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Transcriptomic profiling of dental epithelium and mesenchyme of the third deciduous molar of miniature pigs at embryonic day 50 and 60

ABSTRACT: Reciprocal epithelial–mesenchymal interactions play a fundamental role in ectodermal organogenesis. The tooth is a typical ectodermal organ for studying epithelial–mesenchymal interactions. The dental epithelium (DE) is considered to possess the original inductive odontogenic signals rather than the dental papilla (DP) derived from mesenchyme. At the bell stage, the outer layer of the DP that is adjacent to the DE starts to differentiate into odontoblasts first, implying potential induction by DE. Epithelial signals are requisite inducers for dental cusp formation and odontoblast differentiation. However, conflicting roles for these signals have been reported. Here, we separated DE and dental mesenchyme at early and late bell stage (embryonic day 50 and 60) of the third deciduous mandibular molars(DM3) in miniature pigs and mRNA sequencing was carried out. KEGG analysis and GO enrichment showed that PI3K/AKT was one of the most enriched pathways in DE from E50 to E60. We screened out 198 genes of secreted proteins that were expressed at higher levels in DE than in DP at E50. Function experiments supported the idea that PI3K/AKT signaling from DE to DP promotes odontoblast differentiation via COL4A6 secretion.

ORGANISM(S): Sus scrofa

PROVIDER: GSE223510 | GEO | 2026/01/19

REPOSITORIES: GEO

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Project description:Transforming growth factor (TGF) plays an important role in tooth morphogenesis and mineralization. During postnatal development, the dental pulp (DP) mesenchyme secretes neurotrophic factors that guide trigeminal nerve fibers into and throughout the DP. This process is tightly linked with dentin formation and mineralization. Our laboratory established a mouse model in which Tgfbr2 was conditionally deleted in DP mesenchyme using an Osterix promoter-driven Cre recombinase (Tgfbr2cko). These mice survived postnatally with significant defects in bones and teeth, including reduced mineralization and short roots. Hematoxylin and eosin staining revealed reduced axon-like structures in the mutant mice. Reporter imaging demonstrated that Osterix-Cre activity within the tooth was active in the DP and derivatives, but not in neurons. Immunofluorescence staining for 3 tubulin (neuronal marker) was performed on serial cryosections from control and mutant molars on postnatal days 7 and 24 (P7, P24). Confocal imaging and pixel quantification demonstrated reduced innervation in Tgfbr2cko first molars at both stages compared to controls, indicating that signals necessary to promote neurite outgrowth were disrupted by Tgfbr2 deletion. We performed mRNA-Sequence (RNA-Seq) and gene onotology analyses using RNA from the DP of P7 control and mutant mice to investigate the pathways involved in Tgfbr2-mediated tooth development. These analyses identified downregulation of several mineralization-related and neuronal genes in the Tgfbr2cko DP compared to controls. Select gene expression patterns were confirmed by quantitative real-time PCR and immunofluorescence imaging. Lastly, trigeminal neurons were co-cultured atop Transwell filters overlying primary Tgfbr2f/f DP cells. Tgfbr2 in the DP was deleted via adenovirus-expressed Cre recombinase. Confocal imaging of axons through the filter pores showed increased axonal sprouting from neurons cultured with Tgfbr2-positive DP cells compared to neurons cultured alone. Axon sprouting was reduced when Tgfbr2 was knocked down in the DP cells. Immunofluorescence of dentin sialophosphoprotein in co-cultured DP cells confirmed reduced mineralization potential in cells with Tgfbr2 deletion. Both our proteomics and RNA-Seq analyses indicate that axonal guidance cues, particularly semaphorin signaling, were disrupted by Tgfbr2 deletion. Thus, Tgfbr2 in the DP mesenchyme appears to regulate differentiation and the cells’ ability to guide neurite outgrowth during tooth mineralization and innervation.

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Transcriptomic profiling of dental epithelium and mesenchyme of the third deciduous molar of miniature pigs at embryonic day 50 and 60

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