DNA methylation is an important epigenetic modification critical to the regulation of gene expression during development. To date, little is known about the role of DNA methylation in tooth development in large animal models. Thus, we carried out a comparative genomic analysis of genome-wide DNA methylation profiles in E50 and E60 tooth germ from miniature pigs using methylated DNA immunoprecipitation-sequencing (MeDIP-seq).

Taking advantage of genetic manipulation tools and accessibility, almost all molecular knowledge on vertebrate tooth development was obtained from rodent models that only have one dentition in their entire lives. Whether the tooth development in other vertebrates such as swine or human follows the same rules remains elusive. Rodent dentitions differ considerably from human dentitions, therefore limiting the application of knowledge from rodent tooth to human tooth. Signal-mediated communication between cells and complex gene and protein regulatory networks are key components of tooth development. By combining isobaric tandem mass tag (TMT) labeling with liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) technology, we constructed the proteomic profile of deciduous molars at embryonic days 40 and 50 in miniature pig (Sus scrofa). During the ten days of prenatal development of the miniature pig, the morphology of the lower deciduous molar moves from the early cap to the bell stage. Thus, we identified proteins that are associated with these developing stages and identified differentially regulated proteins (DRPs) that are potential or novel drivers of tooth morphogenesis. Three candidate proteins were validated via qRT-PCR, western blotting analysis, and the location of those proteins in tooth germ were observed by immunohistochemical staining. Multiple signaling pathways and protein interaction network revealed potential mechanisms of early tooth programming in a large mammal. Bioinformatic analysis also showed that cross interaction of Wnt and Sonic hedgehog pathways may play a key role in deciduous development during cap to bell transition in miniature pig.

Miniature pigs, a valuable alternative model for understanding human tooth development, have deciduous teeth from all four tooth families that are replaced once by permanent molars. The extracellular matrix (ECM) supports cells and maintains the integrity of tooth germs during tooth development. However, details on the role of the ECM in tooth development are poorly understood. Here, we performed long noncoding RNA (lncRNA) and messenger RNA (mRNA) expression profiles in the ECM components of deciduous tooth germs by RNA sequencing in miniature pigs. From the early cap to the late bell stages, we identified 4,562 and 3,238 differentially expressed genes (DEGs) from E40 to E50 and E50 to E60, respectively. In addition, a total of 1,464 differentially expressed lncRNAs from E40 to E50 and 969 differentially expressed lncRNAs from E50 to E60 were obtained. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that DEGs were enriched significantly for multiple signaling pathways, especially for the ECM pathway. We then outlined the detailed dynamic gene expression profiling of ECM components during deciduous molar development. Comparison of the cap and bell stages revealed that the structure and functions of the ECM dynamically changed. The ECM-related genes, including THBS1, COL4A5, COL4A6, COL1A1, CHAD, TNR, GP1BA, and ITGA3, were significantly changed, and some were shown to enrich during the bell stage development. Finally, we outlined the coexpression of lncRNAs and ECM properties during tooth development. We showed that the interplay of key lncRNAs could change ECM processes and influence the ECM establishment of tooth patterns to accomplish full tooth formation. These results might provide information to elucidate the regulation network of the lncRNA and ECM in tooth development.

The canonical Wnt/β-catenin signaling pathway has been shown to play essential roles in tooth initiation and early tooth development. However, the role of Wnt/β-catenin signaling in cusp patterning and crown calcification in large mammals are largely unknown. In our previous study, miniature pigs were used as the animal model due to the similarity of tooth anatomy and replacement pattern between miniature pig and human. Dynamic gene expression of third deciduous molar (DM3) in miniature pig at early stages was profiled using microarray method and expression of Wnt genes was significantly correlate with odontogenesis. In the present study, dynamic expression patterns of Wnt/β-catenin signaling genes of DM3 at cap, early bell and late bell (secretory) stage were identified. We found that Lef1 and Axin2 were expressed in the enamel knot and underlying mesenchyme regions. Meanwhile, Dkk1 was expressed in the peripheral and lower parts of dental papilla, thus forming the potential Wnt signaling gradient. We also found that β-Catenin, Axin2 and Lef1 were expressed strongly in undifferentiated cells of the inner enamel epithelium (IEE), but weakly in differentiated ameloblasts. Furthermore, we found that both Wnt signaling read-out gene Lef1 and the inhibitor Dkk1 were co-expressed in the pre-odontoblasts. In conclusion, the spatiotemporal distribution and potential gradient of Wnt signaling may contribute to cusp patterning and crown calcification. These data may yield insight into future study of precise control of crown morphogenesis and regeneration in large mammals.

The tooth root transmits and balances occlusal forces through the periodontium to the alveolar bone. The periodontium, including the gingiva, the periodontal ligament, the cementum and the partial alveolar bone, derives from the dental follicle (DF), except for the gingiva. In the early developmental stages, the DF surrounds the tooth germ as a sphere and functions to promote tooth eruption. However, the morphological dynamics and factors regulating the differentiation of the DF during root elongation remain largely unknown. Miniature pigs are regarded as a useful experimental animal for modeling in craniofacial research because they are similar to humans with respect to dentition and mandible anatomy. In the present study, we used the third deciduous incisor of miniature pig as the model to investigate the factors influencing DF differentiation during root development. We found that the DF was shaped like a crescent and was located between the root apical and the alveolar bone. The expression levels of WNT5a, β-Catenin, and COL-I gradually increased from the center of the DF (beneath the apical foramen) to the lateral coronal corner, where the DF differentiates into the periodontium. To determine the potential regulatory role of the apical papilla on DF cell differentiation, we co-cultured dental follicle stem cells (DFSCs) with stem cells of the apical papilla (SCAPs). The osteogenesis and fibrogenesis abilities of DFSCs were inhibited when being co-cultured with SCAPs, suggesting that the fate of the DF can be regulated by signals from the apical papilla. The apical papilla may sustain the undifferentiated status of DFSCs before root development finishes. These data yield insight into the interaction between the root apex and surrounding DF tissues in root and periodontium development and shed light on the future study of root regeneration in large mammals.

Renewal of integumentary organs occurs cyclically throughout an organism's lifetime, but the mechanism that initiates each cycle remains largely unknown. In a miniature pig model of tooth development that resembles tooth development in humans, the permanent tooth did not begin transitioning from the resting to the initiation stage until the deciduous tooth began to erupt. This eruption released the accumulated mechanical stress inside the mandible. Mechanical stress prevented permanent tooth development by regulating expression and activity of the integrin β1-ERK1-RUNX2 axis in the surrounding mesenchyme. We observed similar molecular expression patterns in human tooth germs. Importantly, the release of biomechanical stress induced downregulation of RUNX2-wingless/integrated (Wnt) signaling in the mesenchyme between the deciduous and permanent tooth and upregulation of Wnt signaling in the epithelium of the permanent tooth, triggering initiation of its development. Consequently, our findings identified biomechanical stress-associated Wnt modulation as a critical initiator of organ renewal, possibly shedding light on the mechanisms of integumentary organ regeneration.

Retinoic acid (RA) signal is involved in tooth development and osteogenic differentiation of mesenchymal stem cells (MSCs). Dental pulp stem cells (DPSCs) are one of the useful MSCs in tissue regeneration. However, the function of RA in osteo/odontogenic differentiation of DPSCs remains unclear. Here, we investigated the expression pattern of RA in miniature pig tooth germ and intervened in the RA signal during osteo/odontogenic differentiation of human DPSCs. Deciduous canine (DC) germs of miniature pigs were observed morphologically, and the expression patterns of RA were studied by in situ hybridization (ISH). Human DPSCs were isolated and cultured in osteogenic induction medium with or without RA or BMS 493, an inverse agonist of the pan-retinoic acid receptors (pan-RARs). Alkaline phosphatase (ALP) activity assays, alizarin red staining, quantitative calcium analysis, CCK8 assay, osteogenesis-related gene expression, and in vivo transplantation were conducted to determine the osteo/odontogenic differentiation potential and proliferation potential of DPSCs. We found that the expression of RARβ and CRABP2 decreased during crown calcification of DCs of miniature pigs. Activation of RA signal in vitro inhibited ALP activities and mineralization of human DPSCs and decreased the mRNA expression of ALP, osteocalcin, osteopontin, and a transcription factor, osterix. With BMS 493 treatment, the results were opposite. Interference in RA signal decreased the proliferation of DPSCs. In vivo transplantation experiments suggested that osteo/odontogenic differentiation potential of DPSCs was enhanced by inversing RA signal. Our results demonstrated that downregulation of RA signal promoted osteo/odontogenic differentiation of DPSCs and indicated a potential target pathway to improve tissue regeneration.