Real-time quantitative PCR analysis of mouse ameloblasts
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE233825 | GEO | 2023/06/01
REPOSITORIES: GEO
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Project description:Tooth and hair development starts from ectodermal invaginations. However, the determination of organ-specific cell fates is poorly understood. The transcription factor Sox21 is expressed in the epithelium of developing teeth. We discovered that disruption of Sox21 caused severe enamel hypoplasia and ectopic hair formation in the gingiva in Sox21 knockout incisors. We dissected tooth germ from P1 Sox21 KO and control incisors for microarray. In particular, several markers of ameloblast maturity, i.e. amelotin (Amtn), laminin α3 (Lama3), and kallikrein-related peptidase 4 (Klk4), were included among the downregulated molecules in Sox21 KO tooth germ, whereas the expression of Mmp20 and Amel was not changed. On the other hand, numerous keratin-related genes were upregulated in the Sox21 KO tooth germ and WT skin. As in the microarray results, the expression of dental epithelial stem cell marker Sox2, as well as keratin family and hair-related genes such as Lgr5, were increased in Sox21 KO dental epithelium. Taken together, these results reflected the perturbed differentiation of ameloblasts and a cell fate change in the Sox21 KO incisors.
2020-01-01 | GSE99359 | GEO
Project description:Ameloblast differentiation is the most critical stepwise process in amelogenesis, and it is controlled by precise molecular events. To better understand the mechanism controlling pre-ameloblasts (PABs) differentiation into secretory ameloblasts (SABs), a more precise identification of molecules and signaling networks will elucidate the mechanisms governing enamel formation and lay a foundation for enamel regeneration. We analyzed transcriptional profiles of human PABs and SABs. From a total of 28,869 analyzed transcripts, we identified 923 differentially expressed genes (DEGs) with p < 0.05 and Fold-change > 2. Among the DEGs, 647 genes showed elevated expression in PABs compared to SABs. Notably, 38 DEGs displayed more than eight-fold changes. Comparative analysis revealed that highly expressed genes in PABs were involved in cell cycle control, DNA damage repair and apoptosis, while highly expressed genes in SABs were related to cell adhesion and extracellular matrix. Moreover, coexpression network analysis uncovered two highly conserved sub-networks contributing to the differentiation, containing transcription regulators (RUNX2, ETV1 and ETV5), solute carrier family members (SLC15A1 and SLC7A11), enamel matrix protein (MMP20), and a polymodal excitatory ion channel (TRPA1). By combining comparative analysis and coexpression networks, this study provides novel biomarkers and research targets for ameloblast differentiation and the potential for their application in enamel regeneration.
2014-10-01 | GSE59214 | GEO
Project description:Background: Ameloblast differentiation is the most critical stepwise process in amelogenesis and controlled by a precisely molecules synergistically. To better understand the molecular events defining cell differentiation between preameloblasts and secretory ameloblasts during amelogenesis, a more precise identification of molecules and signaling networks would shed light on the mechanisms governing enamel formation and help lay a foundation for enamel regeneration. Results: Gene expression profiles of human preameloblast and secretory ameloblast cells were obtained using human genome microarrays. From a total of 28,869 analyzed transcripts, 923 differentially expressed genes (DEGs) with FDR<0.01 and Fold-change > 2 were obtained. Among them, more than twice DEGs were found enriched in PAB (n = 647) compared with SAB (n = 276). Notably, 38 genes were identified significantly differentially expressed between PAB and SAB (Fold Change > 8). Comparison of transcriptional profiles of PAB and SAB together with KEGG pathway analysis revealed genes enrichment in PAB were chiefly involved in cell cycle control, DNA damage repair and apoptosis, while genes related to cell adhesion and extracellular matrix had elevated expression level in SAB. Two co-expression modules were further identified significantly associated with the ameloblast differentiation process by weighted gene co-expression network analysis (WGCNA).These gene networks seem to contribute to cell adhesion, tissue development, cell signaling and division. Furthermore, the co-expression associations of RunX2 and BMP8A were also observed in these modules. Conclusions: In this study, we uncovered that the differentiation from PAB to SAB may rely on a highly regulated network of interactions between conserved signal transduction pathways, including members of BMP/TGF-β, Notch, MAPK pathways to coordinate all aspects of ameloblast in intracellular processes and their social contexts. Specifically, expression of genes associated with cell cycle control, DNA damage repair, and apoptosis pathways regulates pre-ameloblast maturation during tooth development. And the SAB cells are regulated by several signaling pathways controlling enamel matrix proteins secretion and cell adhesion, which are critical for enamel formation and cell-cell interactions. Apart from showing the transcriptional patterns of PAB and SAB, the application of bioinformatic analysis also explored the potential key genes and gene-associations in ameloblast differentiation. These findings will aid in the design of new strategies to promote ameloblast functional differentiation in the regeneration and tissue engineering of teeth. Human tooth buds (18-22 weeks) were obtained from fetal cadaver tissue within 3 hours after legal abortion. Teeth were dissected from the mandibles under a laminar flow hood, embedded in OCT compound, and cryosectioned at 10-μm thickness. These sections were used for laser capture microdissection (LCM). In total of 3 pre-ameloblasts and 3 secretory ameloblasts pooled samples were used for RNA extraction and hybridization on Affymetrix microarray.
2014-10-01 | E-GEOD-59214 | biostudies-arrayexpress
Project description:Dental enamel, the hardest tissue in the human body, is derived from dental epithelial cell ameloblast-secreted enamel matrices. Enamel mineralization occurs in a strictly synchronized manner along with ameloblast maturation in association with ion transport and pH balance, and any disruption of these processes results in enamel hypomineralization. G-protein coupled receptors (GPCRs) function as transducers of external signals by activating associated G-proteins and regulate cellular physiology. Tissue-specific GPCRs play important roles in organ development, though their activities in tooth development remains poorly understood. The present results show that the adhesion-GPCR Gpr115 (Adgrf4) is highly and preferentially expressed in mature ameloblasts and plays a crucial role during enamel mineralization. To investigate the in vivo function of Gpr115, knockout (Gpr115-KO) mice were created and found to develop hypo-mineralized enamel, with a larger acidic area due to the dysregulation of ion composition. Transcriptomic analysis also revealed that deletion of Gpr115 disrupted pH homeostasis and ion transport processes in enamel formation. In addition, in vitro analyses using the dental epithelial cell line Cervical Loop-Derived Dental Epithelial (CLDE) cell demonstrated that Gpr115 is indispensable for the expression of carbonic anhydrase 6 (Car6), which has a critical role in enamel mineralization. Furthermore, an acidic condition induced Car6 expression under the regulation of Gpr115 in CLDE cells. Thus, we concluded that Gpr115 plays an important role in enamel mineralization via regulation of Car6 expression in ameloblasts. The present findings indicate a novel function of Gpr115 in ectodermal organ development and clarify the molecular mechanism of enamel formation.
2020-08-31 | GSE155641 | GEO
Project description:Dental epithelial stem cells give rise to all dental epithelial cell types, inner enamel epithelium, outer enamel epithelium, ameloblasts, stratum intermedium and stellate reticulum to form enamel. These all types of epithelial cells have distinct roles and are essential for enamel formation. These cell types are conventionally classified by their cell shape, however, the transcriptome and biological roles of each cell types are not fully understood. Here, we used single-cell RNA-sequencing to clarify the heterogeneity of dental epithelial cell types. Unbiased clustering of 6260 single-cells from post-natal day 7 mouse incisors are classified into 4 dental epithelial and 2 mesenchymal populations; 2 clusters of ameloblast, IEE/OEE and SI/SR clusters. Secretory-stage of ameloblasts were divided into Dspp+ or Ambn+ ameloblast. Pseudo-time analysis indicated that Dspp+ ameloblast differentiate into Ambn+ ameloblast. Further, Dspp and Ambn would be stage-specific markers of ameloblast. Gene Ontology (GO) analyses of each clusters indicate potent roles of cell types; OEE in regulation of tooth size and SR in transport of nutrition. Moreover, we identified novel dental epithelial cell marker genes, Pttg1, Atf3, Cldn10 and Krt15. These results provide a resource of transcriptome data in dental cells and contribute further molecular analyses of enamel formation.
2020-09-23 | GSE146855 | GEO
Project description:We used RNA-seq to identify differentially expressed genes across ameloblast stages in rats. We found expression patterns of enamel matrix proteins, ion channels and transporters, and endocytosis regulators such as clathrin to match those found in previous experimental studies on ameloblasts. We hypothesized that ameloblasts change their endocytotic pathways as they progress from stage and stage, and we sought to identify novel patterns in expression of genes at the endocytosis/cytoskeleton interface. We found that maturation ameloblasts preferentially express synaptojanin 1 and intersectin 2 and secretory ameloblasts preferentially express Numb protein and amphiphysin. We also uncovered differential alternative splicing of ECI genes across ameloblast populations. In pointing to endocytotic genes hitherto unexplored in ameloblasts that also regulate cell morphology, this bioinformatics study suggests a mechanism by which cervical loop, secretory, and maturation ameloblasts switch modes of endocytosis in order to express genes that better suit the distinct morphology of each cell stage.
2021-07-14 | GSE133330 | GEO
Project description:DLX3 is a homeodomain transcription factor involved in ameloblast differentiation and enamel formation. Mutations in DLX3 in human lead to defects in enamel. However, the downstream targets of Dlx3 transcriptional activity in the enamel organ have not been identified yet. In this study, we compared the transcriptome of enamel organs where Dlx3 has been deleted to control tissues. Total RNA was extracted from enamel organs (mandibular incisors) from Dlx3-WT (N=4) and Dlx3-K14cKO (N=4) mice at P10. cDNA libraries were generated using NEBnext and NuGen kits. Sequencing was performed on the HiSeq2000.
2015-05-14 | E-GEOD-57984 | biostudies-arrayexpress
Project description:Tooth enamel secreted by ameloblasts is the hardest material in the human body, acting as a shield protecting the teeth. However, enamel is gradually damaged or partially lost in over 90% of adults and cannot be regenerated due to a lack of ameloblasts in erupted teeth. Here we use sci-RNA-seq to establish a spatiotemporal single cell atlas for the developing human tooth and identify regulatory mechanisms controlling the differentiation process of human ameloblasts. We reveal key signaling pathways involved between the support cells and ameloblasts during fetal development and recapitulate those findings in a novel human ameloblast in vitro differentiation from iPSCs. We furthermore develop a mineralizing enamel organ-like 3D organoid system. These studies pave the way for future regenerative dentistry and therapies toward genetic diseases affecting enamel formation.
2023-01-11 | GSE184749 | GEO
Project description:Coordinated mineralization of soft tissue is central to organismal form and function, while dysregulated mineralization underlies several human pathologies. Oral epithelial derived ameloblasts are polarized, secretory cells responsible for generating enamel, the most mineralized substance in the human body. Defects in ameloblast development result in enamel anomalies, including amelogenesis imperfecta. Identifying proteins critical in ameloblast development can provide insight into specific pathologies associated with enamel related disorders or more broadly, mechanisms of mineralization. Previous studies identified a role for MEMO1 in bone mineralization; however, whether MEMO1 functions in the generation of additional mineralized structures remains unknown. Here, we identify a critical role for MEMO1 in enamel mineralization. First, we identified that Memo1 is expressed in ameloblasts and that conditional deletion of Memo1 from ameloblasts results in enamel defects, characterized by a decline in mineral density and tooth integrity. Molecular profiling of ameloblasts and their progenitors in Memo1 oral epithelial mutants revealed a disruption to cytoskeletal associated genes and a reduction in late stage ameloblast markers, relative to controls. Histology revealed that the molecular defects correlated with a disruption of the apical Tome’s process and the basolateral interacting, papillary layer, in Memo1 mutant ameloblasts. Finally, deletion of Memo1 from an oral epithelial ameloblast cell line, followed by cellular and molecular analyses, revealed altered cytoskeletal networks, relative to control cells. Collectively, our findings integrate MEMO1 into an emerging network of molecules important for ameloblast development and provide both in vivo and in vitro systems to further interrogate the relationship of cytoskeletal and amelogenesis-related defects.
2023-08-03 | GSE235869 | GEO
Project description:Little is known about the role of cell-cell adhesion in the development of mineralized tissues. Here we report that PERP, a tetraspan membrane protein essential for epithelial integrity, is a critical regulator of enamel formation. Perp is necessary for proper cell attachment and gene expression during tooth development, and its expression is controlled by P63, a master regulator of stratified epithelial development. During enamel formation, PERP is localized to the interface between the enamel-producing ameloblasts and the stratum intermedium (SI), a layer of cells subjacent to the ameloblasts. Perp-null mice display dramatic enamel defects, which are caused, in part, by the detachment of ameloblasts from the SI. Microarray analysis comparing gene expression in teeth of wild-type and Perp-null mice identified several differentially expressed genes during enamel formation. Analysis of these genes in ameloblast-derived LS8 cells upon knock down of PERP confirmed the role for PERP in the regulation of gene expression. Together, our data show that PERP is necessary for the integrity of the ameloblast-SI interface and that the lack of Perp causes downregulation of genes that are critical for proper enamel formation. Two-condition experiment, RNA isolated from WT vs. Perp-null lower 1st molars. Biological replicates: 3 control replicates, 3 mutant replicates.
2011-01-22 | E-GEOD-26796 | biostudies-arrayexpress