In situ expression of the mitochondrial ATPase6 gene in the developing tooth germ of the mouse lower first molar

We previously performed cDNA subtraction between the mouse mandibles on embryonic day 10.5 (E10.5) in the pre-initiation stage of the odontogenesis and E12.0 in the late initiation stage to identify genes expressed at its beginning. Adenosine triphosphate synthase subunit a (Atpase6) is one of the highly expressed genes in the E12.0 mandible including tooth germs. In situ hybridization was conducted using the mouse mandibular first molar from E10.5 to E18.0 to determine the precise expression patterns of Atpase6 mRNA in the developing tooth germ. Atpase6 mRNA was strongly expressed in the presumptive dental epithelium and the underlying mesenchyme at E10.5, and in the thickened dental epithelium at E12.0 and E13.0. Strong in situ signals were observed in the epithelium at E14.0, and in the enamel organ excluded the area of the primary enamel knot at E15.0. Atpase6 was strongly expressed in the inner enamel epithelium, the adjacent stratum intermedium, and the outer enamel epithelium in the cervical loops from E16.0 to E18.0. In addition, strong Atpase6 signals were coincidently demonstrated in various developing cranio-facial organs. These results suggest that Atpase6 participates in the high energy-utilizing functions of the cells related to the initiation and the development of the tooth germ as well as those of the other cranio-facial organs.     

In situ expression of 15 kDa interferon alpha responsive gene in the developing tooth germ of the mouse lower first molar

We previously performed cDNA subtraction between the mouse mandibles at embryonic day 10.5 (E10.5) and E12.0 to make a profile of the regulator genes for odontogenesis. Fifteen kDa interferon alpha responsive gene (Ifrg15) is one of several highly-expressed genes in the E12.0 mandible. The current study examined the precise expression patterns of Ifrg15 mRNA in the mouse mandibular first molar by in situ hybridization to evaluate the possible functional roles of this gene in odontogenesis. Ifrg15 mRNA was expressed in the epithelial and mesenchymal tissues of the mandible at E10.5 and E12.0. The Ifrg15 in situ signal was detected in the epithelial bud and the surrounding mesenchyme at E14.0, and was present in the enamel organ including the primary enamel knot, and in the underlying mesenchyme at E15.0. The in situ signal was restricted in the inner and outer enamel epithelia and the stratum intermedium at E16.0. The signal of Ifrg15 mRNA was further restricted to the inner enamel epithelium and the adjacent stratum intermedium at E17.0 and E18.0. Consequently, the expression of Ifrg15 mRNA was localized in the ameloblasts and odontoblasts at postnatal days 1.0 to 3.0. However, the in situ signal was markedly weaker than at the embryonic period. The expression of Ifrg15 mRNA was coincidently observed in various craniofacial organs as well as in the tooth germ. These results suggest that Ifrg15 is closely related to odontogenesis, especially the differentiation of the ameloblasts and odontoblasts, and to the morphogenesis of the craniofacial organs.     

Possible functional involvement of thymosin beta 4 in developing tooth germ of mouse lower first molar

We examined the detailed in situ expression pattern of thymosin beta 4 (Tβ4) in the developing mouse mandibular first molar. Tβ4 mRNA was expressed in the presumptive dental epithelium at embryonic day 10.5 (E10.5) and in the thickened dental epithelium at E12. An in situ signal was observed in the invaginated epithelial bud at E13, in the enamel organ at E14 and E14.5, and in the primary enamel knot (PEK) at E14.5. The signal was localized in the epithelial cells of the outer layer of the enamel organ at E15 and E15.5. No signal was found in the PEK at these stages. Tβ4 mRNA was expressed in the inner enamel epithelium, cervical loop and dental lamina at E16 and E17. The expression of Tβ4 mRNA was observed in the polarized inner epithelial cells at E18, newborn day 1 (N1) and N2. However, the signal intensity decreased markedly at N3. We herein report for the first time that Tβ4 is distinctly expressed in developing tooth germ, and it may also play functional roles in the initiation, growth and differentiation of tooth germ.     

Expression of Set-α during Morphogenesis of Mouse Lower First Molar

The detailed in situ expression pattern of the Set-α gene has been studied. Previously we showed that Set-α is a differentially expressed gene in the embryonic mouse mandible at day 10.5 (E10.5) gestational age. Cells expressing Set-α were widely distributed in both the epithelial and underlying ectomesenchymal cells at E10.5. At E12, they were slightly aggregated in an area where tooth germ of the lower first molar is estimated to be formed. At E13.5, Set-α was strongly expressed in the tooth germ. At the cap stage, Set-α was expressed in the enamel organ and dental papilla. At the bell stage, Set-α was distinctly expressed in the inner enamel epithelial and dental papilla cells facing the inner enamel epithelial layer, which were intended to differentiate into ameloblasts and odontoblasts, respectively. Interestingly, Set-α was also expressed in several embryonic craniofacial tissues derived from the ectoderm. This study is the first report that Set-α is distinctly expressed in the developing tooth germ, and suggests that Set-α plays an important role in both the initiation and the growth of the tooth germ, as well as in the differentiation of ameloblasts and odontoblasts.     

Expression of TNF-receptor-associated factor genes in murine tooth development

Tumor necrosis factor receptor-associated factors (TRAFs) belong to a family of intracellular adaptor proteins that mediate signaling downstream of various cell surface receptors. We carried out comparative in situ hybridization analysis of five Traf genes Traf1, Traf2, Traf3, Traf4 and Traf6 during murine odontogenesis from the formation of the epithelial thickening to the early bell stage. Traf2, Traf3 and Traf6 showed weak expression in the thickened epithelium. Expression of Traf1, Traf2 and Traf6 were observed in the outer edges of the bud epithelium whereas Traf3 was strongly expressed at the tip of the bud epithelium. Expression of Traf1, Traf4 and Traf6 were detected in the dental papilla mesenchyme. Traf2 showed restricted expression in the internal enamel epithelium of the bell stage while expression of Traf1, Traf3, Traf4 and Traf6 were observed in both the internal and the external enamel epithelium. During early odontogenesis, all five genes show dynamic spatiotemporal expression patterns.     

Expression of BMP2/4/7 during the odontogenesis of deciduous molars in miniature pig embryos

Bone morphogenetic proteins (BMPs) play important roles in tooth development. However, their expression has not been studied in miniature pigs, which have many anatomical similarities in oral and maxillofacial region compared to human. This study investigated BMP2/4/7 expression patterns during deciduous molar development in miniature pigs on embryonic days (E) 40, 50, and 60. The mandibles were fixed, decalcified, and embedded before sectioning. H&E staining, immunohistochemistry, in situ hybridization using specific radionuclide-labeled cRNA probes, and real-time PCR were used to detect the BMP expression patterns during morphogenesis of the third deciduous molar. H&E staining showed that for the deciduous third molar, E40 represented the cap stage, E50 represented the early bell stage, and E60 represented the late bell stage or secretory stage. BMP2 was expressed in both the enamel organ and in the dental mesenchyme on E40 and E50 and was expressed mainly in pre-odontoblasts on E60. BMP7 expression was similar to BMP2 expression, but BMP7 was also expressed in the inner enamel epithelium on E60. On E40, BMP4 was expressed mainly in the epithelium, with some weak expression in the mesenchyme. On E50, BMP4 expression was stronger in the mesenchyme but weaker in the epithelium. On E60, BMP4 was expressed mainly in the mesenchyme. These data indicated that BMP2/4/7 showed differential spatial and temporal expression during the morphogenesis and odontogenesis of deciduous molars, suggesting that these molecules were associated with tooth morphogenesis and cell differentiation.     

In situ expression of heat shock proteins,Hsc73, Hsj2 and Hsp86 in the developing tooth germ of mouse lower first molar

This study examined the detailed gene expression pattern of three different heat shock proteins (HSPs), Hsc73, Hsj2, and Hsp86, by means of an in situ hybridization method. Hsc73, Hsj2, and Hsp86 were shown in our previous study to be differentially expressed in the mouse embryonic mandible at day 10.5 (E10.5) gestational age. These HSP genes showed similar expression patterns during development of the mouse lower first molar. HSPs-expressing cells were widely distributed in both the epithelial and underlying ectomesenchymal cells at E10.5, and then were slightly localized at E12 in an area where the tooth germ of the lower first molar is estimated to be formed. A strong expression of HSPs was observed in the tooth germ at E13.5. At the cap stage, HSPs were expressed in the enamel organ and dental papilla. At the bell stage, HSPs were distinctly expressed in the inner enamel epithelium and dental papilla cells facing the inner enamel epithelial layer, which later differentiate into ameloblasts and odontoblasts, respectively. This study is the first report in which Hsc73, Hsj2, and Hsp86 were distinctly expressed in the developing tooth germ, thus suggesting these HSPs are related to the development and differentiation of odontogenic cells.     

Expression of Wnt signalling pathway genes during tooth development.

We have carried out comparative in situ hybridisation analysis of six Wnt genes Wnts-3, -4, -5a, -6, -7b, and 10b together with Wnt receptor MFz6 and receptor agonist/antagonists MFrzb1 and Mfrp2 during murine odontogenesis from the earliest formation of the epithelial thickening to the early bell stage. Expression of Wnt-4, Wnt-6, and one Wnt receptor MFz6 was observed in the facial, oral and dental epithelium. Wnt10b was localised specifically to the presumptive dental epithelium. Wnts-3 and -7b were expressed in oral epithelium but showed no expression in the presumptive dental epithelium. Wnt-3 also showed no expression in the epithelial cells of the molar bud stage tooth germs, but showed restricted expression in the enamel knots which are signalling centres believed to be involved in regulating tooth shape. Wnts -6, -10b and MFz6 were also detected in the primary and secondary enamel knots. Wnt-5a and agonist/antagonists MFrzb1 and Mfrp2 were expressed in a graded proximo-distal (P-D) manner in mesenchymal cells during the early stages of tooth development with no overlying expression in the oral or dental epithelium. Wnt-5a and MFrzb1 show strong expression in the dental papilla mesenchyme.     

The Drosophila Fry protein interacts with Trc and is highly mobile in vivo

Background

Protogenin (Prtg) has been identified as a gene which is highly expressed in the mouse mandible at embryonic day 10.5 (E10.5) by a cDNA subtraction method between mandibles at E10.5 and E12.0. Prtg is a new member of the deleted in colorectal carcinoma (DCC) family, which is composed of DCC, Neogenin, Punc and Nope. Although these members play an important role in the development of the embryonic central nervous system, recent research has also shed on the non-neuronal organization. However, very little is known regarding the fetal requirement of the non-neuronal organization for Prtg and how this may be associated with the tooth germ development. This study examined the functional implications of Prtg in the developing tooth germ of the mouse lower first molar.

Results

Ptrg is preferentially expressed in the early stage of organogenesis. Prtg mRNA and protein were widely expressed in the mesenchymal cells in the mandible at E10.5. The oral epithelial cells were also positive for Prtg. The expression intensity of Prtg after E12.0 was markedly reduced in the mesenchymal cells of the mandible, and was restricted to the area where the tooth bud was likely to be formed. Signals were also observed in the epithelial cells of the tooth germ. Weak signals were observed in the inner enamel epithelial cells at E16.0 and E18.0. An inhibition assay using a hemagglutinating virus of Japan-liposome containing Prtg antisense-phosphorothioated-oligodeoxynucleotide (AS-S-ODN) in cultured mandibles at E10.5 showed a significant growth inhibition in the tooth germ. The relationship between Prtg and the odontogenesis-related genes was examined in mouse E10.5 mandible, and we verified that the Bmp-4 expression had significantly been decreased in the mouse E10.5 mandible 24 hr after treatment with Prtg AS-S-ODN.

Conclusion

These results indicated that the Prtg might be related to the initial morphogenesis of the tooth germ leading to the differentiation of the inner enamel epithelial cells in the mouse lower first molar. A better understanding of the Prtg function might thus play a critical role in revealing a precious mechanism in tooth germ development.     

Pleiotrophin expression during odontogenesis

Pleiotrophin (PTN) is an extracellular matrix-associated growth factor and chemokine expressed in mesodermal and ectodermal cells. It plays an important role in osteoblast recruitment and differentiation. There is limited information currently available about PTN expression during odontoblast differentiation and tooth formation, and thus the authors aimed to establish the spatiotemporal expression pattern of PTN during mouse odontogenesis. Immortalized mouse dental pulp (MD10-D3, MD10-A11) and odontoblast-like (M06-G3) and ameloblast-like (EOE-3M) cell lines were grown and samples prepared for immunocytochemistry, Western blot, and conventional and quantitative PCR analysis. Effects of BMP2, BMP4, and BMP7 treatment on PTN expression in odontoblast-like M06-G3 cells were tested by quantitative PCR. Finally, immunohistochemistry of sectioned mice mandibles and maxillaries at developmental stages E16, E18, P1, P6, P10, and P28 was performed. The experiments showed that PTN, at both the mRNA and protein level, was expressed in all tested epithelial and mesenchymal dental cell lines and that the level of PTN mRNA was influenced differentially by the bone morphogenetic proteins. The authors observed initial expression of PTN in the inner enamel epithelium with prolonged expression in the ameloblasts and odontoblasts throughout their stages of maturation and strong expression in the terminally differentiated and enamel matrix-secreting ameloblasts and odontoblasts of the adult mouse incisors and molars.     

Spatial and temporal expression of histone demethylase,Kdm2a,during murine molar development

The histone demethylase, lysine (K)-specific demethylase 2A (Kdm2a), is highly conserved and expressed ubiquitously. Kdm2a can regulate cell proliferation and osteo/dentinogenic, adipogenic and chondrogenic differentiation of mesenchymal stem cells (MSCs) derived from dental tissue. We used quantitative real-time RT-PCR analysis and immunohistochemistry to detect Kdm2a expression during development of the murine molar at embryonic days E12, E14, E16 and E17 and postnatal days P3 and P14. Immunohistochemistry results showed no positive staining of Kdm2a at E12. At E14, Kdm2a was expressed weakly in the inner enamel epithelium, stellate reticulum cells and dental sac. At E16, Kdm2a was expressed mainly in the inner and outer enamel epithelium, stratum intermedium and dental sac, but weaker staining was found in cervical loop and dental papilla cells adjacent to the basement membrane. At E17, the strongest Kdm2a staining was detected in the ameloblasts and stronger Kdm2a staining also was detected in the stratum intermedium, outer enamel epithelium and dental papilla cells compared to the expression at E16. Postnatally, we found that Kdm2a was localized in secretory and mature ameloblasts and odontoblasts, and dentin was unstained. Real-time RT-PCR showed that Kdm2a mRNA levels in murine germ cells increased from E12 to E14 and from E14 to E16; no significant change occurred at E16, E17 or P3, then the levels decreased at P14 compared to P3. Kdm2a expression may be closely related to cell proliferation, to ameloblast and odontoblast differentiation and to the secretion of extracellular enamel and dentin during murine tooth development.     

Immunolocalization of BMP-2/-4, FGF-4, and WNT10b in the developing mouse first lower molar.

Intercellular signaling controls all steps of odontogenesis. The purpose of this work was to immunolocalize in the developing mouse molar four molecules that play major roles during odontogenesis: BMP-2, -4, FGF-4, and WNT10b. BMP-2 and BMP-4 were detected in the epithelium and mesenchyme at the bud stage. Staining for BMP-2 markedly increased at the cap stage. The relative amount of BMP-4 strongly increased from E14 to E15. At E15, BMP-4 was detected in the internal part of the enamel knot where apoptosis was intense. In contrast to TGFbeta1, BMP-2 and -4 did not show accumulation at the epithelial-mesenchymal junction where the odontoblast started differentiation. When odontoblasts became functional, BMP-2 and BMP-4 were detected at the apical and basal poles of preameloblasts. BMP-2, which induces ameloblast differentiation in vitro, may also be involved physiologically. The decrease in FGF-4 from E14 to E15 supports a possible role for the growth factor in the control of mesenchymal cell proliferation. The relative amount of FGF-4 was maximal at E17. The subsequent decrease at E19 showed correlation with the withdrawal of odontoblasts and ameloblasts from the cell cycle. WNT10b might also stimulate cell proliferation. At E14-15, WNT10b was present in the mesenchyme and epithelium except for the enamel knot, where the mitotic activity was very low. At E19 there was a decreasing gradient of staining from the cervical loop where cells divide to the tip of the cusp in the inner dental epithelium where cells become postmitotic. The target cells for FGF-4 and WNT10b appeared different.     

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.     

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.     

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.