Murine Molar Tooth Research Articles

Expression of Clu and Tgfb1 during murine tooth development: effects of in‐vivo transfection with anti‐miR‐214

Expression of clusterin (Clu) in the murine first molar tooth germ was markedly increased at postnatal developmental stages. The time-course of expression of this gene paralleled those of other genes encoding proteins involved during the secretory phase of odontogenesis, as described previously. Immunohistochemical studies of clusterin in murine molar tooth germs suggested this protein to be located in outer enamel epithelium, regressing enamel organ, secretory ameloblasts, and the dental epithelium connecting the tooth to the oral epithelium at an early eruptive stage. Immunolabelling of transforming growth factor beta-1 (TGF-β1) revealed it to be located close to clusterin. The levels of expression of Clu and Tgfb1 were markedly decreased following in-vivo transfection with anti-miR-214. In contrast, the expression of several genes associated with regulation of growth and development were increased by this treatment. We suggest that clusterin has functions during secretory odontogenesis and the early eruptive phase. Bioinformatic analysis after treatment with anti-miR-214 suggested that, whilst cellular activities associated with tooth mineralization and eruption were inhibited, activities associated with an alternative developmental activity (i.e. biosynthesis of contractile proteins) appeared to be stimulated. These changes probably occur through regulation mediated by a common cluster of transcription factors and support suggestions that microRNAs (miRNAs) are highly significant as regulators of differentiation during odontogenesis.

MiR-145 and miR-143 Regulate Odontoblast Differentiation through Targeting Klf4 and Osx Genes in a Feedback Loop

Dentin tissue is derived from mesenchymal cells induced into the odontoblast lineage. The differentiation of odontoblasts is a complex process regulated by several transcriptional factor signaling transduction pathways. However, post-translational regulation of these factors during dentinogenesis remains unclear. To further explore the mechanisms, we investigated the role of microRNA (miRNA) during odontoblast differentiation. We profiled the miRNA expression pattern during mouse odontoblast differentiation using a microarray assay and identified that miR-145 and miR-143 were down-regulated during this process. In situ hybridization verified that the two miRNAs were gradually decreased during mouse odontoblast differentiation. Loss-of-function and gain-of-function experiments revealed that down-regulation of miR-145 and miR-143 could promote odontoblast differentiation and increased Dspp and Dmp1 expression in mouse primary dental pulp cells and vice versa. We found that miR-145 and miR-143 controlled odontoblast differentiation through several mechanisms. First, KLF4 and OSX bind to their motifs in Dspp and Dmp1 gene promoters and up-regulate their transcription thereby inducing odontoblast differentiation. The miR-145 binds to the 3'-UTRs of Klf4 and Osx genes, inhibiting their expression. Second, KLF4 repressed miR-143 transcription by binding to its motifs in miR-143 regulatory regions as detected by ChIP assay and dual luciferase reporter assay. Third, miR-143 regulates odontoblast differentiation in part through miR-145 pathway. Taken together, we for the first time showed that the miR-143 and miR-145 controlled odontoblast differentiation and dentin formation through KLF4 and OSX transcriptional factor signaling pathways.

Role of perlecan, a basement membrane-type heparan sulfate proteoglycan, in enamel organ morphogenesis

Abstract Perlecan is a multifunctional heparan sulfate proteoglycan that controls cell-signaling events by interacting with several growth factors, cytokines, and other signaling molecules. Perlecan was thought to be localized only in the basement membrane, but recently, intraepithelial localization of perlecan has been demonstrated in some pathophysiological situations. Therefore, perlecan is expected to modulate epithelial cell behavior. Our recent study demonstrated that perlecan accumulates in the stellate reticulum of the enamel organ of murine molar tooth germs in a stage-specific manner. To understand the function of perlecan in the enamel organ, we generated transgenic ( Tg ) mice that overexpressed perlecan in epithelial cells by using the keratin 5 promoter. Perlecan Tg molars had dull-ended crowns and outward-curved tooth roots, and their enamel was poorly crystallized. The constant overexpression of perlecan and the accompanying disorganized distribution of perlecan-related molecules in the enamel organ resulted in irregular tooth morphology. These results indicate that the time schedule of intraepithelial perlecan expression appears to be critically controlled during enamel organ development. In this brief review, we have described the dynamics of perlecan and its receptors and the timing of cleavage of heparan sulfate chains in odontogenesis, focusing on enamel organ development, and discussed the role of perlecan in enamel organ morphogenesis.

FGF-9 accelerates epithelial invagination for ectodermal organogenesis in real time bioengineered organ manipulation

BackgroundEpithelial invagination is important for initiation of ectodermal organogenesis. Although many factors regulate ectodermal organogenesis, there is not any report about their functions in real-time study. Electric cell-substrate impedance sensing (ECIS), a non-invasive, real-time surveillance system, had been used to detect changes in organ cell layer thickness through quantitative monitoring of the impedance of a cell-to-microelectrode interface over time. It was shown to be a good method for identifying significant real-time changes of cells. The purpose of this study is to establish a combined bioengineered organ-ECIS model for investigating the real time effects of fibroblast growth factor-9 (FGF-9) on epithelial invagination in bioengineered ectodermal organs. We dissected epithelial and mesenchymal cells from stage E14.5 murine molar tooth germs and identified the real-time effects of FGF-9 on epithelial-mesenchymal interactions using this combined bioengineered organ-ECIS model.ResultsMeasurement of bioengineered ectodermal organ thickness showed that Fibroblast growth factor-9 (FGF-9) accelerates epithelial invagination in reaggregated mesenchymal cell layer within 3 days. Gene expression analysis revealed that FGF-9 stimulates and sustains early Ameloblastin and Amelogenin expression during odontogenesis.ConclusionsThis is the first real-time study to show that, FGF-9 plays an important role in epithelial invagination and initiates ectodermal organogenesis. Based on these findings, we suggest FGF-9 can be applied for further study in ectodermal organ regeneration, and we also proposed that the ‘FGF-BMP balancing system’ is important for manipulating the morphogenesis of ectodermal organs. The combined bioengineered organ-ECIS model is a promising method for ectodermal organ engineering and regeneration research.

MicroRNAs during Murine Molar Tooth Development

MicroRNAs (miRNAs) may have essential functions as regards regulation of gene expression, and are likely involved also during development. We have mapped miRNAs during development of the murine first molar tooth germ. The miRNA expression profile was found highly dynamic. During late odontogenesis miR‐214 is highly expressed, and was selected for further studies using antagmomir‐214. Antagomir‐214 was given as mandibular injections (0–100 pmol) at postnatal day 1 (P0). First molar tooth germs were removed at various times. Levels of miRNAs and mRNAs were measured using microarrays. A dosage of 50 pg was found to be optimal. At 24 h after injection the level of miR‐214 was about 0.02‐fold of control level, the level of expression returning close to the control at 72 h. Using a dosage of antagomir‐214 of 50 pmol about 1200 mRNAs and 8 miRNAs were differentially expressed in the tooth germ 24 hrs after injection of antagomir‐214. The microarray results were validated using RT‐PCR. Western‐blotting data was in agreement with changes mRNA levels. Differentially expressed genes were subjected to bioinformatic analysis, suggesting that cellular functions and signaling pathways were significantly altered. We conclude that miR‐214 may have significant regulatory functions during late odontogenesis. Project funding from Dental Faculty, University of Oslo.

Effects of in vivo transfection with anti-miR-214 on gene expression in murine molar tooth germ

MicroRNAs (miRNAs) are an abundant class of noncoding RNAs that are believed to be important in many biological processes through regulation of gene expression. Little is known of their function in tooth morphogenesis and differentiation. MicroRNA-214 (miR-214), encoded by the polycistronic Dnm30os gene, is highly expressed during development of molar tooth germ and was selected as a target for silencing with anti-miR-214. Mandibular injection of 1-100 pmol of anti-miR-214 close to the developing first molar in newborn mice resulted in significant decrease in expression of miR-214, miR-466h, and miR-574-5p in the tooth germ. Furthermore, levels of miR-199a-3p, miR-199a-5p, miR-690, miR-720, and miR-1224 were significantly increased. Additionally, the expression of 863 genes was significantly increased and the expression of 305 genes was significantly decreased. Among the genes with increased expression was Twist-1 and Ezh2, suggested to regulate expression of miR-214. Microarray results were validated using real-time RT-PCR and Western blotting. Among genes with decreased expression were Amelx, Calb1, Enam, and Prnp; these changes also being reflected in levels of corresponding encoded proteins in the tooth germ. In the anti-miR-214-treated molars the enamel exhibited evidence of hypomineralization with remnants of organic material and reduced surface roughness after acid etching, possibly due to the transiently decreased expression of Amelx and Enam. In contrast, several genes encoding contractile proteins exhibited significantly increased expression. mRNAs involved in amelogenesis (Ambn, Amelx, Enam) were not found among targets of miRNAs that were differentially expressed following treatment with anti-miR-214. It is therefore suggested that effects of miR-214 on amelogenesis are indirect, perhaps mediated by the observed miR-214-dependent changes in levels of expression of numerous transcription factors.

Heparanase, heparan sulfate and perlecan distribution along with the vascular penetration during stellate reticulum retraction in the mouse enamel organ

Objective Immunohistochemical and gene expression profiles of heparanase were determined in murine molar tooth germs from their embryonic to postnatal stages, paying special attention to neovascularization within the enamel organ, which is poorly vascularized before birth. Design Protein and gene expression profiles of heparanase, heparan sulfate (HS), vascular endothelial growth factor (VEGF), transforming growth factor-β1 (TGF-β1), and perlecan were comparatively examined by immunohistochemistry and in-situ hybridization, respectively, in mouse mandibular molar tooth germs from embryonic day 11.5 to postnatal day 6. At the same time, their mRNA expression levels were also confirmed by reverse transcriptase-polymerase chain reaction using laser-captured microdissection of enamel organ tissues. Results Stellate reticulum cells highly expressed perlecan but only slightly expressed heparanase and HS in their embryonic days. On and after postnatal day 1, the expressions of heparanase became dramatically higher in the stellate reticulum, while HS disappeared leaving the immunopositivity for perlecan core protein. Immunohistochemically, HS was enhanced around blood vessels which were newly formed after birth within the enamel organs, whose volume was also regressive. Similar expression patterns were obtained for VEGF and TGF-β1. Conclusions Such synchronized expression modes among the HS metabolism-related molecules suggested that heparanase plays an important role in degradation of HS chains, which is closely related to vascular penetration into the stellate reticulum, which may be one of the driving forces for the postnatal regression of the enamel organ.

MicroRNA expression profiling of the developing murine molar tooth germ and the developing murine submandibular salivary gland

Using microarrays, miRNA expression profiles have been established at selected times during development (E15.5, P0 and P5) of the murine first molar mandibular tooth germ and the right submandibular salivary gland (E15.5, P0, P5 and P25). Microarray data was validated using real-time PCR, also facilitating RT-PCR profiling of nine selected miRNAs. In general, good agreement between microarray data and real-time PCR data was found. Further, miRNA expression profiles of foetal and adult liver were also investigated, and found to agree with published data. In tooth germ and salivary gland up to 88 different miRNAs were detected. In all tissues examined miRNA expression was highly dynamic; miRNA profiles changing extensively with time of development. Additionally, the expression of some miRNAs was tissue-specific. Bioinformatic analysis of clusters of miRNAs was attempted using the miRGate software, the results suggesting miRNAs to be involved in the regulation of essential developmental processes, e.g., epithelical cell proliferation, mesodermal cell fate determination and salivary gland morphogenesis.

Changes in gene-expression during development of the murine molar tooth germ

In a matter of a few days the murine tooth germ develops into a complex, mineralized, structure. Murine 30 K microarrays were used to examine gene expression in the mandibular first molar tooth germs isolated at 15.5 dpc and at 2 DPN. Microarray results were validated using real-time RT-PCR. The results suggested that only 25 genes (3 without known functions) exhibited significantly higher expression at 15.5 dpc compared to 2 DPN. In contrast, almost 1400 genes exhibited significantly ( P < 0.015) higher expression at 2 DPN compared to 15.5 dpc, about half of which were genes with unknown functions. More than 50 of the 783 known genes exhibited higher than 10-fold increase in expression at 2 DPN, amongst these were genes coding for enamel matrix proteins which were expressed several 100-fold higher at 2 DPN. GO and KEGG analysis showed highly significant associations between families of the 783 known genes and cellular functions relating to energy metabolism, protein metabolism, regulation of cell division, cell growth and apoptosis. The use of bioinformatics analysis therefore yielded a functional profile in agreement with known differences in tissue morphology and cellular composition between these two stages. Such data is therefore useful in directing attention towards genes, or cellular activities, which likely are worthy of further studies as regards their involvement in odontogenesis.

Developmentally regulated changes in phospholipid composition in murine molar tooth.

In order to explore the possibility that phospholipids are differently expressed during the cascade of events leading to tooth formation, we decided to carry out simultaneous biochemical, histological and electron histochemical studies. High performance thin-layer chromatography and gas-liquid chromatography were used to compare the composition of embryonic mouse first molar tooth germs at day 18 of gestation (E18) and at birth (D1), erupting teeth at day 7 (D7) and erupted molars at day 21 (D21). For the latter, non-demineralized and EDTA-demineralized lipid extracts were analysed separately. Moreover, an ultrahistochemical study was carried out using the iodoplatinate reaction which retains and visualizes phospholipids. Developmentally regulated changes occurred and were closely correlated with an increase in cell membrane phospholipids. Gradual accumulation of phospholipids was identified in the extracellular matrix, at an early stage of tooth germ development within the basement membrane and later, as predentine/dentine and enamel components participating in mineralization processes. Matrix vesicles transiently present in dentine were partly responsible for the lipids that were detected. A first group of phospholipids including phosphatidylcholine as the major membrane-associated phospholipid and phosphatidylinositol as the intracellular second messenger increased by a factor of 2.3 between E18 and D21. This increase is probably associated with cell lengthening and was relatively modest compared with the higher increase detected for a second group of phospholipids, namely phosphatidylethanolamine (x4.8), phosphatidylserine (x 5.9) and sphingomyelin (x5.4). This second group of extracellular matrix-associated phospholipids constituted 68% of the demineralized lipid extract and, therefore, contributes to the mineralization of dental tissues.

Msx1 controls inductive signaling in mammalian tooth morphogenesis.

Members of the Msx homeobox family are thought to play important roles in inductive tissue interactions during vertebrate organogenesis, but their precise developmental function has been unclear. Mice deficient for Msx1 exhibit defects in craniofacial development and a failure of tooth morphogenesis, with an arrest in molar tooth development at the E13.5 bud stage. Because of its potential for experimental manipulation, the murine molar tooth germ provides a powerful system for studying the role of Msx genes in inductive signaling during organogenesis. To further analyze the role of Msx1 in regulating epithelial-mesenchymal interactions during tooth morphogenesis, we have examined the expression of several potential Msx1 downstream genes in Msx1 mutant tooth germs and we have performed functional experiments designed to order these genes into a pathway. Our results show that expression of Bone Morphogenetic Protein 4 (BMP4), the HMG box gene Lef1 and the heparan sulfate proteoglycan syndecan-1 is specifically reduced in Msx1 mutant dental mesenchyme, while expression of the extracellular matrix protein tenascin is unaffected. BMP4 soaked beads can induce Bmp4 and Lef1 expression in explanted wild-type dental mesenchymes, but only Lef1 expression in Msx1 mutant dental mesenchyme. We thus conclude that epithelial BMP4 induces its own expression in dental mesenchyme in a manner that requires Msx1. In turn, we show that addition of BMP4 to Msx1 deficient tooth germs bypasses the requirement for Msx1 and rescues epithelial development from the bud stage to the E14.5 cap stage. Lastly, we show that FGFs induce syndecan-1 expression in dental mesenchyme in a manner that also requires Msx-1. These results integrate Msx1 into a regulatory hierarchy in early tooth morphogenesis and demonstrate that Msx1 is not only expressed in dental mesenchyme in response to epithelial signals, but also in turn regulates the reciprocal expression of inductive signals in the mesenchyme which then act back upon the dental epithelium. We propose that Msx genes function repetitively during vertebrate organogenesis to permit inductive signaling to occur back and forth between tissue layers.

Role of two mineral-associated adhesion molecules, osteopontin and bone sialoprotein, during cementogenesis.

Adhesion molecules and their cell membrane receptors are known to play important regulatory roles in cell differentiation. Consequently, the following experiments were conducted to determine the role of two adhesion molecules, bone sialoprotein (BSP) and osteopontin (OPN) in tooth root formation. Developing murine molar tooth germs at sequential stages of development (developmental days 21-42) were analyzed using immunohistochemical and in situ hybridization techniques. While BSP was localized to alveolar bone and odontoblasts early in development, BSP was distinctly localized to the cemental root surface at latter periods coincident with the initiation of root formation and cementogenesis. Conversely, OPN was distributed in a nonspecific fashion throughout the PDL and the eruption pathway of the forming tooth. In situ hybridization confirmed that cells lining the root surface express BSP. The fact that BSP is specifically localized to the cemental surface suggests that this protein is involved in cementoblast differentiation and/or early mineralization of the cementum matrix. Localization of OPN to non-mineralized tissues further suggests that OPN functions as an inhibitor of mineralization during periodontal ligament formation. These findings collectively suggest that BSP and OPN are intimately involved in the sequence of cellular and molecular events accompanying cementogenesis.

Epithelial-mesenchymal interactions are required for msx 1 and msx 2 gene expression in the developing murine molar tooth

Duplication of the msh-like homeobox gene of Drosophila may be related to the evolution of the vertebrate head. The murine homologues of this gene, msx 1 and msx 2 are expressed in the developing craniofacial complex including the branchial arches, especially in regions of epithelial-mesenchymal organogenesis including the developing tooth. By performing in vitro recombination experiments using homochronic dental and non-dental epithelial and mesenchymal tissues from E10 to E18 mouse embryos, we have found that the maintenance of homeobox gene expression in the tooth is dependent upon tissue interactions. In homotypic recombinants, dental-type tissue interactions occur, leading to expression of both genes in a manner similar to that seen during in vivo development. msx 1 is expressed exclusively in mesenchyme, both in the dental papilla and follicle. msx 2 is expressed in the dental epithelium and only in the mesenchyme of the dental papilla. In heterotypic recombinants, the dental epithelium is able to induce msx 1 expression in non-dental mesenchyme, this potential being lost at the bell stage. In these recombinants msx 2 was induced by presumptive dental epithelium prior to the bud stage but not thereafter. The expression of msx 1 and msx 2 in dental mesenchyme requires the presence of epithelium until the early bell stage. However, whereas non-dental, oral epithelium is capable of maintaining expression of msx 1 in dental mesenchyme throughout tooth development, induction of msx 2 was temporally restricted suggesting regulation by a specific epithelial-mesenchymal interaction related to the inductive events of tooth formation. msx 1 and msx 2, as putative transcription factors, may play a role in regulating the expression of other genes during tooth formation. We conclude that expression of msx 1 in jaw mesenchyme requires a non-specific epithelial signal, whereas msx 2 expression in either epithelium or mesenchyme requires reciprocal interactions between specialized dental cell populations.

Immunofluorescent lectin binding patterns and glycoprotein co-localization in the developing murine molar tooth

Fluorescein-conjugated lectins were used in conjunction with antibodies to laminin, tenascin and amelogenin to investigate saccharide expression in the developing tooth germ. At the bud stage, peanut agglutinin (PNA) binding demonstrated residues that may be d-galactose-(β1 → 3) dGalNAc, and this staining occurred after the expression of tenascin. Only the cap-stage enamel organ suprabasal cells and the enamel knot stained intensely with Ulex europeus agglutinin-I, but not Lotus tetragonolobus agglutinin, implying the transient presence of blood group H type I oligosaccharides. At the late stages of amelogenesis, enamel synthesis is preceded by en bloc loss of inner enamel basement membrane components. Before this, Bandeiraea (Griffonia) simplicifolia — I (BSL-I) staining was lost from post-mitotic ameloblasts, suggesting that a glycosylated species is initially removed. Additionally, PNA was co-localized with amelogenin protein, suggesting that it may express β- d-galactosyl sequences. These results indicate that the glycosylation patterns of matrix components during odontogenesis may be important as they vary in a manner similar to that of the well-known glycoproteins.

Developmental expression of alkaline phosphatase in murine molar tooth germs : Thomas, H.F., Walker, W.A., Indelicato, P.G. and Majeska, R.J. University of Connecticut Health Center, Farmington, CT., U.S.A.

Developmental expression of alkaline phosphatase in murine molar tooth germs : Thomas, H.F., Walker, W.A., Indelicato, P.G. and Majeska, R.J. University of Connecticut Health Center, Farmington, CT., U.S.A.