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.     

In situ expression of ribosomal protein L21 in 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) in the pre-initiation stage of the odontogenesis and E12.0 in the late initiation stage to investigate the key regulator genes in odontogenesis. Ribosomal protein L21 (Rpl21) is one of differentially expressed genes in the E12.0 mandible. This study examined the precise expression pattern of Rpl21 mRNA in the mouse mandibular first molar by in situ hybridization. Rpl21 mRNA was expressed in the presumptive dental epithelium and the underlying mesenchyme at E10.5, and in the thickened dental epithelium at E12.0. Strong in situ signals were observed in the epithelial bud at E14.0, and in the enamel organ at E15.0. However, either no (E14.0) or only a weak (E15.0) in situ signal was found in the primary enamel knot at these gestational days. Rpl21 was strongly expressed in the inner enamel epithelium, cervical loop and dental lamina from E16.0 to E18.0. In addition, Rpl21 mRNA was also demonstrated in various developing cranio-facial organs. These results suggest that Rpl21 participates in the synthesis of various polypeptides which might be related to the initiation and the development of such tooth germ, and also in the synthesis of enamel components in the presecretory stage of the ameloblast. Rpl21 for protein synthesis might also be related to the morphogenesis of the developing cranio-facial organs.     

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.     

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.     

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 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.     

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.     

Functional implication of nucleolin in the mouse first molar development

We examined the functional implication of nucleolin in the mouse first molar development. Both the nucleolin mRNA and protein expressions were demonstrated in the odontogenic epithelial cells in the early stage and in the inner enamel epithelial layer in the late stage. The expression pattern of nucleolin corresponded to the proliferating cells in the tooth germ, thus showing that nucleolin could possibly be related to cell proliferation. No in situ signal of nucleolin was found in the primary enamel knot (PEK). Furthermore, nucleolin protein was demonstrated in the PEK by immunohistochemistry. The existence of nucleolin protein in the PEK may possibly be related to the apoptosis in the PEK cells. An inhibition assay using the hemagglutinating virus of Japan-liposome containing nucleolin antisense phosphorothioated oligonucleotide (AS S-ODN) in cultured mouse mandibles at embryonic day (E) 11.0 showed a marked growth inhibition of tooth germ. Moreover, no developmental arrest was found in the cultured tooth germ at E15.0 treated with nucleolin AS S-ODN. Real time PCR was performed to examine the mRNA expression of nucleolin-related genes, and a significant reduction in the midkine mRNA expression was thus observed in the mouse mandible after being treated with nucleolin AS S-ODN. This inhibition assay indicated that nucleolin could thus be involved in the early stage of tooth germ initiation and morphogenesis, possibly by regulating the midkine expression.     

Glycolytic enzyme Pgk1 is strongly expressed in the developing tooth germ of the mouse lower first molar

This study examined detailed in situ expression patterns and possible functional roles of phosphoglycerate kinase 1 (Pgk1) gene in the developing tooth germ of the mouse lower first molar. The strong expression of Pgk1 mRNA was seen in the odontogenic epithelial cells and surrounding mesenchymal cells of the tooth germ from embryonic day 10.5 (E10.5) to E18.0. Western blotting analysis demonstrated that Pgk1 protein formed 84-kDa protein complex in these embryonic organs. The results of immunoprecipitation-western blotting also suggested this complex to be formed with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Moreover, the immunofluorescence expression of those proteins was shown to overlap each other in the tooth germ at E15.0. A strong immunofluorescence expression of both Pgk1 and GAPDH also corresponded to the in situ expression of those mRNAs. These results suggested that Pgk1 plays some functional roles in the development of tooth germ and other embryonic organs by forming protein complex with GAPDH.     

Genome-wide analysis of gene expression in human embryonic tooth germ

While numerous genes that play important regulatory roles during tooth development in mice have been identified, little is known about gene expression profile and their function during human odontogenesis. To unveil expression profile of odontogenic genes in humans, we conducted genome-wide gene expression analysis by microarray assays to analyze differential gene expression between tooth germ and lip tissue from 11-week old human fetuses. We identified 167 genes that are strongly expressed in the cap stage tooth germ as compared to the lip tissue. Among them, 145 genes were further identified by gene ontology enrichment analysis that are highly represented in multiple gene ontology classes, include extracellular components, sequence-specific DNA binding proteins, Wnt-protein binding molecules, system development, organogenesis, and cell differentiation. Sixty-seven genes that are known to be associated with mammalian tooth development and tooth abnormalities were identified. Real-time PCR was further employed to validate microarray data. Moreover, in situ hybridization assay demonstrated tooth type specific expression of ISL1 and BARX1 in the incisor, canine, and molar respectively, consistent with microarray results. Our results represent a set of reliable data that could provide a solid base for future elaboration of molecular mechanisms underlying human tooth development.     

Expression of Set-alpha during morphogenesis of mouse lower first molar

The detailed in situ expression pattern of the Set- gene has been studied. Previously we showed that Set- is a differentially expressed gene in the embryonic mouse mandible at day 10.5 (E10.5) gestational age. Cells expressing Set- 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- was strongly expressed in the tooth germ. At the cap stage, Set- was expressed in the enamel organ and dental papilla. At the bell stage, Set- 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- was also expressed in several embryonic craniofacial tissues derived from the ectoderm. This study is the first report that Set- is distinctly expressed in the developing tooth germ, and suggests that Set- 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.     

EMMPRIN (basigin/CD147) is involved in the morphogenesis of tooth germ in mouse molars

The pattern of gene expression for extracellular matrix metalloproteinase inducer (EMMPRIN) was revealed in the tooth germ of mouse mandibular molars using quantitative real-time PCR. In situ hybridization and immunohistochemical study demonstrated the characteristic distribution of EMMPRIN in the different stages of tooth germ development. To investigate the functional role played by EMMPRIN in tooth germ development, EMMPRIN siRNA interference approach was carried out in cultured mouse mandibles at embryonic day 11.0 (E11.0). The results showed that EMMPRIN siRNA-treated explants exhibited a marked growth inhibition of tooth germ compared to the control and scrambled siRNA-treated explants. Meanwhile, a significant increase in MT1-MMP mRNA expression and a reduction in MMP-2, MMP-3, MMP-9, MMP-13 and MT2-MMP mRNA expression were observed in the mouse mandibles following EMMPRIN abrogation. The current results indicate that EMMPRIN could thus be involved in the early stage of tooth germ development and morphogenesis, possibly by regulating the expression of MMP genes.     

EGF antisense oligodeoxynucleotides block murine odontogenesis in vitro

The initiation of odontogenesis depends on the site-specific proliferation of mandibular epithelium beginning at Day 11 in embryonic mice. We have previously reported that the local expression of epidermal growth factor mRNA in the murine mandible is developmentally regulated, expressed at Days 9 and 10 immediately prior to the initiation of tooth bud formation at Day 11. Exposure of Day 9 mandibular explants to antisense oligomers of epidermal growth factor blocks the initiation of odontogenesis. These results are the first demonstration of the involvement of epidermal growth factor in the inductive specification of a complex epithelial derivative.     

Determination of genes involved in the early process of molar root development initiation in rat by modified subtractive hybridization

Rat molars and incisors have different root patterning mechanisms: the former form a multirooted pattern while the latter form a single-rooted analogue. But the genetic signals that control root patterning are poorly understood. In this study, to identify the special molecular signals which may lead to the molar root development, we firstly observed that at embryonic day 19 the molar and the incisor began differentially developing: the molar formed double-layered cells of the root sheath while the incisor formed a cervical loop. By using the subtractive hybridization method, we successfully constructed a subtraction cDNA library of the rat molar and incisor tissues. Differentially expressed gene clones were evaluated by dot-blot and sequencing. Sel1l, Nfic, Edar, GATA6, and some novel genes were found differentially expressed, which were strongly related the tooth root patterning. It is anticipated that further study of genes identified will provide insights into their specific roles in the tooth root patterning.