Identification of important genes for tooth development using single cell RNA-sequence datasets of sequential developing mouse molars
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE260975 | GEO | 2025/12/24
REPOSITORIES: GEO
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Project description:The organ development is dictated by the genes preferentially expressed in tissue or cell types uniquely. The clarification of gene expression profile and identification of specific genes in organ provide detailed understanding of organogenesis. Toward this purpose, the genome-wide analysis is a growing powerful tool for understanding development processes in organogenesis. Tooth is composed from enamel, dentin and cementum and these tissues are identical from other organs. The tooth-forming cell types are therefore unique and have complexed organization. The mechanisms of signal induction during tooth development is complicated because of the function of each cell types are still unclear. Previously, we performed the CAGE (Cap Analysis of Gene Expression) using mouse tooth germ to identify the genes preferentially expressed in tooth. The CAGE has advantage in quantification because it counts short reads of 5’ end without the bias of transcript length. Single-cell RNA-sequence (scRNA-seq) is a suitable tool revealing gene expression of each cells. The fundamental question of this study is how we can identify the tooth-specific and cell-type specific genes. In this study, we approach this using combination of scRNA-seq and CAGE (Cap Analysis of Gene Expression) as bioinformatics analyses. We obtained the scRNA-seq datasets of 12,212 cells from postnatal-day (P) 1 mouse molars, and the CAGE-seq datasets from P1 molars. scRNA-seq analysis revealed the spatio-temporal expression of tooth-related genes and CAGE determined whether these genes are truly tooth-specific or are expressed in ubiquitously. Furthermore, we identified candidate genes as novel tissue- and cell type- specific markers. Our results show that the integration of scRNA-seq and CAGE-seq improves the highlight genes important for tooth development from numerous gene profiles. These findings contribute resolving the mechanism of tooth development and establish the basis of tooth regeneration in future.
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Project description:Throughout the various stages of tooth development, reciprocal epithelial-mesenchymal interactions are the driving force, for instance crucially involved in the differentiation of mature enamel-forming ameloblasts and dentin-producing odontoblasts. Here we established mouse tooth ‘assembloids’, comprised of tooth organoid-derived dental epithelial cells (from mouse molars and incisors) cultured together with molar dental pulp stem cells (DPSCs), to mimic these developmental interactions. Assembloids from both tooth types were grown both in basal- and differentiation-inducing conditions. Single cell transcriptomics analysis was applied to in detail characterize and validate the newly developed mouse tooth assembloid model and evaluate the induced differentiation processes.
2023-04-19 | E-MTAB-12544 | biostudies-arrayexpress
Project description:Lipid rafts are membrane microdomains rich in cholesterol, sphingolipids, glycosylphosphatidylinositol-anchored proteins (GPI-APs), and receptors. They are localized at the plasma membrane and are essential for signal transmission and organogenesis. However, few reports on the specific effects of lipid rafts on organogenesis have been reported. Here, we focused on the odontoblast differentiation that secretes the dentin matrix and generates dentin. We found the GPI-AP, lymphocyte antigen-6 (Ly6)/Plaur domain-containing1 (Lypd1), specifically expressed in the dental papilla, especially preodontoblasts, using single-cell RNA sequence (scRNA-seq). Depletion experiments of lipid rafts and Lypd1 using ex vivo culture and cell cultures showed that differentiation of tooth germ and dental mesenchymal cells was inhibited. Furthermore, the C-terminal structural domain containing the omega site, which is necessary to localize the plasma membrane, of Lypd1 is necessary for odontoblast differentiation and the morphological change like odontoblasts. Stimulating downstream Bone morphogenetic protein (Bmp) signaling by BMP2 caused it to activate the phosphorylation of Smad1/5/8 and promote the expression of odontoblast differentiation markers such as Pannexin 3 (Panx3), Alkaline phosphatase (Alp), and Dentin sialophosphoprotein (Dspp). In contrast, suppression of Lypd1 inhibited the phosphorylation of Smad1/5/8. In addition, the expression of Lypd1 predates any other marker of odontoblast differentiation previously described. That implies that Lypd1 could be employed as a novel earlier differentiation marker of odontoblasts. These results suggest that Lypd1 as GPI-AP of lipid rafts is important for tooth organogenesis and plays a pivotal role in odontoblast differentiation by regulating phosphorylation of Smad1/5/8.
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Project description:Equine odontoclastic tooth resorption and hypercementosis (EOTRH) is a relatively recently described dental disorder, commonly found in horses above the age of 15 years old (Carsten Staszyk et al. 2008). The disease was first categorised as EOTRH in 2008, following the identification of clinical cases involving severe resorptive lesions and cementomas forming on the lower portion of equine incisors (Kreutzer et al. 2007) (Baratt 2007). The two main disease indicators are resorptive lesions caused by odontoclasts and hypercementosis, characterised by reparative/irregular cementum deposition, forming bulbous-like features (Schätzle, Tanner, and Bosshardt 2005). Horses with severe EOTRH often present dysphagia, pain whilst eating and behavioral changes, which may lead to further unrelated complications (Earley and Rawlinson 2013). Some suggested that Warmbloods and Thoroughbreds are more commonly diagnosed (Sykora et al, 2014; Smedley et al, 2015; Lorello et al, 2016; Earley et al, 2017), although others state there are no appar¬ent breed associations (James, 2022; Tretow et al, 2023). Furthermore, in a limited group of Icelandic horses, a diagnosis was established in 72.2% of cases (Tretow, Hain, and Bienert-Zeit 2023). While radiography is the primary diagnostic method for EOTRH after clinical examination, recent studies have indicated that this approach may lack sensitivity for early disease detection, potentially impeding treatment if the condition has advanced significantly (Albers, Bienert-Zeit, and Staszyk 2022). A comparable ailment observed in cats is Feline Odontoclastic Resorptive Lesions (FORL). In this species there was a prevalence of 29% , and similar to EOTRH, it exhibited an elevated risk associated with age (Gorrel 2014). The underlying causes of EOTRH are not yet fully understood with many potential factors considered, including age, sex, breed, diet and environment (Pearson et al. 2013). One of the first theories suggest a sequence of events beginning with periodontal inflammation leading to resorptive lesions and subsequent cementum production (Carsten Staszyk et al. 2008). Other studies found gram-negative bacteria (Treponema, Tannerella) were more commonly identified in the gingival crevicular fluid of diseased horses, highlighting the potential importance of the oral microbiome in disease aetiology (Sykora et al. 2013). Current treatment involves extraction of affected teeth. However there is an increased likelihood of neighboring teeth developing EOTRH (Rahmani et al. 2019). Presently there are no reported disease modifying treatments available to treat EOTRH and symptom management and pain reduction are the current gold standard approaches (Foster 2013). Whilst the cementum of teeth is the most effected dental tissue other types of dental tissues include dentine, enamel, pulp and the periodontium. The latter consists of cementum, periodontal ligament and alveolar bone. Compositionally cementum is similar to bone with 55% organic material and 45% inorganic hydroxyapatite (Tziafas 2005). It covers the entire length of equine teeth, potentially explaining the hyperactivity of cementum-producing cells (cementoblasts) in EOTRH [C. Staszyk, Suske, and Pöschke (2015)](Bosshardt 2005). Coronal cementum acts as a support to enamel, whilst cementum nearer to the roots is primarily an interface between tooth and the periodontal ligament (PDL). Finally a third type of cementum is present within the infundibulum near the coronal surface of the tooth. Irregular cementum has been shown to be deposited during EOTRH at both resorbed and non-resorbed surfaces along the tooth-PDL interface. Proteomic tools have been utilised to analyse clinical samples in both normal and disease-specific states. Specifically in dental tissues proteomics has been used to map proteomes of enamel, dentin, cementum and pulp in human teeth [Green et al. (2019)](Widbiller et al. 2019)[Giovani et al. (2020)](Eckhardt et al. 2014). As well as this human dental diseases, such as gingivitis, caries and periodontal disease have all been investigated with a similar approach [Gonçalves et al. (2011)](Wang et al. 2018)(Hartenbach et al. 2020). There have been no studies performed to identify potential pathological distinctions between the cementum tissues from healthy horses and those with EOTRH. We hypothesised that global protein changes in horses with and without EOTRH would increase the understanding of underlying pathways and disease mechanisms enabling insight into aetiologies. In addition, by understanding the pathophysiological mechanisms related to EOTRH this could provide treatment targets to delay or prevent disease progression. To investigate protein profiles we used unbiased label-free mass spectrometry-based proteomics.
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Project description:Cementoblasts are cells that line the surface of the tooth root and produce a mineralized tissue known as cementum. Cementum is critical for attachment of the tooth to the adjacent periodontal ligament and has a similar biochemical composition to bone, however it differs in that it lacks vasculature and innervation, and does not undergo remodelling. It has been difficult to define markers that specifically identify cementoblasts. In order to compare the transcriptome of fully developed cementoblasts with neural crest derived (calvarial) osteoblasts in vivo, we used osteocalcin-GFP (OC-GFP) mice. Cells were isolated by digestion of calvaria (osteoblasts) or extracted molars (cementoblasts) and purified by FACS sorting for GFP positive cells before RNA extraction and microarray analysis.
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Project description:Establishment of human teeth relies on coordinated formation of dentin-pulp complex for nurturing and periodontal tissues for anchoring in alveolar bone. The exact cell origin of dentin-pulp complex and periodontal tissues, and the integrative developmental process remain elusive. Here, we identified a bilaminar core of Cd24a+ and Pax9+ stem cells that governs the tooth establishment and persists into adulthood. Specifically, Cd24a+ stem cells gave rise to the dentin-pulp complex while Pax9+ stem cells mainly generated periodontal tissues as well as partial dental pulp. DTA-mediated cell ablation of the Cd24a+/Pax9+ stem cells significantly compromised tooth establishment. Moreover, during development, the Cd24a+/Pax9+ bilaminar core concentrated on the apical region, collectively migrated and contributed to the newly formed dental root, potentially guided by PDGF-B derived from the alveolar bone. Integrated multi-omic analysis and spatial mapping further revealed lineage-associated key signaling pathways in Cd24a+/Pax9+ stem cells and the unique organization of different cell compositions. Finally, the CD24+/PAX9+ bilaminar core was also detected in human teeth at different stages, suggesting it a conserved developmental mechanism. Together, our work identified a unique bilaminar core of bona fide dental stem cells governing tooth establishment and might guide the future regenerative therapy to treat pulpitis, pulp necrosis and periodontal diseases.
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Project description:Notumis a direct target of Wnt/β-catenin signaling and plays a crucial role as a Wnt inhibitor within a negative feedback loop. In the tooth, Notum is known to be expressed in odontoblasts, and severe dentin defects and irregular tooth roots have been reported in Notum-deficient mice. However, the precise expression pattern of Notum in early tooth development, and the role of Notum in crown and root patterns remain elusive. In the present study, we identified a novel Notum expression in primary enamel knot (EK), secondary EKs, and dental papilla during tooth development. Notum-deficient mice exhibited enlarged secondary EKs, resulting in broader cusp tips, altered cusp patterns, and reduced concavity in crown outline. These alterations in crown outline led to a reduction in cervical tongue length, thereby inducing root fusion in Notum-deficient mice. Overall, these results suggest that the size of secondary EK size, regulated by the Wnt/Notum negative feedback loop, has a significant impact on the patterns of crown and root during tooth morphogenesis.
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Project description:Dental mesenchymal cells (dMCs) differentiate into dentin-secreting odontoblasts and are utilized in the majority of tooth regeneration studies to produce an entire tooth organ. An in vitro culture platform of dMCs is useful in generating an adequate quantity of cells for experiments and regenerative therapy. However, culturing techniques may induce functional modification in dMCs. Here, we examined the transcriptomic alterations induced by in vitro culture and investigated the biological properties of cultured dMCs. Hierarchical clustering analysis and principal components analysis reveal an overall transcriptional similarity between the first and second passages that differed significantly from freshly obtained cells. Following in vitro culture, the biological processes of cell adhesion, chromatin organization, cell differentiation and development are significantly altered. Components of signaling pathways, including p53, transforming group factor β/bone morphogenetic protein (TGFβ/BMP), Hedgehog, and Wnt, as well epigenetic regulators are involved. Furthermore, the osteogenic potential of cultured dMCs is also impaired. Our study provides valuable insights for the modification of the current culturing methods to maintain the characteristics of in vivo dMCs.
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