucose, N-acetylglucosamine, β-D-galactosamine, D-galactose, and sialic acid, were detectable in some components of the tooth germs. The most conspicuous developmental change was increased binding of Con A and WGA. These lectins showed, at the cap stage, moderate binding to the (pre)-ameloblasts and (pre)-odontoblasts. With further development to the late bell stage, but prior to the achievement of well-defined morphological-functional characteristics, the odontoblasts and ameloblasts displayed considerable amounts of α-D-mannose, α-D-glucose as well as β-D-acetylglucosamine and sialic acid. Appropriate control studies confirmed the specificity of the binding of the lectins. Two lectins (DBA and PNA) with known specificity for N-acetylgalactosamine groups were bound by the basement membranes in tooth germs at the cap stage. A third lectin (RCA) with the same specificity did not produce any detectable staining in the same material. Further studies must be planned to determine the specific functions and significance of lectin-HRP-binding glycoconjugates in odontogenesis. © 1996 Wiley-Liss, Inc.     

Detection of sugar residues in rabbit embryo teeth with lectin-horseradish peroxidase conjugate: II. A light microscopal study

The cellular distribution and changes of sugar residues during tooth development in embryos of the rabbit Oryctolagus cuniculus were investigated by using horseradish peroxidase-conjugated lectins (lectin-HRP). The lectins SBA, ECA, and LTA show no binding to any region of the dental cap and bell stages, whereas BS-1 and UEA-1 bind to dental cells at both stages. Appropriate control studies confirmed the specificity of the binding of the lectins. At cap stage, the lectins BS-1 and UEA-1 show moderate binding to the (pre)-ameloblast and (pre)-odontoblast cells. These results suggest that the acetylgalactosamine and α-L-fucose residues present in (pre)-ameloblasts and (pre)-odontoblasts, respectively, are common to determined but relatively undifferentiated cells capable of forming matrices of hard tissues. Since the odontoblast and ameloblast express dentin and enamel, respectively, it can be speculated that the abundance of these residues in these cells might be associated with the maintenance of the capacity of the cells to produce such matrices. At the bell stage, the odontoblasts display considerable amounts of α-L-fucose, whereas α-L-fucose is poorly localized in ameloblasts. However, ameloblasts contain significant quantities of N-acetylgalactosamine, whereas only a diffuse positivity for this carbohydrate is apparent in odontoblasts. The marked changes of the glycosylation pattern of these glycoconjugates might indicate that they play a role during the cell-to-cell interaction and might also be involved in the odontoblastic and ameloblastic functional activity. Such a possibility is entirely speculative until specific in vitro experiments are conducted. J. Morphol. 231:175–184, 1997. © 1997 Wiley-Liss, Inc.     

Leptin and vascular endothelial growth factor regulate angiogenesis in tooth germs

Leptin, a 16 kDa non-glycolated polypeptide of 146 amino acids produced by the ob gene, has a variety of physiological roles not only in lipid metabolism, hematopoiesis, thermogenesis and ovarian function, but also in angiogenesis. This study focuses to investigate the possibility that leptin, as an angiogenic factor, may regulate the angiogenesis during tooth development. We firstly studied the expression of leptin and vascular endothelial growth factor (VEGF) during tooth development immunohistochemically. This investigation revealed that leptin is expressed in ameloblasts, odontoblasts, dental papilla cells and stratum intermedium cells. This expression pattern was similar to that of VEGF, one of the most potent angiogenic factors. Interestingly, more leptin-positive cells were observed in the upper third portion of dental papilla, which is closest to odontoblastic layer, compared to middle and lower thirds. Moreover, in the dental papilla, more CD31 and/or CD34-positive vascular endothelial cells were observed in the vicinity of ameloblasts and odontoblasts expressing leptin and VEGF. These findings strongly suggest that ameloblasts, odontoblasts and dental papilla cells induce the angiogenesis in tooth germs by secretion of leptin as well as VEGF.     

A modification of tooth germ cultivation in vitro and in ovo

Mandibular molar anlages excised from 17-day mouse foetuses were cultured in vitro or in ovo (on the chorioallantoic membrane). In both cases, the explants were underlain either with a Millipore filter or with a piece of fibrin foam. Tooth germs were harvested after 7 days of cultivation and processed histologically. Spatial arrangement was highly preserved in the tooth germs cultured in vitro on fibrin foam. In vitro cultures on Millipore filters revealed significant flattening of tooth germs, caused especially by the collapse of enamel organ and the pulp. The cytodifferentiation of tooth germs cultured in vitro on both substrates (Millipore filter, fibrin foam) was characterized by the presence of odontoblasts, polarizing ameloblasts and predentine. The cytodifferentiation of tooth germs cultured in ovo on Millipore filters placed on chorioallantoic membrane was characterized by the presence of odontoblasts, ameloblasts, predentine, dentine and enamel. However, the flattening of these explants was identical with the changes of the explants cultured on Millipore filters in vitro. In ovo cultivation on the fibrin foam failed to bring satisfactory results.     

Immunocytochemical and biochemical detection of EMMPRIN in the rat tooth germ: differentiation-dependent co-expression with MMPs and co-localization with caveolin-1 in membrane rafts of dental epithelial cells

In tooth development matrix metalloproteinases (MMPs) are under the control of several regulatory mechanisms including the upregulation of expression by inducers and downregulation by inhibitors. The aim of the present study was to monitor the occurrence and distribution pattern of the extracellular matrix metalloproteinase inducer (EMMPRIN), the metalloproteinases MMP-2 and MT1-MMP and caveolin-1 during the cap and bell stage of rat molar tooth germs by means of immunocytochemistry. Strong EMMPRIN immunoreactivity was detected on the cell membranes of ameloblasts and cells of the stratum intermedium in the bell stage of the enamel organ. Differentiating odontoblasts exhibited intense EMMPRIN immunoreactivity, especially at their distal ends. Caveolin-1 immunoreactivity was evident in cells of the internal enamel epithelium and in ameloblasts. Double immunofluorescence studies revealed a focal co-localization between caveolin-1 and EMMPRIN in ameloblastic cells. Finally, western blotting experiments demonstrated the expression of EMMPRIN and caveolin-1 in dental epithelial cells (HAT-7 cells). A substantial part of EMMPRIN was detected in the detergent-insoluble caveolin-1-containing low-density raft membrane fraction of HAT-7 cells suggesting a partial localization within lipid rafts. The differentiation-dependent co-expression of MMPs with EMMPRIN in the enamel organ and in odontoblasts indicates that EMMPRIN takes part in the induction of proteolytic enzymes in the rat tooth germ. The localization of EMMPRIN in membrane rafts provides a basis for further investigations on the role of caveolin-1 in EMMPRIN-mediated signal transduction cascades in ameloblasts.     

Influence of colchicine on the addition of a sugar to the enamel protein in secretory ameloblasts of cultured germs of rat molar tooth by 3H-galactose radioautography

Summary The influence of colchicine on the addition of ³H-galactose to the enamel protein in secretory amelloblasts of cultured germs of rat molar tooth was investigated by light- and electron-microscopic radioautography. In tooth germs cultured without colchicine, the reaction products of ³H-galactose were observed over Golgi cisternae at early chase times and then localized over the enamel with time. In tooth germs cultured with colchicine, the silver grains were seen over the Golgi cisternae, condensing granules and accumulated secretory granules. Some grains also appeared with time over the pale granular material precipitated in the intercellular space with colchicine treatment. In quantitative analysis with light microscopic radioautography, values of silver grain counts over the unit area (100 m²) on ameloblasts and enamel of colchicine-treated tooth germs were significantly lower at both 0 min and 30 min chase after 30 min pulse than those of control tooth germs, respectively. This finding indicates that colchicine diminished the incorporation of ³H-galactose into the secretory ameloblast of cultured tooth germs. It is suggested that colchicine decreases the activity of the Golgi apparatus with regared to the addition of sugar to the synthesizing glycoprotein in the secretory ameloblast.     

Ultrastructure of secretory ameloblasts in the house musk shrew, Suncus murinus, Insectivora

Tooth germs from neonatal house musk shrews, Suncus murinus, were used for the study. The tooth morphogenesis was compared electron microscopically to that of Primates. In the tooth germ at the bell stage, the ameloblast was 3 x 50 microns in size, columnar in shape and had several tubular-type Golgi apparatus which were at the distal end of the cell. Most mitochondria were noted at the proximal end of ameloblasts. Tomes' processes were 1 micron in width, protruded 10 microns from the ameloblast and had many dense bodies and two kinds of vesicles. They were morphologically different from human ameloblasts and enamel rods.     

GTPases RhoA and Rac1 are important for amelogenin and DSPP expression during differentiation of ameloblasts and odontoblasts

Morphogenesis and cytodifferentiation are distinct processes in tooth development. Cell proliferation predominates in morphogenesis; differentiation involves changes in form and gene expression. The cytoskeleton is essential for both processes, being regulated by Rho GTPases. The aim of this study was to verify the expression, distribution, and role of Rho GTPases in ameloblasts and odontoblasts during tooth development in correlation with actin and tubulin arrangements and amelogenin and dentin sialophosphoprotein (DSPP) expression. RhoA, Rac1, and Cdc42 were strongly expressed during morphogenesis; during cytodifferentiation, RhoA was present in ameloblasts and odontoblasts, Rac1 and its effector Pak3 were observed in ameloblasts; and Cdc42 was present in all cells of the tooth germ and mesenchyme. The expression of RhoA mRNA and its effectors RockI and RockII, Rac1 and Pak3, as analyzed by real-time polymerase chain reaction, increased after ameloblast and odontoblast differentiation, according to the mRNA expression of amelogenin and DSPP. The inhibition of all Rho GTPases by Clostridium difficile toxin A completely abolished amelogenin and DSPP expression in tooth germs cultured in anterior eye chamber, whereas the specific inhibition of the Rocks showed only a partial effect. Thus, both GTPases are important during tooth morphogenesis. During cytodifferentiation, Rho proteins are essential for the complete differentiation of ameloblasts and odontoblasts by regulating the expression of amelogenin and DSPP. RhoA and its effector RockI contribute to this role. A specific function for Rac1 in ameloblasts remains to be elucidated; its punctate distribution indicates its possible role in exocytosis/endocytosis.     

Ultrastructural localization of osteocalcin in rat tooth germs by immunogold staining

Summary Osteocalcin was localized by indirect immunogold staining of thin frozen sections of rat tooth germs which had been fixed by different methods. Acrolein fixation proved to be satisfactory considering the preservation of fine structure and antigenicity. In odontoblasts, osteocalcin was found to be localized in the cisternae of the rough endoplasmic reticulum and Golgi apparatus. Few positive transport vesicles were found. Staining for osteocalcin in odontoblastic processes was only observed after strong fixation and was intense in odontoblasts engaged in early dentine formation. Predentine was slightly positive in the neighbourhood of positive processes. Matrix vesicles were negative and strong osteocalcin labeling of dentine seemed to appear after the onset of mineralization.     

Ultrastructural localization of osteocalcin in rat tooth germs by immunogold staining

Osteocalcin was localized by indirect immunogold staining of thin frozen sections of rat tooth germs which had been fixed by different methods. Acrolein fixation proved to be satisfactory considering the preservation of fine structure and antigenicity. In odontoblasts, osteocalcin was found to be localized in the cisternae of the rough endoplasmic reticulum and Golgi apparatus. Few positive transport vesicles were found. Staining for osteocalcin in odontoblastic processes was only observed after strong fixation and was intense in odontoblasts engaged in early dentine formation. Predentine was slightly positive in the neighbourhood of positive processes. Matrix vesicles were negative and strong osteocalcin labeling of dentine seemed to appear after the onset of mineralization.     

Possible involvement of maspin in tooth development

Maspin is a 42 kDa serine protease inhibitor that possesses tumor suppressive and anti-angiogenic activities. Despite of a huge amount of data concerning the expression pattern of maspin in various tissues and its relevance to the biological properties of a variety of human cancer cells, little is known on the maspin expression in skeletal and tooth tissues. Recently, we reported that maspin may play an important role in extracellular matrix formation in bone by enhancing the accumulation of latent TGF-β in the extracellular matrix. This study was performed to elucidate the possible role of maspin in tooth development. First, an immunohistochemical analysis for human tooth germs at the late bell stage showed the expression of maspin by active ameloblasts and odontoblasts that were forming enamel and dentin, respectively. During rat tooth development, maspin expression was observed for the first time in inner and outer enamel epithelial cells and dental papilla cells at early bell stage. The neutralizing anti-maspin antibody inhibited the proper dental tissue formation in organ cultures of mandibular first molars obtained from 21-day-old rat embryos. In addition, the proliferation of HAT-7 cells, a rat odontogenic epithelial cell line, and human dental papilla cells were suppressed in a dose-dependent manner with anti-maspin antibody. Moreover, RT-PCR analysis showed that the expression of mRNA for tooth-related genes including dentin matrix protein 1, dentin sialophosphoprotein and osteopontin in human dental papilla cells was inhibited when treated with anti-maspin antibody. These findings suggest that maspin expressed in ameloblasts and odontoblasts plays an important physiological role in tooth development through the regulation of matrix formation in dental tissues.     

Comparative enzyme histochemical studies on the development of teeth in man and mouse

Summary On the basis of a material consisting of primordia of deciduous teeth from 17 human foetuses and tooth germs from 20 newborn mice and 4 mouse foetuses, the histochemical distribution of succinic dehydrogenase, LDH, BDH, glucose-6-phosphate-dehydrogenase, NADH₂ cytochrome C reductase, and NADPH₂ diaphorase was studied.The investigation comprised partly mapping of the above-mentioned oxidative enzymes in human tooth germs during the period from bud stage to bell stage with incipient hard-tissue formation, and partly a comparative enzyme histochemical investigation of the dental development in man and in the mouse.In both materials there was an increase of all the demonstrated enzyme systems in the ameloblasts in early amelogenesis. On the other hand, the ameloblasts of the two materials showed differences during the period of matrix production: In the human ameloblasts there was activity of succinic dehydrogenase basally as well as apically, while the non-human ameloblasts showed such activity only basally.The odontoblasts of both materials showed a definite reaction for glucose-6-phosphatedehydrogenase, LDH, and BDH, but slight activity of succinic dehydrogenase. This indicates the existence of the pentose shunt as the source of energy in dentinogenesis. On the basis of the enzyme reactions studied, neither material showed a subodontoblastic layer or any reaction in the hard tissues.Material supplied by the Association for the Aid of the Crippled Children, New York. Aided by grants from the Danish State Research Foundation and the Rask-Ørsted Foundation.     

Localization of Beclin1 in mouse developing tooth germs: possible implication of the interrelation between autophagy and apoptosis

Our previous study identified the appearance of autophagy in developing tooth germs, and suggested its possible association with apoptosis in odontogenesis. Beclin1 was recently indicated to play a central role in bridging autophagy and apoptosis, and occupied a key position in the process of development. This study hypothesized that Beclin1 may be involved, and act as the molecular basis of the connection between autophagy and apoptosis in odontogenesis. Immunohistochemical analysis showed the spatiotemporal expression pattern of Beclin1 in odontogenesis from embryonic (E) day 13.5 to postnatal (P) day 5.5. At E stages, Beclin1 was mainly immunolocalized in the cytoplasm of the cells in the enamel organ. Meanwhile, the nucleus localization of Beclin1 was detected in part of the stellate reticulum, outer and inner enamel epithelium, especially at E16.5 and E18.5. At P stages, Beclin1 was detected in the cytoplasm of the odontoblasts, besides the dental epithelium cells. Triple immunofluorescence analysis showed the partial colocalization of Beclin1, autophagic marker LC3, or activated caspase-3 in the E14.5 tooth germs, especially the Beclin1⁺LC3⁺Caspase-3⁺ cells in the PEK. Furthermore, western blot analysis revealed that the full-length (60 kDa) and/or cleaved (50, 37, and 35 kDa) Beclin1 in the developing tooth germs. Taken together, our findings indicate that Beclin1 is involved, and might be responsible for the crosstalk between autophagy and apoptosis in mouse odontogenesis.     

Lectin-binding patterns in the developing tooth

Summary The lectin-binding patterns of the cells involved in amelogenesis and dentinogenesis in developing teeth of rats, were studied. Undifferentiated odontogenic epithelia exhibited very slight staining with almost all of the lectins examined. The lectin-staining affinities of secretory ameloblasts could be divided into two categories: Concanavalin-A (Con-A), Wheat germ agglutinin (WGA) and Soybean agglutinin (SBA) binding occurred from the middle to apical cytoplasm, whereas Ricinus communis agglutinin-I (RCA-I) and Ulex europeus I (UEA-I) binding predominated in the basal regions. The cells of the stratum intermedium exhibited relatively stranges lectin staining, which appeared to be dependent on ameloblastic maturation. The basement membranes in undifferentiated epithelia were markedly positive for lectin binding. Odontoblasts showed moderate Con-A staining on the apical side of the cells, as well as slight-to-moderate reactions with WGA and SBA. Pulp cells and dental papillae showed slight-to-moderate lectin staining, and predentin and dentin were also moderately positive for Con-A and RCA-I binding and slightly so for WGA and SBA. The lectin-binding affinities were enhanced during the formation of enamel and dentin, and appeared to be dependent on the degree of cellular differentiation in ameloblasts and odontoblasts.     

An ultrastructural study of dentinogenesis and amelogenesis in rat molar tooth germs cultured in vitro

Summary Molar tooth germs from three-day-old rats were cultured successfully for fourteen days, permitting the study of the development in vitro of both extracellular matrix and cellular elements such as odontoblasts and ameloblasts. The ultrastructure of the cultured tooth germs was compared with the ultrastructure of tooth germs in vivo at a comparable developmental stage. Progenitor cells of odontoblasts and ameloblasts were found to differentiate in vitro. Odontoblasts seemed to contain more lysosome-like bodies and fewer secretory granules than in vivo. They formed normally mineralizing dentine or a thick layer of dense, unmineralized predentine with incidentally some amorphous, extracellular material. Enamel was exclusively present opposite well developed dentine. It was often hyperor hypomineralized and enamel rods were not as regularly shaped as in vivo. In places where no enamel formation had taken place, large amounts of amorphous extracellular material were sometimes seen. From these observations it can be concluded that cellular development in cultured tooth germs appeared more or less normal, but extracellular matrix formation and mineralization were sometimes disturbed.     

Immunohistochemical localization of Pax6 in the developing tooth germ of mice

Recent studies have reported that supernumerary teeth were observed in the maxillary incisor area in several Pax6 homozygous mutant mouse and rat strains. To date, it remains unknown whether Pax6 is expressed during tooth development in any species. The study aimed to analyze the expression of Pax6 during mouse incisor and molar development. C57BL/6J mouse embryos on days E12.5, E13.5, E14.5, E16.5 and E18.5 were produced. Heads from these embryos, as well as from P1.5 mice, were processed for paraffin wax embedding (N ≥ 3 for each stage) and prepared for immunohistochemistry. Pax6 immunostaining was found in all tooth germs examined. At the E12.5 dental placode, E13.5 bud stage, E14.5 cap stage and E16.5 early bell stage, Pax6 was expressed in ectodermally derived tissues of tooth germs and oral epithelia adjacent to the tooth germs. Cells in the underlying dental ectomesenchyme that showed Pax9 expression were Pax6 negative. At E18.5 and P1.5, Pax6 was expressed in more differentiated ameloblasts and cells of the stratum intermedium and stellate reticulum that were derived from the oral epithelium, as well as in mesenchyme-derived differentiated odontoblasts. Pax6 expression was also observed in the submandibular gland, tongue filiform papilla and hair follicle at E16.5 and P1.5. The present study demonstrated that Pax6 was expressed in incisor and molar germs during mouse tooth development. The results provide a basis for exploring the function of Pax6 during tooth development.     

Effects of suramin,a polyanionic drug inducing lysosomal storage disorders on tooth germs in vitro

Summary— Suramin, a potent inhibitor of lysosomal enzymes, is commonly employed as a tool for inducing experimental mucopolysaccharidosis and lipidosis. The effects of the drug on embryonic mouse molars were analysed. Presecretory ameloblasts and odontoblasts were loaded with lysosome-like vacuoles. Staining with MC22-33F, an antibody to choline phospholipids and sphingomyelin, was completely reversed in the suramin-treated germs, in that it stained only presecretory ameloblasts (versus odontoblasts and some pulpal cells in the control group), according to a developmentally regulated pattern. The suramin-induced cytoplasmic changes were reminiscent of the features of mucopolysaccharidoses and lipidoses. The basement membrane, separating the enamel organ from the dental papilla, displayed suramin-induced patches, and in predentin collagen fibrillogenesis was found to be disturbed. Furthermore, autoradiography was employed to reveal uptake and distribution of [³H] suramin in the cells and predentin. Finally, a suramin-induced disturbance of the metabolism of sulphated macromolecules was found. The results imply that suramin effects in vitro on tooth germs can be used as a useful experimental model with to study both the action of the drug as well as cell and extracellular matrix perturbations in a mucopolysaccharidosis-like condition.     

Regulation of proliferation in developing human tooth germs by MSX homeodomain proteins and cyclin-dependent kinase inhibitor p19INK4d

Before the secretion of hard dental tissues, tooth germs undergo several distinctive stages of development (dental lamina, bud, cap and bell). Every stage is characterized by specific proliferation patterns, which is regulated by various morphogens, growth factors and homeodomain proteins. The role of MSX homeodomain proteins in odontogenesis is rather complex. Expression domains of genes encoding for murine Msx1/2 during development are observed in tissues containing highly proliferative progenitor cells. Arrest of tooth development in Msx knockout mice can be attributed to impaired proliferation of progenitor cells. In Msx1 knockout mice, these progenitor cells start to differentiate prematurely as they strongly express cyclin-dependent kinase inhibitor p19^INK4d. p19^INK4d induces terminal differentiation of cells by blocking the cell cycle in mitogen-responsive G1 phase. Direct suppression of p19^INK4d by Msx1 protein is, therefore, important for maintaining proliferation of progenitor cells at levels required for the normal progression of tooth development. In this study, we examined the expression patterns of MSX1, MSX2 and p19^INK4d in human incisor tooth germs during the bud, cap and early bell stages of development. The distribution of expression domains of p19^INK4d throughout the investigated period indicates that p19^INK4d plays active role during human tooth development. Furthermore, comparison of expression domains of p19^INK4d with those of MSX1, MSX2 and proliferation markers Ki67, Cyclin A2 and pRb, indicates that MSX-mediated regulation of proliferation in human tooth germs might not be executed by the mechanism similar to one described in developing tooth germs of wild-type mouse.     

Itm2a Expression in the Developing Mouse First Lower Molar,and the Subcellular Localization of Itm2a in Mouse Dental Epithelial Cells

Itm2a is a type II transmembrane protein with a BRICHOS domain. We investigated the temporospatial mRNA and protein expression patterns of Itm2a in the developing lower first molar, and examined the subcellular localization of Itm2a in murine dental epithelial (mDE6) cells. From the initiation to the bud stage, the in situ and protein signals of Itm2a were not detected in either the dental epithelial or mesenchymal cells surrounding the tooth bud. However, at the bell stage, these signals of Itm2a were primarily observed in the inner enamel epithelium of the enamel organ. After the initiation of the matrix formation, strong signals were detected in ameloblasts and odontoblasts. Itm2a showed a punctate pattern in the cytoplasm of the mDE6 cells. The perinuclear-localized Itm2a displayed a frequent overlap with the Golgi apparatus marker, GM130. A tiny amount of Itm2a was colocalized with lysosomes and endoplasmic reticulum. Minimal or no overlap between the Itm2a-EGFP signals with the other organelle markers for endoplasmic reticulum, lysosome and mitochondria used in this study noted in the cytoplasm. These findings suggest that Itm2a may play a role in cell differentiation during odontogenesis, rather than during the initiation of tooth germ formation, and may be related to the targeting of proteins associated with enamel and dentin matrices in the secretory pathway.