Single-cell Transcriptome Landscape of DNA Methylome Regulators Associated with Orofacial Clefts in the Mouse Dental Pulp.

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.

Single-Cell Transcriptome Analysis Defines Expression of Kabuki Syndrome-Associated KMT2D Targets and Interacting Partners.

Objectives. Kabuki syndrome (KS) is a rare genetic disorder characterized by developmental delay, retarded growth, and cardiac, gastrointestinal, neurocognitive, renal, craniofacial, dental, and skeletal defects. KS is caused by mutations in the genes encoding histone H3 lysine 4 methyltransferase (KMT2D) and histone H3 lysine 27 demethylase (KDM6A), which are core components of the complex of proteins associated with histone H3 lysine 4 methyltransferase SET1 (SET1/COMPASS). Using single-cell RNA data, we examined the expression profiles of Kmt2d and Kdm6a in the mouse dental pulp. In the incisor pulp, Kmt2d and Kdm6a colocalize with other genes of the SET1/COMPASS complex comprised of the WD-repeat protein 5 gene (Wdr5), the retinoblastoma-binding protein 5 gene (Rbbp5), absent, small, and homeotic 2-like protein-encoding gene (Ash2l), nuclear receptor cofactor 6 gene (Ncoa6), and Pax-interacting protein 1 gene (Ptip1). In addition, we found that Kmt2d and Kdm6a coexpress with the downstream target genes of the Wingless and Integrated (WNT) and sonic hedgehog signaling pathways in mesenchymal stem/stromal cells (MSCs) at different stages of osteogenic differentiation. Taken together, our results suggest an essential role of KMT2D and KDK6A in directing lineage-specific gene expression during differentiation of MSCs.

The chromatin accessibility landscape in the dental pulp of mouse molars and incisors.

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.

Fibroblast Growth Factor 2 High Molecular Weight Isoforms in Dentoalveolar Mineralization.

Transgenic mice overexpressing human high molecular weight fibroblast growth factor 2 (HMWFGF2) isoforms in osteoblast and odontoblast lineages (HMWTg) exhibit decreased dentin and alveolar bone mineralization, enlarged pulp chamber, and increased fibroblast growth factor 23 (FGF23). We examined if the alveolar bone and dentin mineralization defects in HMWTg mice resulted from increased FGF23 expression and whether an FGF23 neutralizing antibody could rescue the hypomineralization phenotype. HMWTg and VectorTg control mice were given subcutaneous injections of FGF23 neutralizing antibody twice/week starting at postnatal day 21 for 6 weeks. Since Calcitriol (1,25D) have direct effects in promoting bone mineralization, we also determined if 1,25D protects against the defective dentin and alveolar bone mineralization. Therefore, HMWTg mice were given subcutaneous injections of 1,25D daily or concomitantly with FGF23 neutralizing antibody for 6 weeks. Our results showed that HMWTg mice displayed thickened predentin, alveolar bone hypomineralization, and enlarged pulp chambers. FGF23 neutralizing antibody and 1,25D monotherapy partially rescued the dentin mineralization defects and the enlarged pulp chamber phenotype in HMWTg mice. 1,25D alone was not sufficient to rescue the alveolar bone hypomineralization. Interestingly, HMWTg mice treated with both FGF23 neutralizing antibody and 1.25D further rescued the enlarged pulp chamber size, and dentin and alveolar bone mineralization defects. We conclude that the dentin and alveolar bone mineralization defects in HMWTg mice might result from increased FGF23 expression. Our results show a novel role of HMWFGF2 on dentoalveolar mineralization.

Single-cell transcriptomics defines Dot1L interacting partners and downstream target genes in the mouse molar dental pulp.

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.

Single-cell transcriptome analysis defines mesenchymal stromal cells in the mouse incisor dental pulp.

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.

Genome-wide distribution of 5hmC in the dental pulp of mouse molars and incisors.

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.

The Development of Dentin Microstructure Is Controlled by the Type of Adjacent Epithelium.

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. © 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).

Dental cell type atlas reveals stem and differentiated cell types in mouse and human teeth.

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.

Generation and characterization of DSPP-Cerulean/DMP1-Cherry reporter mice.

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.