Mouse Tooth Germs Research Articles

Misexpression of Genes in Mouse Tooth Germs Using In Vitro Electroporation

Misexpression of Genes in Mouse Tooth Germs Using In Vitro Electroporation

Misexpression of Genes in Mouse Tooth Germs Using In Vitro Electroporation

Mouse mandible primodia in vitro explant cultures are an excellent model for investigating tooth development. Mandibular arch epithelial-mesenchymal tissue recombinations and implantation of beads soaked in signaling proteins have revealed much about the early events of odontogenesis. These approaches do, however, suffer from several disadvantages, in particular the nonphysiological nature of beads soaked in very high concentrations of proteins and the inability to directly manipulate receptor and transcription factor gene expression. We have utilized the technique of DNA electroporation to deliver targeted gene expression to defined areas of mandibular arch epithelium or mesenchyme. This approach is being used to (a) ectopically express epithelial signals such as Shh and Bmp-4 , (b) inhibit BMP and FGF signaling by expression of dominant negative receptors and antagonists, and (c) misexpress transcriptional factors in the mesenchyme. The technical aspects of electroporation are discussed, as well as preliminary findings.

マウス歯胚における p63 の局在

マウス歯胚における p63 の局在

Inositol hexasulphate, a casein kinase inhibitor, alters the distribution of dentin matrix protein 1 in cultured embryonic mouse tooth germs.

Immunohistochemical studies using a polyclonal antibody, raised against the recombinant form of dentin matrix protein 1 (DMP1), show that DMP1 was detected mainly in odontoblasts in cultured mouse embryonic tooth germs. However, in restricted areas, DMP1 staining was also observed in secretory ameloblasts, in the stratum intermedium and stellate reticulum, but only when the odontoblasts located in front of them were unstained. When the embryonic tooth germs were cultured in the presence of inositol hexasulfate, a casein kinase I and II inhibitor, staining of odontoblasts was weak or nil, whereas, in contrast, ameloblasts and enamel organ were strongly immunolabelled, suggesting an enhanced translocation of DMP1 after secretion to the secretory ameloblasts and/or stratum intermedium and stellate reticulum. Moreover, DMP1--was shown to be a good substrate for gelatinase A (MMP-2), but not to gelatinase B (MMP- 9). We hypothesized that DMP1--or the sub-fractions cleaved by the MMP--could behave as diffusible signaling molecule (s) rather than as a true dentin extracellular matrix component.

TNF Signaling via the Ligand–Receptor Pair Ectodysplasin and Edar Controls the Function of Epithelial Signaling Centers and Is Regulated by Wnt and Activin during Tooth Organogenesis

Ectodermal dysplasia syndromes affect the development of several organs, including hair, teeth, and glands. The recent cloning of two genes responsible for these syndromes has led to the identification of a novel TNF family ligand, ectodysplasin, and TNF receptor, edar. This has indicated a developmental regulatory role for TNFs for the first time. Our in situ hybridization analysis of the expression of ectodysplasin (encoded by the Tabby gene) and edar (encoded by the downless gene) during mouse tooth morphogenesis showed that they are expressed in complementary patterns exclusively in ectodermal tissue layer. Edar was expressed reiteratively in signaling centers regulating key steps in morphogenesis. The analysis of the effects of eight signaling molecules in the TGFβ, FGF, Hh, Wnt, and EGF families in tooth explant cultures revealed that the expression of edar was induced by activinβA, whereas Wnt6 induced ectodysplasin expression. Moreover, ectodysplasin expression was downregulated in branchial arch epithelium and in tooth germs of Lef1 mutant mice, suggesting that signaling by ectodysplasin is regulated by LEF-1-mediated Wnt signals. The analysis of the signaling centers in tooth germs of Tabby mice (ectodysplasin null mutants) indicated that in the absence of ectodysplasin the signaling centers were small. However, no downstream targets of ectodysplasin signaling were identified among several genes expressed in the signaling centers. We conclude that ectodysplasin functions as a planar signal between ectodermal compartments and regulates the function, but not the induction, of epithelial signaling centers. This TNF signaling is tightly associated with epithelial–mesenchymal interactions and with other signaling pathways regulating organogenesis. We suggest that activin signaling from mesenchyme induces the expression of the TNF receptor edar in the epithelial signaling centers, thus making them responsive to Wnt-induced ectodysplasin from the nearby ectoderm. This is the first demonstration of integration of the Wnt, activin, and TNF signaling pathways.

Inositol Hexasulphate, a Casein Kinase Inhibitor, Alters Enamel Formation in Cultured Embryonic Mouse Tooth Germs

Post-translational modification of enamel proteins is regulated by casein kinases (CK) and results in binding sites for calcium ions that subsequently play a key role during the initial stages of mineralization. Phosphorylation may also influence the secretion and extracellular organization of enamel proteins. Previous studies indicated that inositol hexasulphate inhibited the activity of CK-I and/or CK-II in mouse tooth germs (Torres-Quintana et al., 1998). We hypothesized that inositol hexasulphate would also inhibit the activity of the specific casein kinase(s) identified in secretory ameloblasts, and would prove useful for determination of the extent to which phosphorylation might influence the organization of enamel proteins at early stages of enamel formation. To test this hypothesis, we dissected mandibular first molars from 18-day-old mouse embryos and cultured them for 11 days in the presence of 0-0.1 mM inositol hexasulphate. Ultastructural analysis revealed that the formation of enamel was largely impaired at an inhibitor concentration > or = 0.08 mM. Quantitative radioautographic analysis of [33P]phosphate incorporation indicated that radiolabeled phosphate normally secreted into forming enamel was retained within ameloblasts. In contrast, no significant difference was observed between control and inositol-hexasulphate-treated tooth germs when cultures were labeled with [3H]serine and [3H]proline. SDS-PAGE and Western blot analysis confirmed that while inositol hexasulphate inhibited CK-mediated phosphorylation, it did not significantly alter protein synthesis. We conclude that impairment of phosphorylation leads to intracellular accumulation of [3H]phosphate-containing material by ameloblasts. We also conclude that when non-phosphorylated enamel matrix proteins are secreted, they are either unable to form an enamel matrix that supports mineralization, or they diffuse throughout a poorly mineralized dentin.

A new function of BMP4: dual role for BMP4 in regulation of Sonic hedgehog expression in the mouse tooth germ

The murine tooth development is governed by sequential and reciprocal epithelial-mesenchymal interactions. Multiple signaling molecules are expressed in the developing tooth germ and interact each other to mediate the inductive tissue interactions. Among them are Sonic hedgehog (SHH), Bone Morphogenetic Protein-2 (BMP2) and Bone Morphogenetic Protein-4 (BMP4). We have investigated the interactions between these signaling molecules during early tooth development. We found that the expression of Shh and Bmp2 is downregulated at E12.5 and E13.5 in the dental epithelium of the Msx1 mutant tooth germ where Bmp4 expression is significantly reduced in the dental mesenchyme. Inhibition of BMP4 activity by noggin resulted in repression of Shh and Bmp2 in wild-type dental epithelium. When implanted into the dental mesenchyme of Msx1 mutants, beads soaked with BMP4 protein were able to restore the expression of both Shh and Bmp2 in the Msx1 mutant epithelium. These results demonstrated that mesenchymal BMP4 represents one component of the signal acting on the epithelium to maintain Shh and Bmp2 expression. In contrast, BMP4-soaked beads repressed Shh and Bmp2 expression in the wild-type dental epithelium. TUNEL assay indicated that this suppression of gene expression by exogenous BMP4 was not the result of an increase in programmed cell death in the tooth germ. Ectopic expression of human Bmp4 to the dental mesenchyme driven by the mouse Msx1 promoter restored Shh expression in the Msx1 mutant dental epithelium but repressed Shh in the wild-type tooth germ in vivo. We further demonstrated that this regulation of Shh expression by BMP4 is conserved in the mouse developing limb bud. In addition, Shh expression was unaffected in the developing limb buds of the transgenic mice in which a constitutively active Bmpr-IB is ectopically expressed in the forelimb posterior mesenchyme and throughout the hindlimb mesenchyme, suggesting that the repression of Shh expression by BMP4 may not be mediated by BMP receptor-IB. These results provide evidence for a new function of BMP4. BMP4 can act upstream to Shh by regulating Shh expression in mouse developing tooth germ and limb bud. Taken together, our data provide insight into a new regulatory mechanism for Shh expression, and suggest that this BMP4-mediated pathway in Shh regulation may have a general implication in vertebrate organogenesis.

Different Histogenesis of Experimental Odontogenic Cysts by the Renal Subcapsular Transplantation of Tooth Germs of Mice.

The tooth germs of first mandibular molars of 17.5-day-old C3H mouse embryos were transplanted into the renal subcapsular spaces of 3-month-old syngenic male mice. Grafts were harvested at timed sequences from 1, 2, 3, 4 weeks and 2, 3, 4, 5 months, and were examined histopathologically by total serial sections. Of 32 cases of grafts, keratocyst-like lesions were formed in 13 cases, and dentigerous cyst-like lesions were formed in 7 cases. None of the cases developed two cysts at the same time. In the keratocyst-like lesions the cavity developed from the enamel epithelium in the early stage after transplantation, and in the dentigerous cyst-like lesion the cavity developed by cystic degeneration of stellate reticular layer of enamel organ in the late stage after transplantation. The present experiment revealed that keratocyst-like lesions and dentigerous cyst-like lesions developed through different histogeneses.

Immunofluorescence detection of cadherins in mouse tooth germs during root development

The distribution of two cell-adhesion molecules, E- and P-cadherin, was studied in relation to morphological changes in Hertwig’s epithelial root sheath. Before root dentinogenesis had started, the root sheath expressed both cadherins. As dentinogenesis proceeded, the sheath fragmented and lost P-cadherin rapidly and E-cadherin slowly, whereas the intact sheath at the apical end continued to express both. These results suggest that the two cadherins play a part in root as well as in crown development, and indicate that the decrease in the amount of these molecules and the fragmentation of the epithelial root sheath are interrelated.

P–626 - Expression of mRNAs for FGF and FGFRs during developmental stage in mouse tooth germ

P–626 - Expression of mRNAs for FGF and FGFRs during developmental stage in mouse tooth germ

Effects of inositol hexasulphate, a casein kinase inhibitor, on dentine phosphorylated proteins in organ culture of mouse tooth germs

To study the effects of impaired protein phosphorylation on dentine formation and mineralization, inositol hexasulphate, an intracellular type I and type II casein kinase inhibitor, was used in an in vitro organotypic culture system. Mandibular first molar tooth germs were dissected from 18-day-old mouse embryos and cultured for 11 days with and without inositol hexasulphate at different concentrations. At 0.04–0.08 mM inhibitor, cellular alterations were not detected. Dentine displayed the characteristic purple-blue colour when Stains all, a specific stain for extracellular phosphoproteins, was used. At 0.1 mM, dentine failed to stain and mineralization did not occur, as seen from the von Kossa method. The presence of numerous lysosome-like vesicles inside cells indicated that the experiment was at the limits of cytotoxicity; higher concentrations induced severe cellular alterations. Therefore, quantitative radioautography was carried out on germs treated or not with the inhibitor at 0.1 mM. [ 33P]-phosphate incorporation showed that grain density in inhibited germs compared with that in control germs was about double in odontoblasts and half in the predentine/dentine compartment. In the presence of inositol hexasulphate the incorporation of [ 3H]serine into odontoblast cell bodies was unchanged between 2 and 24 h while in predentine/dentine, grain density was higher between 1 and 4 h, and reduced at 24 h. Both with [ 33P]phosphate and [ 3H]serine, labelling was seen throughout the porous dentine formed in vitro and not as a band located at the predentine/dentine junction, as is the case in vivo. With [ 3H]proline, in the presence of the inhibitor, a small reduction of grain density occurred in cell bodies, no significant difference was seen between 1 and 4 h in predentine/dentine, and more silver grains were present after 24 h both in cells and in the matrix. The radioautographic data support the view that the inhibitor interacts mostly with post-transductional phosphorylation and does not alter significantly other cell synthetic pathways and functions. Finally, the experiments presented here confirm that phophorylated proteins have a key role in dentine mineralization.

Comparison of the effects of carboxyl-terminal parathyroid hormone peptide[53-84] and aminoterminal peptide[1-34] on mouse tooth germ in vitro

The biological activity of carboxyl-terminal parathyroid hormone peptide[53-84] (PTH[53-84]) and aminoterminal peptide [1-34] (PTH[1-34]) during cultivation of the lower molar tooth germ from mouse embryos was examined. PTH[1-34] increased alkaline phosphatase activity in the tooth germ at an early developmental stage (early to advanced bell stage), and decreased it at a late developmental stage (advanced to late bell stage). On the other hand, PTH[53-84] decreased alkaline phosphatase activity at the early stage and increased it at the late stage. Thus, both PTH fragments had distinct effects on tooth development in vitro, which were confirmed by histological observation of their marked effects on the formation of dentine and enamel. These results show that PTH[53-84] has biological activity in the murine tooth germ, opposite to the action of PTH[1-34].

P-263 - In vitro effect of carboxyl-terminal parathyroid hormone peptide [53-84] in mouse tooth germ; comparison with the effect of amino-terminal peptide [1-34

P-263 - In vitro effect of carboxyl-terminal parathyroid hormone peptide [53-84] in mouse tooth germ; comparison with the effect of amino-terminal peptide [1-34

The Influence of Low Power Laser Local Irradiation on the Mineralization of Tooth Germ in Mice

Previously, we demonstrated the suppressive effect of low power laser (He-Ne laser) irradiation on mineralization in molar tooth germ using the organ culture system. In the present study, we examined the in vivo effect of the laser on the development of mandibular first and second molars in neonatal mouse. The laser was irradiated at developmentally different stages and the chemical and histological changes in these molars were examined. At an early developmental stage of tooth germ showing cell proliferation and differentiation, the laser irradiation caused decreases of alkaline phosphatase activity, of calcium content and the formation of dentin and enamel in tooth germ. However, at a late developmental stage of tooth germ showing additional mineralization, these decreases were not observed at the time of the laser irradiation. From the in vivo experiment, we confirmed that He-Ne laser irradiation suppressed tooth formation and mineralization at an early developmental stage.(J. Jpn. Soc. Laser Dent. 7: 30-36, 1996 Reprint requests to Dr. Tsuboi)

The Influence of Low Power Laser Local Irradiation on the Mineralization of Tooth Germ in Mice

To elucidate the in vivo effect of low power diode laser irradiation on mineralization in molar tooth germ, we examined the effect of the laser on the development of mandibular first and second molars in neonatal mouse. In a newborn mouse, the mandibular first molar showed differentiated odontoblast, differentiated ameloblast, slight dentin and enamel formation. The second molar was an early bell stage. In a two day-old neonatal mouse, the developmental stage of the second molar corresponded to the stage of the first molar in a newborn mouse. The laser (50mW, 5min/day) was irradiated to the mandibular tooth germ at twice and the chemical and histological changes in these molars were examined. In the second molar showing an early developmental stage (cell proliferation and differentiation), the laser irradiation increased alkaline phosphatase activity, calcium content and the formation of both dentin and enamel. Also, in the first molar showing a late developmental stage (additional mineralization), the laser irradiation also increased the mineralization in both dentin and enamel. From the in vivo experiment, we confirmed that diode laser irradiation accelerated tooth formation and mineralization at all developmental stages observed in the present study.(J. Jpn. Soc. Laser Dent. 7: 22-29, 1996 Reprint requests to Dr. Okumura)

A role for mesenchyme-derived tachykinins in tooth and mammary gland morphogenesis.

Tachykinin peptides such as substance P (SP) function as neurotransmitters and neuromodulators in the mammalian central and peripheral nervous systems. Here, we provide evidence that they may also play an important role in the morphogenesis of some nonneural organs where epithelial-mesenchymal interactions are involved. We show the following. (1) mRNA encoding tachykinin precursor proteins is expressed transiently in condensing mesenchyme during the development of mouse tooth germ, mammary gland, limb bud, external auditory meatus and genital tubercle. (2) In developing tooth germ and mammary gland; mRNA encoding the neutral endopeptidase (NEP) that degrades secreted tachykinins is spatially and temporally co-expressed with tachykinin precursor mRNA. (3) SP and the mRNA encoding SP receptors are also expressed in the developing tooth germ. (4) Tooth development in explant cultures is blocked both by tachykinin-precursor-specific antisense oligonucleotide and by an SP receptor antagonist: in both cases the block is relieved by exogenous SP. Together, these findings suggest a surprising new role for tachykinins in tooth and mammary gland morphogenesis, and possibly also in limb, ear and external genitalia morphogenesis.

S2-5 - Pharmacological study of the mineralization of cultured tooth germ in mice

S2-5 - Pharmacological study of the mineralization of cultured tooth germ in mice

Effects of Low Power Laser Irradiation on the Mineralization of Tooth Germ in Mice

Effects of Low Power Laser Irradiation on the Mineralization of Tooth Germ in Mice

Distal-less and other homeobox genes in the development of the dentition.

The mammalian tooth develops through an interaction between two tissue layers of different embryologic origin. A number of transcription factors and as well as two members of the Msx class of homeobox genes have been shown to be involved in the histogenesis of the mammalian tooth. This raised the possibility that other homeobox genes might be involved in dental morphogenesis. We have amplified mouse tooth germ cDNA from three different gestational ages by the polymerase chain reaction with degenerate primers for 18 classes of homeobox genes. Members of several classes have been isolated, including the Msx genes, two Dlx genes, and the Dbx, MHox, Mox2A genes. One of the Dlx genes, Dlx-7, had not previously been reported in mammals, and some details are presented of its cDNA sequence. This work plus that of other investigators has shown that at least six Dlx genes are expressed in developing teeth or in first branchial arches, suggesting the possibility that these genes are involved in specifying complexity within or between teeth. The screening approach with degenerate primers is a successful way to identify new as well as previously known regulatory genes expressed in developing tooth embryos.

Inhibition of in vitro mineralization in osteoblastic cells and in mouse tooth germ by phosphatidylinositol-specific phospholipase C

We have examined the effect of phosphatidylinositol-specific phospholipase C (PIPLC) on the in vitro mineralization during cultivation of both osteoblast-like MC3T3-E1 cells and the lower molar tooth germ from mouse embryo. PIPLC not only caused the release of alkaline phosphatase (ALP) from cell membranes into the culture medium but also produced a disturbance of in vitro mineralization in MC3T3-E1 cells and in mouse embryonic tooth germ. These findings strongly suggest that ALP, functioning as an ectoenzyme, is involved in the physiological mineralization process of both bone and teeth.