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*

Epithelial-mesenchymal interactions (EMIs) are critical for tooth development. Molecular mechanisms mediating these interactions in root formation is not well understood. Laser capture microdissection (LCM) and subsequent microarray analyses enable large scale in situ molecular and cellular studies of root formation but to date have been hindered by technical challenges of gaining intact histological sections of non-decalcified mineralized teeth or jaws with well-preserved RNA. Here,we describe a new method to overcome this obstacle that permits LCM of dental epithelia,adjacent mesenchyme,odontoblasts and cementoblasts from mouse incisors and molars during root development. Using this method,we obtained RNA samples of high quality and successfully performed microarray analyses. Robust differences in gene expression,as well as genes not previously associated with root formation,were identified. Comparison of gene expression data from microarray with real-time reverse transcriptase polymerase chain reaction (RT-PCR) supported our findings. These genes include known markers of dental epithelia,mesenchyme,cementoblasts and odontoblasts,as well as novel genes such as those in the fibulin family. In conclusion,our new approach in tissue preparation enables LCM collection of intact cells with well-preserved RNA allowing subsequent gene expression analyses using microarray and RT-PCR to define key regulators of tooth root development.
Animals ; Dental Cementum ; cytology ; metabolism ; Epithelial-Mesenchymal Transition ; physiology ; Gene Expression Regulation, Developmental ; Laser Capture Microdissection ; Mice ; Mice, Inbred Strains ; Odontoblasts ; metabolism ; Oligonucleotide Array Sequence Analysis ; Reverse Transcriptase Polymerase Chain Reaction ; Tooth Germ ; metabolism ; Tooth Root ; growth & development

Generation of bio-engineered teeth by using stem cells will be a major approach for bioengineered implantation. Previous studies have demonstrated that dissociated tooth germ cells are capable of generating a tooth after reaggregation in vitro. However, the cellular and molecular mechanisms underlying this tooth regeneration are not clear. In this study, we dispersed E13.5 molar germ into single cells, immediately reaggregated them into cell pellet, then grafted the reaggregates under mouse kidney capsule for various times of culture. We investigated the morphogenesis and the expression of several developmental genes in dental epithelial cells in reaggregates of tooth germ cells. We found that dissociated tooth germ cells, after reaggregation, recapitulated normal tooth developmental process. In addition, dissociated dental epithelial cells retained the expression of Fgf8, Noggin, and Shh during reaggregation and tooth regeneration processes. Our results demonstrated that, despite of under dissociated status, dental epithelial cells maintained their odontogenic fate after re-aggregation with dental mesenchymal cells. These results provided important information for future in vitro generation of bio-engineered teeth from stem cells.
Animals ; Cell Culture Techniques ; methods ; Cell Differentiation ; Embryo, Mammalian ; Epithelial Cells ; cytology ; metabolism ; Female ; Gene Expression Regulation, Developmental ; genetics ; Male ; Mice ; Odontogenesis ; genetics ; Tooth Germ ; cytology ; physiology

AIMTo detect the expression of HSP25 in rat dental follicles both in vivo and vitro, and explore the underlying mechanism of HSP25 on the proliferation and differentiation of rat dental follicle cells (DFCs).

METHODOLOGYImmunohistochemistry was performed to detect the expression of HSP25 in mandibles of postnatal rats on days 1, 3, 5, 7, 9 and 11 in vivo. In vitro, the expression of HSP25 in DFCs was detected by an indirect immunofluorescence assay. Thiazolyl blue tetrazolium bromide (MTT) assay, flow cytometry and alkaline phosphatase (ALP) assay were used to identify the time-course effect mediated by different concentrations of recombinant murine HSP25 of 0, 1, 10, 50 and 100 ng/mL on rat DFCs.

RESULTSExpression of HSP25 was not detected in dental follicles of the rats until day 5 after birth, but became up-regulated in a time-dependent manner till day 11. HSP25 was detected in the cytoplasm of cultured rat DFCs. No significant difference could be observed in the proliferation of DFCs after stimulation with different concentrations of HSP25 on days 1, 2 and 3 (P > 0.05). HSP25 at concentrations of 50 ng/mL and 100 ng/mL up-regulated the ALP activity of DFCs on day 9 (P < 0.05).

CONCLUSIONHSP25-immunoreactivity increased chronologically during the development of dental follicles. The protein had no significant effect on cell proliferation but may play a role in cementoblast/osteoblast differentiation of DFCs.

Alkaline Phosphatase ; analysis ; Ameloblasts ; cytology ; Animals ; Cell Culture Techniques ; Cell Differentiation ; physiology ; Cell Proliferation ; Coloring Agents ; Cytoplasm ; ultrastructure ; Dental Sac ; cytology ; growth & development ; Flow Cytometry ; Fluorescent Antibody Technique, Indirect ; HSP27 Heat-Shock Proteins ; analysis ; physiology ; Odontoblasts ; cytology ; Rats ; Rats, Sprague-Dawley ; Tetrazolium Salts ; Thiazoles ; Tooth Germ ; cytology ; Up-Regulation ; physiology

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 study the distribution and expression of fibromodulin, decorin and biglycan in developing normal periodontal tissues, so as to understand its role in periodontal tissue formation.

METHODSThirty six BALB/c mice in different developing stages were killed and their bilateral mandibular first molars with surrounding alveolar bones and gingival tissues were taken out, Power Vision two steps immunohistochemical method with anti-fibromodulin, anti-decorin and anti-biglycan was used to detect the tissue distribution and cellular localization of fibromodulin and related proteoglycans, decorin and biglycan.

RESULTSFibromodulin was strongly expressed in the subcutaneous gingival connective tissue, periodontal ligament, mainly in gingival and periodontal fibroblasts as well as their matrices. Strong expression was also noted in the area close to the interfaces of periodontal ligament-alveolar bone and periodontal ligament-cementum. Decorin was strongly expressed in the area of gingival connective tissue, periodontal ligament and the surface of alveolar bone, while biglycan was stained evidently in gingival connective tissue throughout the period of investigation, but negative in the surface of alveolar bone and osteoblasts.

CONCLUSIONSFibromodulin may interact with decorin and biglycan to regulate the network formation of gingival connective tissues and periodontal collagen fibers, and may be involved in mineralization of the alveolar bone and cementum.

Alveolar Process ; cytology ; growth & development ; Animals ; Biglycan ; Decorin ; Extracellular Matrix Proteins ; analysis ; Fibromodulin ; Gingiva ; chemistry ; growth & development ; Immunohistochemistry ; Mice ; Mice, Inbred BALB C ; Osteoblasts ; chemistry ; Periodontal Ligament ; chemistry ; growth & development ; Proteoglycans ; analysis ; Tooth Germ ; chemistry

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

OBJECTIVETo investigate the expression and biological effect of Shh during late bell stage by morphological and semi-quantitative analysis.

METHODSTooth germs were selected from new born Bal b/c mouse (P1, P2, P3, P5, P7). Semi-quality of Shh was measured by Western Blot and the expression place and strength of Shh were observed by in situ hybridization.

RESULTSShh was expressed in the ameloblast layer during late bell stage; the expression strength was high in secretive period and decreased with development; the active N-section was detectable before P3.

CONCLUSIONShh expresses specially in the ameloblast layer in late bell stage, and expression quality is related to the function of ameloblasts.

Ameloblasts ; metabolism ; Animals ; Blotting, Western ; Gene Expression ; Hedgehog Proteins ; In Situ Hybridization ; In Vitro Techniques ; Mice ; Mice, Inbred BALB C ; Tooth Germ ; cytology ; metabolism ; Trans-Activators ; biosynthesis ; genetics