RESUMO
Evidence has shown that miRNAs could play a role in dental fluorosis, but there is no study has investigated the global expression miRNA profiles of fluoride-exposed enamel organ. In this study, we analysed the differentially expressed (DE) miRNAs between fluoride-treated and control enamel organ for the first time and found several candidate miRNAs and signaling pathways worthy of further research. Thirty Wistar rats were randomly distributed into three groups and exposed to drinking water with different fluoride contents for 10 weeks and during the gestation. The three groups were a control group (distilled water), medium fluoride group (75 mg/L NaF), and high fluoride group (150 mg/L NaF). On the embryonic day 19.5, the mandible was dissected for histological analysis, and the enamel organ of the mandibular first molar tooth germ was collected for miRNA sequencing (miRNA-seq) and quantitative real-time PCR analysis (qRT-PCR). Typical dental fluorosis was observed in the incisors of the prepregnant rats. In addition to the disorganized structure of enamel organ cells, 39 DE miRNAs were identiï¬ed in the ï¬uoride groups compared with the control group, and good agreement between the miRNA-seq data and qRT-PCR data was found. The functional annotation of the target genes of 39 DE miRNAs showed significant enrichment in metabolic process, cell differentiation, calcium signaling pathway, and mitogen-activated protein kinase(MAPK) signaling pathway terms. This study provides a theoretical reference for an extensive understanding of the mechanism of fluorosis and potential valuable miRNAs as therapeutic targets in fluorosis.
Assuntos
Órgão do Esmalte/efeitos dos fármacos , Fluoretos/toxicidade , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , MicroRNAs , Animais , Embrião de Mamíferos , Órgão do Esmalte/embriologia , Órgão do Esmalte/metabolismo , Feminino , Fluorose Dentária , Ratos Wistar , Transcriptoma/efeitos dos fármacosRESUMO
We herein report that deletion of mTOR in dental epithelia caused defective development of multiple cell layers of the enamel organ, which culminated in tooth malformation and cystogenesis. Specifically, cells of the stellate reticulum and stratum intermedium were poorly formed, resulting in cystic changes. The pre-ameloblasts failed to elongate along the apical-basal axis and persisted vigorous expression of Sox2 and P63, which are normally downregulated during cytodifferentiation. Expression of amelogenic markers was also attenuated in mutants. Cell proliferation and cell sizes in mutants were significantly reduced over time. Importantly, we found reduced amounts and aberrant aggregations of cytoskeletal components in mutants, along with attenuated expression of cytoskeleton regulator Cdc42, whose epithelial deletion causes a similar phenotype. Moreover, disruption of actin assembly in an organ culture system affected cell proliferation and cytodifferentiation of tooth germs, supporting a causative role of mTOR-regulated cytoskeleton dynamics for the observed phenotype of mTOR mutant mice. In further support of this view, we showed that mTOR overactivation caused increased cytoskeletal component synthesis and assembly, along with accelerated cytodifferentiation in the enamel organ. Finally, we demonstrated that mTOR regulated enamel organ development principally through the mTORC1 pathway.
Assuntos
Citoesqueleto/metabolismo , Órgão do Esmalte/embriologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Citoesqueleto/genética , Órgão do Esmalte/citologia , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Camundongos , Camundongos Transgênicos , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Serina-Treonina Quinases TOR/genética , Transativadores/genética , Transativadores/metabolismo , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Ligação ao GTP/metabolismoRESUMO
Tooth enamel is manufactured by the inner enamel epithelium of the multilayered enamel organ. Msx2 loss-of-function mutation in a mouse model causes an abnormal accumulation of epithelial cells in the enamel organ, but the underlying mechanism by which Msx2 regulates amelogenesis is poorly understood. We therefore performed detailed histological and molecular analyses of Msx2 null mice. Msx2 null ameloblasts and stratum intermedium (SI) cells differentiated normally in the early stages of amelogenesis. However, during subsequent developmental stages, the outer enamel epithelium (OEE) became highly proliferative and transformed into a keratinized stratified squamous epithelium that ectopically expressed stratified squamous epithelium markers, including Heat shock protein 25, Loricrin, and Keratin 10. Moreover, expression of hair follicle-specific keratin genes such as Keratin 26 and Keratin 73 was upregulated in the enamel organ of Msx2 mutants. With the accumulation of keratin in the stellate reticulum (SR) region and subsequent odontogenic cyst formation, SI cells gradually lost the ability to differentiate, and the expression of Sox2 and Notch1 was downregulated, leading to ameloblast depolarization. As a consequence, the organization of the Msx2 mutant enamel organ became disturbed and enamel failed to form in the normal location. Instead, there was ectopic mineralization that likely occurred within the SR. In summary, we show that during amelogenesis, Msx2 executes a bipartite function, repressing the transformation of OEE into a keratinized stratified squamous epithelium while simultaneously promoting the development of a properly differentiated enamel organ competent for enamel formation.
Assuntos
Órgão do Esmalte/metabolismo , Epitélio/metabolismo , Proteínas de Homeodomínio/metabolismo , Ameloblastos/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Cistos/embriologia , Cistos/metabolismo , Microanálise por Sonda Eletrônica , Órgão do Esmalte/embriologia , Epitélio/embriologia , Genótipo , Hibridização In Situ , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Microtomografia por Raio-XRESUMO
Cdc42, a Rho family small GTPase, regulates cytoskeleton organization, vesicle trafficking, and other cellular processes in development and homeostasis. However, Cdc42's roles in prenatal tooth development remain elusive. Here, we investigated Cdc42 functions in mouse enamel organ. Cdc42 showed highly dynamic temporospatial patterns in the developing enamel organ, with robust expression in the outer enamel epithelium, stellate reticulum (SR), and stratum intermedium layers. Strikingly, epithelium-specific Cdc42 deletion resulted in cystic lesions in the enamel organ. Cystic lesions were first noted at embryonic day 15.5 and progressively enlarged during gestation. At birth, cystic lesions occupied the bulk of the entire enamel organ, with intracystic erythrocyte accumulation. Ameloblast differentiation was retarded upon epithelial Cdc42 deletion. Apoptosis occurred in the Cdc42 mutant enamel organ prior to and synchronously with cystogenesis. Transmission electron microscopy examination showed disrupted actin assemblies, aberrant desmosomes, and significantly fewer cell junctions in the SR cells of Cdc42 mutants than littermate controls. Autophagosomes were present in the SR cells of Cdc42 mutants relative to the virtual absence of autophagosome in the SR cells of littermate controls. Epithelium-specific Cdc42 deletion attenuated Wnt/ß-catenin and Shh signaling in dental epithelium and induced aberrant Sox2 expression in the secondary enamel knot. These findings suggest that excessive cell death and disrupted cell-cell connections may be among multiple factors responsible for the observed cystic lesions in Cdc42 mutant enamel organs. Taken together, Cdc42 exerts multidimensional and pivotal roles in enamel organ development and is particularly required for cell survival and tooth morphogenesis.
Assuntos
Cistos/embriologia , Órgão do Esmalte/embriologia , Epitélio/embriologia , Proteínas rho de Ligação ao GTP/metabolismo , Actinas/metabolismo , Ameloblastos/metabolismo , Animais , Apoptose , Autofagossomos/metabolismo , Western Blotting , Diferenciação Celular , Proteínas do Citoesqueleto , Marcação In Situ das Extremidades Cortadas , Junções Intercelulares/metabolismo , Camundongos , Microscopia Eletrônica de Transmissão , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Tooth is made of an enamel-covered crown and a cementum-covered root. Studies on crown dentin formation have been a major focus in tooth development for several decades. Interestingly, the population prevalence for genetic short root anomaly (SRA) with no apparent defects in crown is close to 1.3%. Furthermore, people with SRA itself are predisposed to root resorption during orthodontic treatment. The discovery of the unique role of Nfic (nuclear factor I C; a transcriptional factor) in controlling root but not crown dentin formation points to a new concept: tooth crown and root have different control mechanisms. Further genetic mechanism studies have identified more key molecules (including Osterix, ß-catenin, and sonic hedgehog) that play a critical role in root formation. Extensive studies have also revealed the critical role of Hertwig's epithelial root sheath in tooth root formation. In addition, Wnt10a has recently been found to be linked to multirooted tooth furcation formation. These exciting findings not only fill the critical gaps in our understanding about tooth root formation but will aid future research regarding the identifying factors controlling tooth root size and the generation of a whole "bio-tooth" for therapeutic purposes. This review starts with human SRA and mainly focuses on recent progress on the roles of NFIC-dependent and NFIC-independent signaling pathways in tooth root formation. Finally, this review includes a list of the various Cre transgenic mouse lines used to achieve tooth root formation-related gene deletion or overexpression, as well as strengths and limitations of each line.
Assuntos
Odontogênese/fisiologia , Transdução de Sinais , Raiz Dentária/embriologia , Animais , Cemento Dentário/embriologia , Dentina/embriologia , Órgão do Esmalte/embriologia , Proteínas Hedgehog/metabolismo , Humanos , Camundongos , Fatores de Transcrição NFI/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Odontogênese/genética , Fator de Transcrição Sp7 , Fatores de Transcrição/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismoRESUMO
OBJECTIVE: Ghrelin, an appetite-stimulating hormone, plays diverse regulatory functions in cell growth, proliferation, differentiation and apoptosis during mammalian development. There is limited information currently available regarding Ghrelin expression during mammalian tooth development, thus we aimed to establish the spatiotemporal expression of Ghrelin during murine molar odontogenesis. DESIGN: Immunohistochemistry was performed to detect the expression pattern of Ghrelin in mandible molar from E15.5 to PN7 during murine tooth development. RESULTS: The results showed that Ghrelin initially expressed in the inner enamel epithelium and the adjacent mesenchymal cells below, further with persistent expression in the ameloblasts and odontoblasts throughout the following developmental stages. In addition, Ghrelin was also present in Hertwig's epithelial root sheath at the beginning of tooth root formation. CONCLUSIONS: These results suggest that Ghrelin was present in tooth organs throughout the stages of tooth development, especially in ameloblasts and odontoblasts with little spatiotemporal expression differences. However, the potential regulatory roles of this hormone in tooth development still need to be validated by functional studies.
Assuntos
Grelina/biossíntese , Grelina/metabolismo , Dente Molar/metabolismo , Ameloblastos/citologia , Ameloblastos/metabolismo , Animais , Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Esmalte Dentário/citologia , Esmalte Dentário/embriologia , Esmalte Dentário/metabolismo , Órgão do Esmalte/embriologia , Órgão do Esmalte/crescimento & desenvolvimento , Órgão do Esmalte/metabolismo , Epitélio/embriologia , Epitélio/metabolismo , Feminino , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos ICR , Dente Molar/citologia , Dente Molar/efeitos dos fármacos , Dente Molar/crescimento & desenvolvimento , Odontoblastos/citologia , Odontoblastos/metabolismo , Odontogênese/efeitos dos fármacos , Odontogênese/fisiologia , Gravidez , Germe de Dente/embriologia , Germe de Dente/crescimento & desenvolvimento , Germe de Dente/metabolismo , Raiz Dentária/embriologia , Raiz Dentária/crescimento & desenvolvimento , Raiz Dentária/metabolismoRESUMO
AIMS: To analyze expression patterns of IGF-1, caspase-3 and HSP-70 in human incisor and canine tooth germs during the late bud, cap and bell stages of odontogenesis. MATERIALS AND METHODS: Head areas or parts of jaw containing teeth from 10 human fetuses aged between 9th and 20th developmental weeks were immunohistochemically analyzed using IGF-1, active caspase-3 and HSP-70 markers. Semi-quantitative analysis of each marker's expression pattern was also performed. RESULTS: During the analyzed period, IGF-1 and HSP-70 were mostly expressed in enamel organ. As development progressed, expression of IGF-1 and HSP-70 became more confined to differentiating tissues in the future cusp tip area, as well as in highly proliferating cervical loops. Few apoptotic bodies highly positive to active caspase-3 were observed in enamel organ and dental papilla from the cap stage onward. However, both enamel epithelia moderately expressed active caspase-3 throughout the investigated period. CONCLUSIONS: Expression patterns of IGF-1, active caspase-3 and HSP-70 imply importance of these factors for early human tooth development. IGF-1 and HSP-70 have versatile functions in control of proliferation, differentiation and anti-apoptotic protection of epithelial parts of human enamel organ. Active caspase-3 is partially involved in formation and apoptotic removal of primary enamel knot, although present findings might reflect its ability to perform other non-death functions such as differentiation of hard dental tissues secreting cells and guidance of ingrowth of proliferating cervical loops.
Assuntos
Caspase 3/biossíntese , Proteínas de Choque Térmico HSP70/biossíntese , Fator de Crescimento Insulin-Like I/biossíntese , Germe de Dente/metabolismo , Diferenciação Celular , Dente Canino/citologia , Dente Canino/embriologia , Dente Canino/metabolismo , Esmalte Dentário/metabolismo , Papila Dentária/citologia , Papila Dentária/embriologia , Papila Dentária/crescimento & desenvolvimento , Papila Dentária/metabolismo , Órgão do Esmalte/citologia , Órgão do Esmalte/embriologia , Órgão do Esmalte/metabolismo , Feto , Humanos , Imuno-Histoquímica , Incisivo/embriologia , Incisivo/metabolismo , Odontogênese , Germe de Dente/citologia , Germe de Dente/embriologiaRESUMO
Histone methylation is one of the most widely studied post-transcriptional modifications. It is thought to be an important epigenetic event that is closely associated with cell fate determination and differentiation. To explore the spatiotemporal expression of histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 27 trimethylation (H3K27me3) epigenetic marks and methylation or demethylation transferases in tooth organ development, we measured the expression of SET7, EZH2, KDM5B and JMJD3 via immunohistochemistry and quantitative polymerase chain reaction (qPCR) analysis in the first molar of BALB/c mice embryos at E13.5, E15.5, E17.5, P0 and P3, respectively. We also measured the expression of H3K4me3 and H3K27me3 with immunofluorescence staining. During murine tooth germ development, methylation or demethylation transferases were expressed in a spatial-temporal manner. The bivalent modification characterized by H3K4me3 and H3K27me3 can be found during the tooth germ development, as shown by immunofluorescence. The expression of SET7, EZH2 as methylation transferases and KDM5B and JMJD3 as demethylation transferases indicated accordingly with the expression of H3K4me3 and H3K27me3 respectively to some extent. The bivalent histone may play a critical role in tooth organ development via the regulation of cell differentiation.
Assuntos
Histonas/metabolismo , Odontogênese/fisiologia , Processamento de Proteína Pós-Traducional/fisiologia , Animais , Diferenciação Celular/fisiologia , Proteínas de Ligação a DNA/análise , Papila Dentária/embriologia , Embrião de Mamíferos , Órgão do Esmalte/embriologia , Proteína Potenciadora do Homólogo 2 de Zeste , Epigênese Genética/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Histona-Lisina N-Metiltransferase/análise , Histona Desmetilases com o Domínio Jumonji/análise , Lisina/metabolismo , Metilação , Camundongos , Camundongos Endogâmicos BALB C , Complexo Repressor Polycomb 2/análise , Germe de Dente/embriologiaRESUMO
During the formation of dental enamel, maturation-stage ameloblasts express ion-transporting transmembrane proteins. The SLC4 family of ion-transporters regulates intra- and extracellular pH in eukaryotic cells by cotransporting HCO3 (-) with Na(+). Mutation in SLC4A4 (coding for the sodium-bicarbonate cotransporter NBCe1) induces developmental defects in human and murine enamel. We have hypothesized that NBCe1 in dental epithelium is engaged in neutralizing protons released during crystal formation in the enamel space. We immunolocalized NBCe1 protein in wild-type dental epithelium and examined the effect of the NBCe1-null mutation on enamel formation in mice. Ameloblasts expressed gene transcripts for NBCe1 isoforms B/D/C/E. In wild-type mice, weak to moderate immunostaining for NBCe1 with antibodies that recognized isoforms A/B/D/E and isoform C was seen in ameloblasts at the secretory stage, with no or low staining in the early maturation stage but moderate to high staining in the late maturation stage. The papillary layer showed the opposite pattern being immunostained prominently at the early maturation stage but with gradually less staining at the mid- and late maturation stages. In NBCe1 (-/-) mice, the ameloblasts were disorganized, the enamel being thin and severely hypomineralized. Enamel organs of CFTR (-/-) and AE2a,b (-/-) mice (CFTR and AE2 are believed to be pH regulators in ameloblasts) contained higher levels of NBCe1 protein than wild-type mice. Thus, the expression of NBCe1 in ameloblasts and the papillary layer cell depends on the developmental stage and possibly responds to pH changes.
Assuntos
Órgão do Esmalte/citologia , Órgão do Esmalte/embriologia , Simportadores de Sódio-Bicarbonato/metabolismo , Ameloblastos/citologia , Ameloblastos/metabolismo , Amelogênese , Animais , Western Blotting , Calcificação Fisiológica/genética , Antiportadores de Cloreto-Bicarbonato/metabolismo , Cricetinae , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Órgão do Esmalte/diagnóstico por imagem , Órgão do Esmalte/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Incisivo/metabolismo , Mandíbula/metabolismo , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Simportadores de Sódio-Bicarbonato/deficiência , Simportadores de Sódio-Bicarbonato/genética , Regulação para Cima/genética , Microtomografia por Raio-XRESUMO
Teeth develop through distinct morphological stages. At the cap stage, a compactly clustered and concentrically arranged cell mass, the enamel knot, appears at the tip of the enamel organ. Cells in this knot express sets of key molecules, and as such have been proposed to act as a signaling center directing tooth morphogenesis and tooth cusp formation. YAP is a transcriptional co-activator of the Hippo signaling pathway that is essential for the proper regulation of organ growth. In this study, we analyzed the tooth phenotype in transgenic mice that overexpressed a constitutively active form of YAP in the dental epithelium. We found that overexpression of YAP resulted in deformed tooth morphogenesis with widened dental lamina. In addition, the enamel knot was mislocated to the upper portion of the enamel organ, where it remained devoid of proliferating cells and contained apoptotic cells with intense Edar transcripts and reduced E-cadherin expression. Interestingly, some signaling molecules, such as Shh, Fgf4, and Wnt10a, were not expressed in this mislocated enamel knot, but remained at the tip of the enamel organ. Analysis of these data suggests that the signaling center is induced by reciprocal epithelial-mesenchymal interactions, and its induction may be independent of the enamel knot.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Órgão do Esmalte/embriologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Odontogênese/genética , Fosfoproteínas/genética , Amelogênese/genética , Animais , Apoptose/genética , Caderinas/análise , Adesão Celular/genética , Proteínas de Ciclo Celular , Receptor Edar/análise , Receptor Edar/genética , Órgão do Esmalte/anormalidades , Células Epiteliais/patologia , Epitélio/embriologia , Fator 4 de Crescimento de Fibroblastos/análise , Proteínas Hedgehog/análise , Via de Sinalização Hippo , Mesoderma/embriologia , Mesoderma/patologia , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/análise , Fenótipo , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais/genética , Anormalidades Dentárias/genética , Coroa do Dente/anormalidades , Coroa do Dente/embriologia , Germe de Dente/anormalidades , Germe de Dente/embriologia , Proteínas Wnt/análise , Proteínas de Sinalização YAPRESUMO
Many ciliopathies have clinical features that include tooth malformations but how these defects come about is not clear. Here we show that genetic deletion of the motor protein Kif3a in dental mesenchyme results in an arrest in odontogenesis. Incisors are completely missing, and molars are enlarged in Wnt1(Cre+)Kif3a(fl/fl) embryos. Although amelogenesis and dentinogenesis initiate in the molar tooth bud, both processes terminate prematurely. We demonstrate that loss of Kif3a in dental mesenchyme results in loss of Hedgehog signaling and gain of Wnt signaling in this same tissue. The defective dental mesenchyme then aberrantly signals to the dental epithelia, which prompts an up-regulation in the Hedgehog and Wnt responses in the epithelia and leads to multiple attempts at invagination and an expanded enamel organ. Thus, the primary cilium integrates Hedgehog and Wnt signaling between dental epithelia and mesenchyme, and this cilia-dependent integration is required for proper tooth development.
Assuntos
Proteínas Hedgehog/fisiologia , Cinesinas/fisiologia , Odontogênese/fisiologia , Proteínas Wnt/fisiologia , Animais , Contagem de Células , Movimento Celular/fisiologia , Proliferação de Células , Cílios/fisiologia , Dentinogênese/fisiologia , Órgão do Esmalte/embriologia , Células Epiteliais/citologia , Epitélio/embriologia , Deleção de Genes , Proteínas Hedgehog/genética , Processamento de Imagem Assistida por Computador , Cinesinas/genética , Mesoderma/citologia , Mesoderma/embriologia , Camundongos , Camundongos Knockout , Crista Neural/citologia , Odontoblastos/citologia , Odontogênese/genética , Transdução de Sinais/fisiologia , Anormalidades Dentárias/genética , Germe de Dente/embriologia , Proteínas Wnt/genética , Microtomografia por Raio-XRESUMO
The tooth works as a functional unit with its surrounding bony socket, the alveolar bone. The growth of the tooth and alveolar bone is co-ordinated so that a studied distance always separates the 2, known as the tooth-bone interface (TBI). Lack of mineralization, a crucial feature of the TBI, creates the space for the developing tooth to grow and the soft tissues of the periodontium to develop. We have investigated the interactions between the tooth and its surrounding bone during development, focusing on the impact of the developing alveolar bone on the development of the mouse first molar (M1). During development, TRAP-positive osteoclasts are found to line the TBI as bone starts to be deposited around the tooth, removing the bone as the tooth expands. An enhancement of osteoclastogenesis through RANK-RANKL signaling results in an expansion of the TBI, showing that osteoclasts are essential for defining the size of this region. Isolation of the M1 from the surrounding mesenchyme and alveolar bone leads to an expansion of the tooth germ, driven by increased proliferation, indicating that, during normal development, the growth of the tooth germ is constrained by the surrounding tissues.
Assuntos
Processo Alveolar/embriologia , Alvéolo Dental/embriologia , Dente/embriologia , Fosfatase Ácida/análise , Animais , Carbocianinas , Proliferação de Células , Corantes , Órgão do Esmalte/embriologia , Corantes Fluorescentes , Isoenzimas/análise , Mesoderma/embriologia , Camundongos , Índice Mitótico , Odontogênese/fisiologia , Técnicas de Cultura de Órgãos , Osteoclastos/fisiologia , Osteogênese/fisiologia , Periodonto/embriologia , Periodonto/fisiologia , Ligante RANK/fisiologia , Receptor Ativador de Fator Nuclear kappa-B/fisiologia , Transdução de Sinais/fisiologia , Fosfatase Ácida Resistente a Tartarato , Germe de Dente/embriologia , Alvéolo Dental/fisiologiaRESUMO
Signals of perlecan, an extracellular matrix molecule, which accumulates within the intercellular spaces of the stellate reticulum of the enamel organ, are mediated by at least two receptors, dystroglycan (DG) and integrin ß1, in a case-dependent manner in various events in embryogenesis and pathogenesis. This study aims to understand the expression profiles of these two perlecan receptors at both protein and gene levels in murine enamel organ development. Before birth, α-DG was immunolocalized in stellate reticulum cells, in which perlecan was colocalized, while integrin ß1 was mainly distributed in the peripheral enamel organ cells as well as the dental mesenchymal cells. On and after postnatal Day 1, the expression of α-DG was dramatically decreased in the stellate reticulum, while integrin ß1 was enhanced around blood vessels within the enamel organ. Furthermore, biosyntheses of α-DG and integrin ß1 by dental epithelial and pulp mesenchymal cells were confirmed in vitro by using immunofluorescence and reverse-transcriptase polymerase chain reaction. The results suggest that DG is a perlecan receptor that specifically functions in the stellate reticulum of the embryonic stage, but that dental epithelial and mesenchymal cells are maturated by capturing perlecan signals differentially through integrin ß1.
Assuntos
Distroglicanas/metabolismo , Órgão do Esmalte/metabolismo , Expressão Gênica , Integrina beta1/metabolismo , Animais , Linhagem Celular , Distroglicanas/genética , Órgão do Esmalte/citologia , Órgão do Esmalte/embriologia , Órgão do Esmalte/crescimento & desenvolvimento , Células Epiteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Incisivo/citologia , Incisivo/embriologia , Incisivo/crescimento & desenvolvimento , Incisivo/metabolismo , Integrina beta1/genética , Mesoderma/citologia , Camundongos Endogâmicos ICR , Dente Molar/citologia , Dente Molar/embriologia , Dente Molar/crescimento & desenvolvimento , Dente Molar/metabolismo , Especificidade de ÓrgãosRESUMO
BACKGROUND AND OBJECTIVE: Some clinical cases of hypoplastic tooth root are congenital. Because the formation of Hertwig's epithelial root sheath (HERS) is an important event for root development and growth, we have considered that understanding the HERS developmental mechanism contributes to elucidate the causal factors of the disease. To find integrant factors and phenomenon for HERS development and growth, we studied the proliferation and mobility of the cervical loop (CL). MATERIAL AND METHODS: We observed the cell movement of CL by the DiI labeling and organ culture system. To examine cell proliferation, we carried out immunostaining of CL and HERS using anti-Ki67 antibody. Cell motility in CL was observed by tooth germ slice organ culture using green fluorescent protein mouse. We also examined the expression of paxillin associated with cell movement. RESULTS: Imaging using DiI labeling showed that, at the apex of CL, the epithelium elongated in tandem with the growth of outer enamel epithelium (OEE). Cell proliferation assay using Ki67 immunostaining showed that OEE divided more actively than inner enamel epithelium (IEE) at the onset of HERS formation. Live imaging suggested that mobility of the OEE and cells in the apex of CL were more active than in IEE. The expression of paxillin was observed strongly in OEE and the apex of CL. CONCLUSION: The more active growth and movement of OEE cells contributed to HERS formation after reduction of the growth of IEE. The expression pattern of paxillin was involved in the active movement of OEE and HERS. The results will contribute to understand the HERS formation mechanism and elucidate the cause of anomaly root.
Assuntos
Órgão do Esmalte/embriologia , Odontogênese/fisiologia , Coroa do Dente/embriologia , Germe de Dente/embriologia , Raiz Dentária/embriologia , Animais , Movimento Celular/fisiologia , Proliferação de Células , Esmalte Dentário/citologia , Esmalte Dentário/embriologia , Esmalte Dentário/crescimento & desenvolvimento , Órgão do Esmalte/citologia , Órgão do Esmalte/crescimento & desenvolvimento , Epitélio/embriologia , Epitélio/crescimento & desenvolvimento , Proteínas de Fluorescência Verde , Antígeno Ki-67/análise , Substâncias Luminescentes , Camundongos , Dente Molar/embriologia , Dente Molar/crescimento & desenvolvimento , Técnicas de Cultura de Órgãos , Paxilina/análise , Coroa do Dente/citologia , Coroa do Dente/crescimento & desenvolvimento , Germe de Dente/citologia , Germe de Dente/crescimento & desenvolvimento , Raiz Dentária/citologia , Raiz Dentária/crescimento & desenvolvimentoRESUMO
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.
Assuntos
Transportador de Glucose Tipo 1/metabolismo , Glucose/farmacocinética , Dente Molar/metabolismo , Odontogênese , Animais , Transporte Biológico/efeitos dos fármacos , Linhagem Celular , Relação Dose-Resposta a Droga , Órgão do Esmalte/embriologia , Órgão do Esmalte/crescimento & desenvolvimento , Órgão do Esmalte/metabolismo , Epitélio/embriologia , Epitélio/crescimento & desenvolvimento , Epitélio/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 2/genética , Transportador de Glucose Tipo 2/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Masculino , Camundongos , Camundongos Endogâmicos ICR , Dente Molar/embriologia , Dente Molar/crescimento & desenvolvimento , Floretina/farmacologia , Gravidez , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Técnicas de Cultura de Tecidos , Germe de Dente/embriologia , Germe de Dente/crescimento & desenvolvimento , Germe de Dente/metabolismoRESUMO
Perlecan, a heparan sulfate proteoglycan, is enriched in the intercellular space of the enamel organ. To understand the role of perlecan in tooth morphogenesis, we used a keratin 5 promoter to generate transgenic (Tg) mice that over-express perlecan in epithelial cells, and examined their tooth germs at tissue and cellular levels. Immunohistochemistry showed that perlecan was more strongly expressed in the enamel organ cells of Tg mice than in wild-type mice. Histopathology showed wider intercellular spaces in the stellate reticulum of the Tg molars and loss of cellular polarity in the enamel organ, especially in its cervical region. Hertwig's epithelial root sheath (HERS) cells in Tg mice were irregularly aligned due to excessive deposits of perlecan along the inner, as well as on the outer sides of the HERS. Tg molars had dull-ended crowns and outward-curved tooth roots and their enamel was poorly crystallized, resulting in pronounced attrition of molar cusp areas. In Tg mice, expression of integrin ß1 mRNA was remarkably higher at E18, while expression of bFGF, TGF-ß1, DSPP and Shh was more elevated at P1. The overexpression of perlecan in the enamel organ resulted in irregular morphology of teeth, suggesting that the expression of perlecan regulates growth factor signaling in a stage-dependent manner during each step of the interaction between ameloblast-lineage cells and mesenchymal cells.
Assuntos
Esmalte Dentário/metabolismo , Órgão do Esmalte/patologia , Regulação da Expressão Gênica , Proteoglicanas de Heparan Sulfato/metabolismo , Odontogênese , Citoesqueleto de Actina/metabolismo , Animais , Diferenciação Celular , Polaridade Celular , Células Cultivadas , Esmalte Dentário/embriologia , Esmalte Dentário/patologia , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/patologia , Desenvolvimento Embrionário , Órgão do Esmalte/embriologia , Órgão do Esmalte/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Proteoglicanas de Heparan Sulfato/genética , Imuno-Histoquímica , Endogamia , Integrina beta1/genética , Integrina beta1/metabolismo , Queratina-15 , Queratina-5/genética , Queratina-5/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Eletrônica , Plasmídeos/genética , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Dente/embriologia , Dente/metabolismo , Dente/patologia , Dente/ultraestrutura , Coroa do Dente/metabolismo , Raiz Dentária/embriologia , Raiz Dentária/metabolismo , Raiz Dentária/patologia , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Transgenes , Microtomografia por Raio-XRESUMO
OBJECTIVE: To investigate and compare the cellular expression of non-secreted Fgf11-14 and secreted Fgf15-18 and -20 mRNAs during tooth formation. MATERIALS AND METHODS: mRNA expression was analyzed from the morphological initiation of the mouse mandibular first molar development to the onset of crown calcification using sectional in situ hybridization. RESULTS: This study found distinct, differentially regulated expression patterns for the Fgf11-13, -15-17 and -20, in particular in the epithelial-mesenchymal interface, whereas Fgf14 and 18 mRNAs were not detected. Fgf11, -15, -16, -17 and -20 were seen in the epithelium, whereas Fgf12 and -13 signals were restricted to the mesenchymal tissue component of the tooth. Fgf11 was observed in the putative epithelial signaling areas, the tertiary enamel knots and enamel free areas of the calcifying crown. Fgf15, Fgf17 and -20 were transiently colocalized in the thickened dental epithelium at E11.5. Later Fgf15 and -20 were exclusively expressed in the epithelial enamel knot signaling centers. In contrast, Fgf13 was present in the dental mesenchyme including odontoblasts cell lineage, whereas Fgf12 appeared transiently in the preodontoblasts. CONCLUSIONS: The expression of the Fgf11-13, -15, -17 and -20 in the epithelial signaling centers and/or epithelial-mesenchymal interfaces at key stages of the tooth formation suggest important functions in odontogenesis. Future analyses of the transgenic mice will help elucidate in vivo functions of the studied Fgfs during odontogenesis and whether any of the functions of the tooth expressed epithelial and mesenchymal Fgfs of different sub-families are redundant.
Assuntos
Fatores de Crescimento de Fibroblastos/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Dente Molar/embriologia , Odontogênese/genética , Ameloblastos/citologia , Animais , Linhagem da Célula , Papila Dentária/embriologia , Órgão do Esmalte/embriologia , Epitélio/embriologia , Fatores de Crescimento de Fibroblastos/análise , Hibridização In Situ , Mesoderma/embriologia , Camundongos , Odontoblastos/citologia , Calcificação de Dente/genética , Coroa do Dente/embriologia , Germe de Dente/embriologiaAssuntos
Proteínas Facilitadoras de Transporte de Glucose/análise , Odontogênese/fisiologia , Dente/embriologia , Ameloblastos/citologia , Animais , Órgão do Esmalte/embriologia , Células Epiteliais/citologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas Facilitadoras de Transporte de Glucose/fisiologia , Transportador de Glucose Tipo 1/análise , Transportador de Glucose Tipo 2/análise , Camundongos , Camundongos Endogâmicos ICR , Odontoblastos/citologia , Técnicas de Cultura de Órgãos , Dente/crescimento & desenvolvimentoRESUMO
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.
Assuntos
Morfogênese/fisiologia , Odontogênese/fisiologia , Dente Decíduo/embriologia , Animais , Membrana Basal/embriologia , Dente Pré-Molar/embriologia , Diferenciação Celular/fisiologia , Proliferação de Células , Dente Canino/embriologia , Dentina/embriologia , Órgão do Esmalte/embriologia , Epitélio/embriologia , Processamento de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Incisivo/embriologia , Mesoderma/embriologia , Modelos Animais , Odontoblastos/citologia , Antígeno Nuclear de Célula em Proliferação/análise , Suínos , Porco Miniatura , Germe de Dente/embriologiaRESUMO
OBJECTIVE: Versican is a large, aggregating chondroitin sulphate proteoglycan. In dental tissue, versican expression occurs primarily in mesenchymal tissue but rarely in epithelial tissue. We investigated the expression, localisation and synthesis of versican in the enamel organ of the developing tooth germ. DESIGN: To elucidate versican localisation in vivo, in situ hybridisation and immunohistochemistry were conducted in foetal ICR mice at E11.5-E18.5. Epithelium and mesenchyme from the lower first molars at E16.0 were enzymatically separated and versican mRNA expression was investigated by semi-quantitative RT-PCR. Organ culture of the separated samples combined with metabolic labelling with [(35)S], followed by gel filtration, was performed to analyse secreted proteoglycans. RESULTS: Versican mRNA was first expressed in the thickened dental epithelium at E12.0 and continued to be expressed in the enamel organ until the bell stage. Versican immunostaining was detected in the stellate reticulum areas from the bud stage to the apposition stage. The enamel organ at E16.0 expressed versican mRNA at a level comparable to that in dental mesenchyme. Furthermore, when compared to dental mesenchyme, about 1/2-3/4 of the [(35)S]-labelled versican-like large proteoglycan was synthesised and released into tissue explants by the enamel organ. CONCLUSIONS: The dental epithelium of developing tooth germ is able to synthesise significant amounts of versican.