The regulatory processes in developmental biology research are significantly influenced by long non-coding RNAs (lncRNAs). However, the dynamics of lncRNA expression during human tooth development remain poorly understood. In this research, we examined the lncRNAs present in the dental epithelium (DE) and dental mesenchyme (DM) at the late bud, cap, and early bell stages of human fetal tooth development through bulk RNA sequencing. Developmental regulators co-expressed with neighboring lncRNAs were significantly enriched in odontogenesis. Specific lncRNAs expressed in the DE and DM, such as PANCR, MIR205HG, DLX6-AS1, and DNM3OS, were identified through a combination of bulk RNA sequencing and single-cell analysis. Further subcluster analysis revealed lncRNAs specifically expressed in important regions of the tooth germ, such as the inner enamel epithelium and coronal dental papilla (CDP). Functionally, we demonstrated that CDP-specific DLX6-AS1 enhanced odontoblastic differentiation in human tooth germ mesenchymal cells and dental pulp stem cells. These findings suggest that lncRNAs could serve as valuable cell markers for tooth development and potential therapeutic targets for tooth regeneration.
Humans ; RNA, Long Noncoding/metabolism* ; Odontogenesis/genetics* ; Tooth Germ/embryology* ; Cell Differentiation ; Gene Expression Regulation, Developmental ; Mesoderm/metabolism* ; Tooth/embryology* ; Gene Expression Profiling ; Sequence Analysis, RNA ; Dental Pulp/cytology*

Positional information plays a crucial role in embryonic pattern formation, yet its role in tooth development remains unexplored. In this study, we investigated the regional specification of lingual and buccal dental mesenchyme during tooth development. Tooth germs at the cap stage were dissected from mouse mandibles, and their lingual and buccal mesenchymal regions were separated for bulk RNA sequencing. Gene ontology analysis revealed that odontogenesis, pattern specification, and proliferation-related genes were enriched in the lingual mesenchyme, whereas stem cell development, mesenchymal differentiation, neural crest differentiation, and regeneration-related genes were predominant in the buccal mesenchyme. Reaggregation experiments using Wnt1^creERT/+; R26R^tdT/+ and WT mouse models demonstrated that lingual mesenchyme contributes to tooth formation, while buccal mesenchyme primarily supports surrounding tissues. Furthermore, only the lingual part of tooth germs exhibited odontogenic potential when cultured in vitro and transplanted under the kidney capsule. Bulk RNA transcriptomic analysis further validated the regional specification of the lingual and buccal mesenchyme. These findings provide novel insights into the molecular basis of positional information in tooth development and pattern formation.
Animals ; Mice ; Odontogenesis/genetics* ; Tooth Germ/cytology* ; Mesoderm/cytology* ; Cell Differentiation ; Mesenchymal Stem Cells ; Tooth/embryology*

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.
Animals ; Cell Differentiation ; physiology ; DNA-Binding Proteins ; analysis ; Dental Papilla ; embryology ; Embryo, Mammalian ; Enamel Organ ; embryology ; Enhancer of Zeste Homolog 2 Protein ; Epigenesis, Genetic ; physiology ; Gene Expression Regulation, Developmental ; Histone-Lysine N-Methyltransferase ; analysis ; Histones ; metabolism ; Jumonji Domain-Containing Histone Demethylases ; analysis ; Lysine ; metabolism ; Methylation ; Mice ; Mice, Inbred BALB C ; Odontogenesis ; physiology ; Polycomb Repressive Complex 2 ; analysis ; Protein Processing, Post-Translational ; physiology ; Tooth Germ ; embryology

Tooth replacement is a common trait to most vertebrates, including mammals. Mammals, however, have lost the capacity for continuous tooth renewal seen in most other vertebrates, and typically have only 1-2 generations of teeth. Here, we review the mechanisms of tooth replacement in reptiles and mammals, and discuss in detail the current and historical theories on control of timing and pattern of tooth replacement and development.
Animals ; Biology ; Humans ; Mammals ; physiology ; Odontogenesis ; genetics ; physiology ; Reptiles ; physiology ; Tooth ; growth & development ; Tooth Germ ; embryology ; Tooth, Deciduous ; growth & development

AIMTo characterize the odontogenic capability of apical bud and phenotypical change of apical bud cells (ABCs) in different microenvironment.

METHODOLOGYIncisor apical bud tissues from neonatal SD rat were dissected and transplanted into the renal capsules to determine their odontogenic capability. Meanwhile ABCs were cultured and purified by repeated differential trypsinization. Then ABCs were cultured with conditioned medium from developing apical complex cells (DAC-CM). Immunocytochemistry, reverse transcriptase polymerase chain reaction (RT-PCR) and scanning electron microscope (SEM) were performed to compare the biological change ofABC treated with or without DAC-CM.

RESULTSFirst we confirmed the ability of apical bud to form crown-like structure ectopically. Equally important, by using the developing apical complex (DAC) conditioned medium, we found the microenvironment created by root could abrogate the "crown" features of ABCs and promote their proliferation and differentiation.

CONCLUSIONABCs possess odontogenic capability to form crown-like tissues and this property can be affected by root-produced microenvironment.

Ameloblasts ; cytology ; Amelogenin ; analysis ; Animals ; Animals, Newborn ; Cell Culture Techniques ; Cell Differentiation ; physiology ; Cell Proliferation ; Cell Transplantation ; Culture Media, Conditioned ; Dental Enamel Proteins ; analysis ; Epithelial Cells ; cytology ; Immunohistochemistry ; Incisor ; cytology ; embryology ; Keratin-14 ; analysis ; Kidney ; surgery ; Microscopy, Electron, Scanning ; Odontogenesis ; physiology ; Phenotype ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Tooth Apex ; cytology ; Tooth Crown ; cytology ; Tooth Germ ; cytology

OBJECTIVETo clone PAK5-N terminal sequence for expression in E. coli to prepare its polyclonal antibody, and examine the role of PAK5 in dental germ cells.

METHODSBased on human PAK5 cDNA sequence, PCR primers were designed to amplify PAK5-N terminal sequence. The PCR product was cloned into the expression vector pGEX-4T-1 EcoRI/XhoI sites, and the recombinant plasmids were identified by agarose gel electrophoresis followed by DNA sequence analysis. The recombinant plasmids were transformed into E. coli BL21 and the expression of GST-fusion protein was induced by IPTG. Glutathione-Sepharose beads were used to purify GST-fusion PAK5-N-terminal fragment. Anti-PAK5 polyclonal antibody was obtained in immunizing rabbits with purified GST-PAK5 N-terminal fusion protein, and the antibodies were purified by protein A beads and used for detection of PAK5 expression in dental germ cells.

RESULTS AND CONCLUSIONSWe successfully cloned PAK5-N terminal gene fragment, and achieved protein expression, purification and production of PAK5-NT polyclonal antibody. The results of Western blotting indicated that PAK5 can be highly expressed in the dental germ cells, suggesting that PAK5 may play an important role in biological function of dental germ cells.

Animals ; Antibodies, Monoclonal ; biosynthesis ; genetics ; immunology ; Base Sequence ; Blotting, Western ; Cloning, Molecular ; Humans ; Molecular Sequence Data ; Rabbits ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Sequence Analysis, DNA ; Tooth Germ ; cytology ; embryology ; enzymology ; p21-Activated Kinases ; biosynthesis ; genetics ; immunology

OBJECTIVETo observe the transcriptional expression of enamelin in developing postnatal rat first mandibular molar germs, for further studies of functions of enamelin in enamel development and mineralization.

METHODSTissue slices of first mandibular molar germ of rat 1, 3, 7, 10, 14 days after birth were prepared. The enamelin mRNA expression was identified by in situ hybridization.

RESULTSEnamelin mRNA was observed in both ameloblast and odontoblast in 1-10 day old rat postnatal first mandibular molar germs. Enamelin mRNA appeared very weakly at 1st day, and increased through 3rd day, reached the maximum at 7th day, and reduced at 10th day and became negative at 14th day postnatally; while the expression of enamelin mRNA in odontoblast maintained lower from 1st to 10th day and negative at 14th day postnatally.

CONCLUSIONEnamelin gene transcriptional expression lasts from preameloblast to maturation ameloblast, which suggests that enamelin may participate in the development of enamel and mantle dentin.

Ameloblasts ; metabolism ; Animals ; Dental Enamel Proteins ; biosynthesis ; genetics ; Gene Expression Regulation ; In Situ Hybridization ; Molar ; embryology ; Odontoblasts ; metabolism ; RNA, Messenger ; analysis ; Rats ; Tooth Germ ; growth & development ; metabolism ; Transcription, Genetic

OBJECTIVETo establish a new culture system for mouse tooth germs in chick embryo.

METHODSThe mandibular first molar germ fragments of 15 embryonic days' Kunming mouse embryo were implanted into the lateral mesenchyme of 4-5 days' chick embryo wing buds in ove. Eggs were reincubated and implanted tissues were examined by histochemistry.

RESULTSThe cultured tooth germ development continued from cap stage to latest bell stage. The ameloblast and the odontoblast all differentiated maturely and secreted matrix.

CONCLUSION4-5 days' wing buds chick embryo could serve as developing the mouse tooth germs and demonstrate well physiological process of differentiation and morphogenesis.

Ameloblasts ; Animals ; Chick Embryo ; Mice ; Molar ; embryology ; Odontoblasts ; Tooth Germ ; embryology

OBJECTIVETo study the expression of MMP-8 in human and rat tooth development.

METHODSImmunohistochemistry was used to detect the localization of MMP-8 protein while in situ hybridization was used to examine the expression of MMP-8 mRNA.

RESULTSThe expression of MMP-8 protein was localized in odontoblast and dentin matrix at the later bell stage in human tooth germ. The dentin was denser close to the pulp cavity. The expression of MMP-8 mRNA was found in very few polarized odontoblast at the early bell stage and all polarized odontoblast at the later bell stage in rat tooth germ.

CONCLUSIONThe results suggested that MMP-8 involved in dentin matrix rebuilding in the process of dentin formation in human and rat dental development.

Animals ; Animals, Newborn ; Dentin ; enzymology ; Embryo, Mammalian ; In Situ Hybridization ; Matrix Metalloproteinase 8 ; biosynthesis ; genetics ; Maxilla ; enzymology ; Odontogenesis ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Sprague-Dawley ; Tooth Germ ; embryology ; enzymology

OBJECTIVETo observe the temporal and spatial expression of Smad7 during human tooth germ development and evaluate the effect of Smad7 on tooth germ development.

METHODSThe expression of Smad7 and its changes at different stages of human tooth germ were detected by using immunohistochemical staining.

RESULTSSmad7 was expressed at all stages of tooth germ, but the distribution patterns at various stages were different. It indicated that temporal and spatial expressing mode of Smad7 during human tooth germ development was specific, which was similar to that of TGF-beta its signal transducer Smad2/3.

CONCLUSIONSmad7 might play an important role in TGF-beta intracellular signaling for modulating the differentiation of ameloblasts and odontoblasts.

Ameloblasts ; cytology ; Cell Differentiation ; DNA-Binding Proteins ; genetics ; metabolism ; physiology ; Fetus ; Humans ; Immunohistochemistry ; Odontoblasts ; cytology ; Odontogenesis ; Signal Transduction ; Smad7 Protein ; Tooth ; growth & development ; Tooth Germ ; embryology ; Trans-Activators ; genetics ; metabolism ; physiology ; Transforming Growth Factor beta ; genetics ; metabolism ; physiology