Late Bell Stage Research Articles

Bivalent Histone Codes on WNT5A during Odontogenic Differentiation

Lineage-committed differentiation is an essential biological program during odontogenesis, which is tightly regulated by lineage-specific genes. Some of these genes are modified by colocalization of H3K4me3 and H3K27me3 marks at promoter regions in progenitors. These modifications, named “bivalent domains,” maintain genes in a poised state and then resolve for later activation or repression during differentiation. Wnt5a has been reported to promote odontogenic differentiation in dental mesenchyme. However, relatively little is known about the epigenetic modulations on Wnt5a activation during tooth development. Here, we investigated the spatiotemporal patterns of H3K4me3 and H3K27me3 marks in developing mouse molars. Associated H3K4me3 methylases (mixed-lineage leukemia [MLL] complex) and H3K27me3 demethylases (JMJD3 and UTX) were dynamically expressed between early and late bell stage of human tooth germs and in cultured human dental papilla cells (hDPCs) during odontogenic induction. Poised WNT5A gene was marked by bivalent domains containing repressive marks (H3K27me3) and active marks (H3K4me3) on promoters. The bivalent domains tended to resolve during inducted differentiation, with removal of the H3K27me3 mark in a JMJD3-dependent manner. When JMJD3 was knocked down in cultured hDPCs, odontogenic differentiation was suppressed. The depletion of JMJD3 epigenetically repressed WNT5A activation by increased H3K27me3 marks. In addition, JMJD3 could physically interact with ASH2L, a component of the MLL complex, to form a coactivator complex, cooperatively modulating H3K4me3 marks on WNT5A promoters. Overall, our study reveals that transcription activities of WNT5A were epigenetically regulated by the negotiated balance between H3K27me3 and H3K4me3 marks and tightly mediated by JMJD3 and MLL coactivator complex, ultimately modulating odontogenic commitment during dental mesenchymal cell differentiation.

Spatiotemporal Expression of Wnt/β-catenin Signaling during Morphogenesis and Odontogenesis of Deciduous Molar in Miniature Pig.

The canonical Wnt/β-catenin signaling pathway has been shown to play essential roles in tooth initiation and early tooth development. However, the role of Wnt/β-catenin signaling in cusp patterning and crown calcification in large mammals are largely unknown. In our previous study, miniature pigs were used as the animal model due to the similarity of tooth anatomy and replacement pattern between miniature pig and human. Dynamic gene expression of third deciduous molar (DM3) in miniature pig at early stages was profiled using microarray method and expression of Wnt genes was significantly correlate with odontogenesis. In the present study, dynamic expression patterns of Wnt/β-catenin signaling genes of DM3 at cap, early bell and late bell (secretory) stage were identified. We found that Lef1 and Axin2 were expressed in the enamel knot and underlying mesenchyme regions. Meanwhile, Dkk1 was expressed in the peripheral and lower parts of dental papilla, thus forming the potential Wnt signaling gradient. We also found that β-Catenin, Axin2 and Lef1 were expressed strongly in undifferentiated cells of the inner enamel epithelium (IEE), but weakly in differentiated ameloblasts. Furthermore, we found that both Wnt signaling read-out gene Lef1 and the inhibitor Dkk1 were co-expressed in the pre-odontoblasts. In conclusion, the spatiotemporal distribution and potential gradient of Wnt signaling may contribute to cusp patterning and crown calcification. These data may yield insight into future study of precise control of crown morphogenesis and regeneration in large mammals.

Spatial signalling mediated by the transforming growth factor-β signalling pathway during tooth formation

Tooth development relies on sequential and reciprocal interactions between the epithelial and mesenchymal tissues, and it is continuously regulated by a variety of conserved and specific temporal-spatial signalling pathways. It is well known that suspensions of tooth germ cells can form tooth-like structures after losing the positional information provided by the epithelial and mesenchymal tissues. However, the particular stage in which the tooth germ cells start to form tooth-like structures after losing their positional information remains unclear. In this study, we investigated the reassociation of tooth germ cells suspension from different morphological stages during tooth development and the phosphorylation of Smad2/3 in this process. Four tooth morphological stages were designed in this study. The results showed that tooth germ cells formed odontogenic tissue at embryonic day (E) 14.5, which is referred to as the cap stage, and they formed tooth-like structures at E16.5, which is referred to as the early bell stage, and E18.5, which is referred to as the late bell stage. Moreover, the transforming growth factor-β signalling pathway might play a role in this process.

Involvement of IGF-2, IGF-1R, IGF-2R and PTEN in development of human tooth germ – an immunohistochemical study

ABSTRACTInsulin-Like Growth Factor 2 (IGF-2) is a peptide hormone essential for prenatal growth and development. IGF-2 exerts its mitogenic effects via Insulin-Like Growth Factor 1 Receptor (IGF-1R), and is eliminated by binding to Insulin-Like Growth Receptor 2 (IGF-2R). IGF-2 is also negatively regulated by Phosphatase and Tensin Homolog (PTEN), a phosphatase mutated in various tumors. Not much is known about the interplay between these factors during human odontogenesis. In this study, expression patterns of IGF-2, IGF-1R, IGF-2R and PTEN were analyzed by double immunofluorescence in incisor human tooth germs during the foetal period of development between the 7th and 20th gestational week. Throughout the investigated period, IGF-2 was mostly expressed in enamel organ, whereas mild to moderate expression of PTEN could be seen in dental papilla and parts of enamel organ. Expression of IGF-1R was ubiquitous and displayed strong intensity throughout the entire enamel organ. In contrast, expression of IGF-2R had rather erratic pattern in enamel organ and dental papilla alike. Expression patterns of IGF-2, IGF-1R, IGF-2R and PTEN in highly proliferative cervical loops, as well as in differentiating pre-ameloblasts and pre-odontoblasts of cusp tip region during the early and late bell stages when enamel organ acquires definitive shape, indicate importance of these factors in crown morphogenesis of human incisor. Taken together, our data suggest the involvement of IGF-2, IGF-1R, IGF-2R and PTEN in temporo-spatial patterning of basic cellular processes (proliferation, differentiation) during normal tooth development. They are also relevant for improving knowledge of molecular basis of human odontogenesis.

Hyaluronan and hyaluronan synthases expression and localization in embryonic mouse molars.

Hyaluronan (HA) and hyaluronan synthases (HASs) have been shown to play critical roles in embryogenesis and organ development. However, there have not been any studies examining HA and HAS expression and localization during tooth development. The present study was designed to investigate the expression of HA and three isoforms of HASs (HAS1, 2, 3) in embryonic mouse molars. The first mandibular embryonic mouse molars were examined by immunohistochemistry at E11.5, E13.5, E14.5, E16.5, and E18.5. PCR and western blot analyses were performed on RNA and proteins samples from E13.5 to E18.5 tooth germs. At the initial stage (E11.5), HA and HASs were expressed in the dental epithelium but not the underlying dental mesenchyme. HA immunostaining gradually increased in the enamel organ from the bud stage (E13.5) to the late bell stage (E18.5), and HA and HASs were highly expressed in the stellate reticulum and stratum intermedium. HA immunostaining was also enhanced in the dental mesenchyme and its derived tissues, but it was not expressed in the ameloblast and odontoblast regions. The three HAS isoforms had distinct expression patterns, and they were expressed in the dental mesenchyme and odontoblast at various levels. Furthermore, HAS1 and HAS2 expression decreased, while HAS3 expression increased from E13.5 to E18.5. These results suggested that HA synthesized by different HASs is involved in embryonic mouse molar morphogenesis and cytodifferentiation.

Facioscapulohumeral muscular dystrophy (FSHD) region gene 1 (FRG1) expression and possible function in mouse tooth germ development.

Abnormal expression of Facioscapulohumeral muscular dystrophy (FSHD) region gene 1 (FRG1) is involved in the pathogenesis of FSHD. FRG1 is also important for the normal muscular and vascular development. Our previous study showed that FRG1 is one of the highly expressed genes in the mandible on embryonic day 10.5 (E10.5) than on E12.0. In this study, we investigated the temporospatial expression pattern of FRG1 mRNA and protein during the development of the mouse lower first molar, and also evaluated the subcellular localization of the FRG1 protein in mouse dental epithelial (mDE6) cells. The FRG1 expression was identified in the dental epithelial and mesenchymal cells at the initiation and bud stages. It was detected in the inner enamel epithelium at the cap and early bell stages. At the late bell and root formation stages, these signals were detected in ameloblasts and odontoblasts during the formation of enamel and dentin matrices, respectively. The FRG1 protein was localized in the cytoplasm in the mouse tooth germ in vivo, while FRG1 was detected predominantly in the nucleus and faintly in the cytoplasm in mDE6 cells in vitro. In mDE6 cells treated with bone morphogenetic protein 4 (BMP4), the protein expression of FRG1 increased in cytoplasm, suggesting that FRG1 may translocate to the cytoplasm. These findings suggest that FRG1 is involved in the morphogenesis of the tooth germ, as well as in the formation of enamel and dentin matrices and that FRG1 may play a role in the odontogenesis in the mouse following BMP4 stimulation.

Gastrin-releasing peptide expression and its effect on the calcification of developing mouse incisor.

Gastrin-releasing peptide (GRP) is considered to be one of the cancer growth factors. This peptide's receptor (GRPR) is known as a G protein-coupled receptor, regulating intracellular calcium storage and releasing signals. This study is the first to investigate the function of GRP during mouse incisor development. We hypothesized that GRP is one of the factors that affects the regulation of calcification during tooth development. To verify the expression pattern of GRP, in situ hybridization was processed during incisor development. GRP was expressed at the late bell stage and hard tissue formation stage in the epithelial tissue. To identify the genuine function of GRP during incisor development, a gain-of-function analysis was performed. After GRP overexpression in culture, the phenotype of ameloblasts, odontoblasts and predentin was altered compared to control group. Moreover, enamel and dentin thickness was increased after renal capsule transplantation of GRP-overexpressed incisors. With these results, we suggest that GRP plays a significant role in the formation of enamel and dentin by regulating ameloblasts and predentin formation, respectively. Thus, GRP signaling is strongly related to calcium acquisition and secretion during mouse incisor development.

Expression and roles of syndecan-4 in dental epithelial cell differentiation.

Syndecan-4 (SDC4), a transmembrane heparan sulfate proteoglycan, acts as a signal transducer. It affects the growth and differentiation of a number of tissues and organs. However, the specific mechanisms through which SDC4 regulates the differentiation of dental epithelial cells (amelogenesis) and tooth development remains largely unknown. In the present study, to identify the SDC4-regulated processes in dental epithelial cells, the SDC4 expression pattern was examined in mouse molar and postnatal incisor tooth germs during the late bell stage of development. Small interfering RNA (siRNA) was designed for this study and used to downregulate SDC4 expression in the rat dental epithelial cell line, HAT-7. The results revealed that SDC4 was mainly present in the oral epithelium, the dental epithelial cells of enamel organs in the molars and the cervical loops in the incisors. When the inner enamel epithelial cells gave rise to ameloblasts, however, the loss of SDC4 expression was evident. SDC4 was also expressed in stratum intermedium(SI) cells in the incisors and in dental mesenchymal cells adjacent to the cervical loops in molars (E18) and postnatal incisors. Fibroblast growth factor10 (FGF10) promoted proliferation and slightly decreased cell differentiation. The knockdown of SDC4 using specific siRNA led to a decrease in cell proliferation and a highly significant increase in amelogenin, ameloblastin, kallikrein4 and matrix metalloproteinase20 expression, molecules that are known to participate in the formation of enamel. These effects were attenuated by FGF10, which upregulated SDC4 expression. Taken together, these results suggest that SDC4 participates in amelogenesis, and FGF10 may modulate dental epithelial cell behaviors through the regulation of SDC4 expression.

Construction of a cDNA library for miniature pig mandibular deciduous molars.

BackgroundThe miniature pig provides an excellent experimental model for tooth morphogenesis because its diphyodont and heterodont dentition resembles that of humans. However, little information is available on the process of tooth development or the exact molecular mechanisms controlling tooth development in miniature pigs or humans. Thus, the analysis of gene expression related to each stage of tooth development is very important.ResultsIn our study, after serial sections were made, the development of the crown of the miniature pigs’ mandibular deciduous molar could be divided into five main phases: dental lamina stage (E33-E35), bud stage (E35-E40), cap stage (E40-E50), early bell stage (E50-E60), and late bell stage (E60-E65). Total RNA was isolated from the tooth germ of miniature pig embryos at E35, E45, E50, and E60, and a cDNA library was constructed. Then, we identified cDNA sequences on a large scale screen for cDNA profiles in the developing mandibular deciduous molars (E35, E45, E50, and E60) of miniature pigs using Illumina Solexa deep sequencing. Microarray assay was used to detect the expression of genes. Lastly, through Unigene sequence analysis and cDNA expression pattern analysis at E45 and E60, we found that 12 up-regulated and 15 down-regulated genes during the four periods are highly conserved genes homologous with known Homo sapiens genes. Furthermore, there were 6 down-regulated and 2 up-regulated genes in the miniature pig that were highly homologous to Homo sapiens genes compared with those in the mouse.ConclusionOur results not only identify the specific transcriptome and cDNA profile in developing mandibular deciduous molars of the miniature pig, but also provide useful information for investigating the molecular mechanism of tooth development in the miniature pig.

Morphometric approach to pulp fibroblast development in tooth germ.

This paper builds a morphometric framework for the analysis of dental pulp fibroblast evolution during tooth development. We investigated 15 tooth germs (cases) organized, by histological criteria, in three groups corresponding to cap, early bell, and late bell stages, respectively. Each group comprised five cases. The morphometric description used the following parameters: area (A), perimeter (P)—automatically extracted by a color segmentation technique, and form factor (FF)—calculated as 4πA/P 2. The designed framework operated at inter- and intragroup levels. The intergroup analysis quantified the differences between groups, in the sense of a relative distance (RD) adequately defined by mean-value scaling. We showed that the stage of early bell is approximately 5 times closer to late bell than to cap. The quantification procedure required concomitant information about A, P parameters (as P versus A dependences, or FF values), whereas the procedure failed for A or P separately used. The intragroup analysis quantified the similarity of the cases belonging to the same stage. We proved that, unlike the intergroup tests, the individual exploitation of all three descriptors A, P, and FF is effective, yielding highly compatible results. Within any group, most cases presented RDs less than 10% from the group mean value, regardless of the descriptor type.

Morphology and chronology of diphyodont dentition in miniature pigs, Sus Scrofa

There is little knowledge about the tooth replacement in large mammals. The aim of this study is to investigate the tooth replacement patterns in Chinese miniature pig (Sus Scrofa). The developmental patterns of mandibular successional and additional teeth from Chinese miniature pig before and after birth were investigated by microanatomy, immunohistochemistry, and cone beam computed tomography. Secondary dental lamina for successional teeth was not visible until its predecessor progressed to late bell stage. Successional teeth reached early cap stage when their predecessor began to erupt. The development patterns and speed varied between anterior and posterior successional teeth. Additional molars, derived from the free end of additional dental lamina, initiated sequentially in mandible ramus, while previous additional molar progressed into late bell stage. Proliferating cells in the permanent primordium were distributed asymmetrically. Our findings identify the characteristic patterns about spatiotemporal morphogenesis of successional teeth in context of their predecessor and cascade initiation of additional molars in miniature pigs. Our study provides a basis toward better understanding the mechanisms underlying diphyodont replacement in human and also assists in tooth regeneration and tooth engineering in large animal.

The expression and function of thymosin beta 10 in tooth germ development

This study presents the expression pattern and functions of thymosin beta 10 (Tbeta10), a Tbeta4 homologue during the development of mouse lower first molars. An in situ signal of Tbeta10 was detected on embryonic day 10.5 (E10.5)-E15.5 mainly in dental mesenchymal cells as well as in dental epithelial cells, while Tbeta4 was expressed in dental epithelial cells. In the late bell stage, preodontoblasts with strong Tbeta10 expression and preameloblasts with strong Tbeta4 expression exhibited face-to-face localization, suggesting that an intimate cell-cell interaction might exist between preodontoblasts and preameloblasts to form dentin and enamel matrices. A strong Tbeta10 signal was found in odontoblasts in the lateral side of the dental pulp and in Hertwig’s epithelial root sheath, thus suggesting that Tbeta10 participates in the formation of the outline of the tooth root. An inhibition assay using Tbeta10-siRNA in E11.0 mandibles showed significant growth inhibition in the tooth germ. The Tbeta10-siRNA-treated E15.0 tooth germ also showed significant developmental arrest. The number of Ki67-positive cells significantly decreased in the Tbeta10-siRNA-treated mandibles. The cellular proliferative activity was also significantly suppressed in Tb10-siRNA-treated cultured mouse dental pulpal and epithelial cells. These results indicate that developmental arrest of the tooth germ might be caused by a reduction in cell proliferative activity. The stage-specific temporal and spatial expression pattern of Tbeta10 in the developing tooth germ is indicative of multiple functions of Tbeta10 in the developmental course from initiation to root formation of the tooth germ.

MicroRNAome and Expression Profile of Developing Tooth Germ in Miniature Pigs

MicroRNAs (miRNAs) play important roles in the regulation of rodent tooth development, but little is known about their role in tooth development in large mammals. We identified 637 unique miRNA sequences in a large-scale screen for miRNA expression profiles in the developing lower deciduous molars of miniature pigs (Sus scrofa) using Illumina Solexa deep sequencing. These candidate miRNAs and another 105 known Sus scrofa miRNAs were included in the custom-designed microarray and used to analyze the miRNA expression profile in the bud, cap, early bell, and late bell stages of tooth development. Microarray analysis revealed 166 transcripts that were differentially expressed in the four stages. Bioinformatic analysis identified 18 key miRNAs, including let-7f, miR-128, miR-200b, and miR-200c, that might play key roles in tooth development. Taken together, our results not only identified the specific microRNAome and expression profile in developing lower deciduous molars of the miniature pig, but they also provided useful information for investigating the molecular mechanism of tooth development in the miniature pig.

MicroRNA miR-34a Regulates Cytodifferentiation and Targets Multi-signaling Pathways in Human Dental Papilla Cells

Odontogenesis relies on the reciprocal signaling interactions between dental epithelium and neural crest-derived mesenchyme, which is regulated by several signaling pathways. Subtle changes in the activity of these major signaling pathways can have dramatic effects on tooth development. An important regulator of such subtle changes is the fine tuning function of microRNAs (miRNAs). However, the underlying mechanism by which miRNAs regulate tooth development remains elusive. This study determined the expression of miRNAs during cytodifferentiation in the human tooth germ and studied miR-34a as a regulator of dental papilla cell differentiation. Using microarrays, miRNA expression profiles were established at selected times during development (early bell stage or late bell stage) of the human fetal tooth germ. We identified 29 differentially expressed miRNAs from early bell stage/late bell stage comparisons. Out of 6 miRNAs selected for validation by qPCR, all transcripts were confirmed to be differentially expressed. miR-34a was selected for further investigation because it has been previously reported to regulate organogenesis. miR-34a mimics and inhibitors were transfected into human fetal dental papilla cells, mRNA levels of predicted target genes were detected by quantitative real-time PCR, and levels of putative target proteins were examined by western blotting. ALP and DSPP expression were also tested by qPCR, western blotting, and immunofluorescence. Findings from these studies suggested that miR-34a may play important roles in dental papilla cell differentiation during human tooth development by targeting NOTCH and TGF-beta signaling.

The expression pattern of FHL2 during mouse molar development

Four and a half LIM domains 2 (FHL2) functions as a transcriptional co-activator or co-repressor in a cell-type-specific manner. As a positive regulator, FHL2 plays an important role in osteoblast differentiation and bone formation. Our previous study showed that FHL2 was expressed in odontoblasts in mature human teeth under normal and pathological conditions. The purpose of this study was to investigate the spatial-temporal expression patterns of FHL2 at different stages of mouse molar development by immunohistochemistry. Our results showed that at the bud and cap stage, FHL2 was expressed both in enamel organ and the underlying mesenchyme. At the early bell stage, FHL2 appeared in the inner and outer enamel epithelium, stratum intermedium and the secondary enamel knot. Positive staining gradually converged at the cusps of dental papilla. At the late bell stage, FHL2 was expressed in the terminal differentiated ameloblasts and odontoblasts and stratum intermedium. At the postnatal day, FHL2 was detected in the secretory and mature ameloblasts and odontoblasts and mature enamel, and gradually appeared at Hertwig's epithelial root sheath and periodontal tissues. The spatial-temporal expression patterns of FHL2 from the bud stage to the postnatal day (13.5) suggested that during tooth development, FHL2 might play an important role in ameloblast and odontoblast differentiation, secretion of enamel and dentin matrix, mineralization of enamel, molar crown morphogenesis, as well as root formation.

Glucose uptake mediated by glucose transporter 1 is essential for early tooth morphogenesis and size determination of murine molars

Glucose is an essential source of energy for body metabolism and is transported into cells by glucose transporters (GLUTs). Well-characterized class I GLUT is subdivided into GLUTs1–4, which are selectively expressed depending on tissue glucose requirements. However, there is no available data on the role of GLUTs during tooth development. This study aims to clarify the functional significance of class I GLUT during murine tooth development using immunohistochemistry and an in vitro organ culture experiment with an inhibitor of GLUTs1/2, phloretin, and Glut1 and Glut2 short interfering RNA (siRNA). An intense GLUT1-immunoreaction was localized in the enamel organ of bud-stage molar tooth germs, where the active cell proliferation occurred. By the bell stage, the expression of GLUT1 in the dental epithelium was dramatically decreased in intensity, and subsequently began to appear in the stratum intermedium at the late bell stage. On the other hand, GLUT2-immunoreactivity was weakly observed in the whole tooth germs throughout all stages. The inhibition of GLUTs1/2 by phloretin in the bud-stage tooth germs induced the disturbance of primary enamel knot formation, resulting in the developmental arrest of the explants and the squamous metaplasia of dental epithelial cells. Furthermore, the inhibition of GLUTs1/2 in cap-to-bell-stage tooth germs reduced tooth size in a dose dependent manner. These findings suggest that the expression of GLUT1 and GLUT2 in the dental epithelial and mesenchymal cells seems to be precisely and spatiotemporally controlled, and the glucose uptake mediated by GLUT1 plays a crucial role in the early tooth morphogenesis and tooth size determination.

Expression patterns of histone acetyltransferases p300 and CBP during murine tooth development

The transcription coactivators p300 and CBP were reported to have indispensable roles in various tissues during the embryonic and fetal stages. Little is known about the activities of these coactivators during the tooth development, so we investigated the temporal expression patterns of p300 and CBP in developing mouse tooth germs. The first molars of fetal BALB/c mice were used to investigate p300 and CBP expression patterns. Immunohistochemistry was performed on a series of sections from embryonic Day(E) 11.5 to E19.5. Laser microdissection and real-time polymerase chain reactions were used to investigate the inner enamel epithelium, stellate reticulum, outer enamel epithelium, and dental papilla between E18.5 and E19.5. P300 had a presence in nearly every region of the tooth germ from E11.5 to E19.5. However, the expression of CBP was first detected at E12.5 and was relatively lower than the expression of p300 until E18.5. Both p300 and CBP expression levels peaked unexpectedly on DayE18.5. Additionally, the expression of p300 was distinctly lower than CBP at E19.5. In the late bell stage, p300 and CBP mRNA was highly expressed in the stellate reticulum and dental papilla, and there was a relatively low amount of CBP mRNA in the outer enamel epithelia and inner enamel epithelia from E18.5 to E19.5. The temporal patterns of p300 and CBP expression were identified during tooth development. The difference in the spatiotemporal expression of p300 and CBP indicated that they play different roles in tooth development.

Amelogenin and enamelysin localization in human dental germs

Odontogenesis is extensively studied in animal models but less understood in human. In early amelogenesis, amelogenin constitutes 90% of enamel organic matrix, which is degraded by enamelysin and replaced by hydroxyapatite crystals. Here, amelogenin and enamelysin distribution changes during amelogenesis were shown by co-localization experiments by confocal microscopy. Early bell stage showed more amelogenin labeling than enamelysin, as free immune-reactive granular patches towards basal membrane between ameloblast and odontoblast. Increased amelogenin expression and secretion towards extracellular matrix formation region was found. Enamelysin distribution was perinuclear in early bell stage. During late bell stage, a decreasing amelogenin labeling in contrast with enamelysin increasing along the cells was found, suggesting specific temporal amelogenin degradation. Enamelysin was located initially around nuclei and later was found in all the ameloblast and stellate reticulum cytoplasm. Amelogenin was observed inside ameloblast, stellate reticulum, and intermediate stratum cells in the enamel as well as in the newly formed dentin extracellular matrix. In contrast, in dentin more amelogenin than enamelysin was found located close to the periphery.

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.

Early morphogenesis of heterodont dentition in minipigs

The minipig provides an excellent experimental model for tooth morphogenesis because its diphyodont and heterodont dentition resemble that of humans. However, little information is available on the processes of tooth development in the pig. The purpose of this study was to classify the early stages of odontogenesis in minipigs from the initiation of deciduous dentition to the late bell stage when the successional dental lamina begins to develop. To analyze the initiation of teeth anlagens and the structural changes of dental lamina, a three-dimensional (3D) analysis was performed. At the earliest stage, 3D reconstruction revealed a continuous dental lamina along the length of the jaw. Later, the dental lamina exhibited remarkable differences in depth, and the interdental lamina was shorter. The dental lamina grew into the mesenchyme in the lingual direction, and its inclined growth was underlined by asymmetrical cell proliferation. After the primary tooth germ reached the late bell stage, the dental lamina began to disintegrate and fragmentize. Some cells disappeared during the process of lamina degradation, while others remained in small islands known as epithelial pearls. The minipig can therefore, inter alia, be used as a model organism to study the fate of epithelial pearls from their initiation to their contribution to pathological structures, primarily because of the clinical significance of these epithelial rests.