Search

Although histone methyltransferases are implicated in many key developmental processes, the contribution of individual chromatin modifiers in dental tissues is not well understood. Using single-cell RNA sequencing, we examined the expression profiles of the disruptor of telomeric silencing 1-like (Dot1L) gene in the postnatal day 5 mouse molar dental pulp. Dot1L is the only known enzyme that methylates histone 3 on lysine 79, a modification associated with gene expression. Our research revealed 15 distinct clusters representing different populations of mesenchymal stromal cells (MSCs), immune cells, pericytes, ameloblasts and endothelial cells. We documented heterogeneity in gene expression across different subpopulations of MSCs, a good indicator that these stromal progenitors undergo different phases of osteogenic differentiation. Interestingly, although Dot1L was broadly expressed across all cell clusters within the molar dental pulp, our analyses indicated specific enrichment of Dot1L within two clusters of MSCs, as well as cell clusters characterized as ameloblasts and endothelial cells. Moreover, we detected Dot1L co-expression with protein interactors involved in epigenetic activation such as Setd2, Sirt1, Brd4, Isw1, Bptf and Suv39h1. In addition, Dot1L was co-expressed with Eed2, Cbx3 and Dnmt1, which encode epigenetic factors associated with gene silencing and heterochromatin formation. Dot1l was co-expressed with downstream targets of the insulin growth factor and WNT signaling pathways, as well as genes involved in cell cycle progression. Collectively, our results suggest that Dot1L may play key roles in orchestrating lineage-specific gene expression during MSC differentiation.

The dental pulp is critical for the production of odontoblasts to create reparative dentin. In recent years, dental pulp has become a promising source of mesenchymal stem cells that are capable of differentiating into multiple cell types. To elucidate the transcriptional control mechanisms specifying the early phases of odontoblast differentiation, we analysed the DNA demethylation pattern associated with 5-hydroxymethylcytosine (5hmC) in the primary murine dental pulp. 5hmC plays an important role in chromatin accessibility and transcriptional control by modelling a dynamic equilibrium between DNA methylation and demethylation. Our research revealed 5hmC enrichment along genes and non-coding regulatory regions associated with specific developmental pathways in the genome of mouse incisor and molar dental pulp. Although the overall distribution of 5hmC is similar, the intensity and location of the 5hmC peaks significantly differs between the incisor and molar pulp genome, indicating cell type-specific epigenetic variations. Our study suggests that the differential DNA demethylation pattern could account for the distinct regulatory mechanisms underlying the tooth-specific ontogenetic programs.

Objective Significant evidence links epigenetic processes governing the dynamics of DNA methylation and demethylation to an increased risk of syndromic and nonsyndromic cleft lip and/or cleft palate (CL/P). Previously, we characterized mesenchymal stem/stromal cells (MSCs) at different stages of osteogenic differentiation in the mouse incisor dental pulp. The main objective of this research was to characterize the transcriptional landscape of regulatory genes associated with DNA methylation and demethylation at a single-cell resolution. Design We used single-cell RNA sequencing (scRNA-seq) data to characterize transcriptome in individual subpopulations of MSCs in the mouse incisor dental pulp. Settings The biomedical research institution. Patients/Participants This study did not include patients. Interventions This study collected and analyzed data on the single-cell RNA expssion in the mouse incisor dental pulp. Main outcome measure(s) Molecular regulators of DNA methylation/demethylation exhibit differential transcriptional landscape in different subpopulations of osteogenic progenitor cells. Results scRNA-seq analysis revealed that genes encoding DNA methylation and demethylation enzymes (DNA methyltransferases and members of the ten-eleven translocation family of methylcytosine dioxygenases), methyl-DNA binding domain proteins, as well as transcription factors and chromatin remodeling proteins that cooperate with DNA methylation machinery are differentially expressed within distinct subpopulations of MSCs that undergo different stages of osteogenic differentiation. Conclusions These findings suggest some mechanistic insights into a potential link between epigenetic alterations and multifactorial causes of CL/P phenotypes.

The dental pulp is a promising source of progenitor cells for regenerative medicine. The natural function of dental pulp is to produce odontoblasts to generate reparative dentin. Stem cells within the pulp tissue originate from the migrating neural crest cells and possess mesenchymal stem cell properties with the ability to differentiate into multiple lineages. To elucidate the transcriptional control mechanisms underlying cell fate determination, we compared the transcriptome and chromatin accessibility in primary dental pulp tissue derived from 5–6-day-old mice. Using RNA sequencing and assay for transposase-accessible chromatin using sequencing (ATAC-seq), we correlated gene expression with chromatin accessibility. We found that the majority of ATAC-seq peaks were concentrated at genes associated with development and cell differentiation. Most of these genes were highly expressed in the mouse dental pulp. Surprisingly, we uncovered a group of genes encoding master transcription factors that were not expressed in the dental pulp but retained open chromatin states. Within this group, we identified key developmental genes important for specification of the neural crest, adipocyte, neural, myoblast, osteoblast and hepatocyte lineages. Collectively, our results uncover a complex relationship between gene expression and the chromatin accessibility landscape in the mouse dental pulp.

Considerable amount of research has been focused on dentin mineralization, odontoblast differentiation, and their application in dental tissue engineering. However, very little is known about the differential role of functionally and spatially distinct types of dental epithelium during odontoblast development. Here we show morphological and functional differences in dentin located in the crown and roots of mouse molar and analogous parts of continuously growing incisors. Using a reporter (DSPP-cerulean/DMP1-cherry) mouse strain and mice with ectopic enamel (Spry2(+/-);Spry4(-/-)), we show that the different microstructure of dentin is initiated in the very beginning of dentin matrix production and is maintained throughout the whole duration of dentin growth. This phenomenon is regulated by the different inductive role of the adjacent epithelium. Thus, based on the type of interacting epithelium, we introduce more generalized terms for two distinct types of dentins: cementum versus enamel-facing dentin. In the odontoblasts, which produce enamel-facing dentin, we identified uniquely expressed genes (Dkk1, Wisp1, and Sall1) that were either absent or downregulated in odontoblasts, which form cementum-facing dentin. This suggests the potential role of Wnt signalling on the dentin structure patterning. Finally, we show the distribution of calcium and magnesium composition in the two developmentally different types of dentins by utilizing spatial element composition analysis (LIBS). Therefore, variations in dentin inner structure and element composition are the outcome of different developmental history initiated from the very beginning of tooth development. Taken together, our results elucidate the different effects of dental epithelium, during crown and root formation on adjacent odontoblasts and the possible role of Wnt signalling which together results in formation of dentin of different quality. (c) 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Odontoblast differentiation is a complex and multistep process regulated by signaling pathways, including the Wnt/β-catenin signaling pathway. Both positive and negative effects of Wnt/β-catenin signaling on dentinogenesis have been reported, but the underlying mechanisms of these conflicting results are still unclear. To gain a better insight into the role of Wnt/β-catenin in dentinogenesis, we used dental pulp cells from a panel of transgenic mice, in which fluorescent protein expression identifies cells at different stages of odontoblast and osteoblast differentiation. Our results showed that exposure of pulp cells to WNT3a at various times and durations did not induce premature differentiation of odontoblasts. These treatments supported the survival of undifferentiated cells in dental pulp and promoted the formation of 2.3GFP+ preodontoblasts and their rapid transition into differentiated odontoblasts expressing DMP1-Cherry and DSPP-Cerulean transgenes. WNT3a also promoted osteogenesis in dental pulp cultures. These findings provide critical information for the development of improved treatments for vital pulp therapy and dentin regeneration.

Understanding cell types and mechanisms of dental growth is essential for reconstruction and engineering of teeth. Therefore, we investigated cellular composition of growing and non-growing mouse and human teeth. As a result, we report an unappreciated cellular complexity of the continuously-growing mouse incisor, which suggests a coherent model of cell dynamics enabling unarrested growth. This model relies on spatially-restricted stem, progenitor and differentiated populations in the epithelial and mesenchymal compartments underlying the coordinated expansion of two major branches of pulpal cells and diverse epithelial subtypes. Further comparisons of human and mouse teeth yield both parallelisms and differences in tissue heterogeneity and highlight the specifics behind growing and non-growing modes. Despite being similar at a coarse level, mouse and human teeth reveal molecular differences and species-specific cell subtypes suggesting possible evolutionary divergence. Overall, here we provide an atlas of human and mouse teeth with a focus on growth and differentiation., Unlike human teeth, mouse incisors grow throughout life, based on stem and progenitor cell activity. Here the authors generate single cell RNA-seq comparative maps of continuously-growing mouse incisor, non-growing mouse molar and human teeth, combined with lineage tracing to reveal dental cell complexity.

The dental pulp is known to be highly heterogenous, comprising distinct cell types including mesenchymal stromal cells (MSCs), which represent neural-crest-derived cells with the ability to differentiate into multiple cell lineages. However, the cellular heterogeneity and the transcriptome signature of different cell clusters within the dental pulp remain to be established. To better understand discrete cell types, we applied a single-cell RNA sequencing strategy to establish the RNA expression profiles of individual dental pulp cells from 5- to 6-day-old mouse incisors. Our study revealed distinct subclasses of cells representing osteoblast, odontoblast, endothelial, pancreatic, neuronal, immune, pericyte and ameloblast lineages. Collectively, our research demonstrates the complexity and diversity of cell subclasses within the incisor dental pulp, thus providing a foundation for uncovering the molecular processes that govern cell fate decisions and lineage commitment in dental pulp-derived MSCs.

Objective In the present study, we compared the in vitro osteogenic and dentinogenic potential of pulp cells from incisors and molars. Design Primary pulp cultures were established from DSPP-Cerulean/DMP1-Cherry and BSP-GFPtpz reporter mouse lines and processed for various assays. Results Our results showed marked differences in dentinogenic and osteogenic potentials of primary cultures from unerupted molars and incisors isolated from 5 to 7 days old pups. While primary cultures from both incisors and molars differentiated into odontoblasts and osteoblasts, cultures from molars differentiated into more DSPP-Cerulean + cells (∼5.5 %) compared to incisor cultures (∼0.7 %) at Day 14 and appear to be more committed to the odontogenic lineage. On the other hand, cultures from incisors show more differentiation into BSP-GFPtpz + cells (∼25 %) compared to molar cultures (∼16 %) and were more committed to the osteogenic lineage. Data were analyzed by Student’s t-test and statistical significance was set at P ≤ 0.05. Conclusion Since in the dental pulp, MSCs are the primary source of progenitors capable of giving rise to osteoblasts and odontoblasts, our results provide evidence for differences in the commitment of MSCs in molars and incisors to the odontogenic and osteogenic lineages, respectively.

Bone sialoprotein (BSP) is a member of the SIBLING family with essential roles in skeletogenesis. In the developing teeth, although the expression and function of BSP in the formation of acellular cementum and periodontal attachment are well documented, there are uncertainties regarding the expression and function of BSP by odontoblasts and dentin. Reporter mice are valuable animal models for biological research, providing a gene expression readout that can contribute to cellular characterization within the context of a developmental process. In the present study, we examined the expression of a BSP-GFPtpz reporter mouse line during odontoblast differentiation, reparative dentinogenesis, and bone. In the developing teeth, BSP-GFPtpz was expressed at high levels in cementoblasts but not in odontoblasts or dentin. In bones, the transgene was highly expressed in osteoblasts at an early stage of differentiation. Interestingly, despite its lack of expression in odontoblasts and dental pulp during tooth development, the BSP-GFPtpz transgene was detected during in vitro mineralization of primary pulp cultures and during reparative dentinogenesis following pulp exposures. Importantly, under these experimental contexts, the expression of BSP-GFPtpz was still exclusive to DSPP-Cerulean, an odontoblast-specific reporter gene. This suggests that the combinatorial use of BSP-GFPtpz and DSPP-Cerulean can be a valuable experimental tool to distinguish osteogenic from dentinogenic cells, thereby providing an avenue to investigate mechanisms that distinctly regulate the lineage progression of progenitors into odontoblasts versus osteoblasts.

Aim: To examine the contribution of perivascular cells expressing αSMA to reactionary dentinogenesis. Methodology: An inducible, Cre- loxP in vivo fate- mapping approach was used to examine the contribution of the descendants of cells expressing the αSMA-CreERT2 transgene to reactionary dentinogenesis in mice molars. Reactionary dentinogenesis was induced by experimental mild injury to dentine without pulp exposure. The Student's t test was used to determine statistical significance at *P ≤ 0.05. Results: The lineage tracing experiments revealed that mild injury to dentine first led to activation of αSMA-tdTomato+ cells in the entire pulp chamber. The percentage of areas occupied by αSMA-tdTomato+ in injured (7.5 ± 0.7%) teeth were significantly higher than in teeth without injury (2 ± 0.5%). After their activation, αSMA- tdTomato+ cells migrated towards the site of injury, gave rise to pulp cells and a few odontoblasts that became integrated into the existing odontoblast layer expressing Col2.3-GFP and Dspp. Conclusion: Mild insult to dentine activated perivascular αSMA-tdTomato+ cells giving rise to pulp cells as well as a few odontoblasts that were integrated into the pre-existing odontoblast layer.

The goal of this study was to examine the effects of early and limited exposure of perivascular cells expressing α (αSMA) to fibroblast growth factor 2 (FGF2) in vivo. We performed in vivo fate mapping by inducible Cre-loxP and experimental pulp injury in molars to induce reparative dentinogenesis. Our results demonstrate that early delivery of exogenous FGF2 to exposed pulp led to proliferative expansion of αSMA-tdTomato cells and their accelerated differentiation into odontoblasts. In vivo lineage-tracing experiments showed that the calcified bridge/reparative dentin in FGF2-treated pulps were lined with an increased number of Dspp odontoblasts and devoid of BSP osteoblasts. The increased number of odontoblasts derived from αSMA-tdTomato cells and the formation of reparative dentin devoid of osteoblasts provide in vivo evidence for the stimulatory effects of FGF signaling on odontoblast differentiation from early progenitors in dental pulp.

To gain a better understanding of the progression of progenitor cells in the odontoblast lineage, we have examined and characterized the expression of a series of GFP reporters during odontoblast differentiation. However, previously reported GFP reporters (pOBCol2.3-GFP, pOBCol3.6-GFP, and DMP1-GFP), similar to the endogenous proteins, are also expressed by bone-forming cells, which made it difficult to delineate the two cell types in various in vivo and in vitro studies. To overcome these difficulties we generated DSPP- Cerulean/DMP1-Cherry transgenic mice using a bacterial recombination strategy with the mouse BAC clone RP24-258g7. We have analyzed the temporal and spatial expression of both transgenes in tooth and bone in vivo and in vitro. This transgenic animal enabled us to visualize the interactions between odontoblasts and surrounding tissues including dental pulp, ameloblasts and cementoblasts. Our studies showed that DMP1-Cherry, similar to Dmp1, was expressed in functional and fully differentiated odontoblasts as well as osteoblasts, osteocytes and cementoblasts. Expression of DSPP-Cerulean transgene was limited to functional and fully differentiated odontoblasts and correlated with the expression of Dspp. This transgenic animal can help in the identification and isolation of odontoblasts at later stages of differentiation and help in better understanding of developmental disorders in dentin and odontoblasts.