Project description:In a systemic effort to survive environmental stress, organ systems fluctuate and adapt to overcome external pressures. The evolutionary drive back towards homeostasis, makes it difficult to determine if an organism experienced a toxic exposure to stress, especially in early prenatal and neonatal periods of development. Previous studies indicate that primary human teeth may provide historical records of experiences related to stressors during that early time window. To assess the molecular effects of early life adversity on enamel formation, we used a limited bedding and nesting (LBN) mouse model of early life adversity (ELA), to assess changes in enamel organ gene expression and enamel mineralization. Enamel of postnatal day 12 (P12) weight-matched ELA mice was more mineralized as compared control enamel. RNAseq showed 724 genes significantly upregulated in ELA enamel organs as compared to controls, and 574 significantly downregulated genes (DESeq2 p-value <= 0.05). Transcripts expressing the enamel matrix proteins amelogenin (Amelx) and enamelin (Enam) were among the top 4 most differentially expressed genes. The most significantly enriched GO and Reactome pathway was Extracellular Matrix Organization, while the most significantly enriched KEGG Pathways were Butanoate metabolism and Synthesis and Degradation of Ketone Bodies. qPCR analysis of weight-matched ELA and control enamel organs confirmed that expression of the ameloblast-specific proteins Amelx and Enam were highly upregulated in ELA mice. When evaluating molecular mechanisms for amelogenin upregulation, we found significantly increased expression of Dlx3, while transcripts for clock genes Per1 and Nrd1, which have also positively associated with amelogenin expression, were downregulated. These findings suggest that the developing enamel organs are sensitive to the pressures of early life adversity and produce structural biomarkers in tooth enamel mineral to reflect these changes. Together these results support the possibility that tooth enamel may contain biomarkers of cellular effects associated with systemic early life adversity.

Project description:The tooth is a convenient experimental model for the study of the basic mechanisms of organ development, including differentiation, cellular interaction, morphogenesis, and production and mineralization of extracellular matrices. The rodent incisor teeth grow continuously and exhibit all stages of tooth development at any time. This characteristic makes them convenient models for the study of enamel formation, which occurs in several distinct stages along the tooth axis. The distribution and structure of mouse incisor enamel resemble that of the rat. The enamel covers only the labial aspect of the tooth, and can be divided into four layers: a thin inner prism-free layer, inner enamel with prism decussation, i.e. transverse rows of prisms with prisms inclined medially and laterally in alternate rows, outer enamel with parallel prisms inclined incisally and a thin superficial prism-free layer. Recently, we have described how this elaborate organization of the enamel is established in the initial enamel formed on the unerupted and unworn incisal tip of the incisors. The very first enamel formed, i.e. the most incisally situated enamel, is always prism-free, corresponding to the superficial layer in fully established enamel, although thicker. Going in apical direction, i.e. in the direction of initiation of new enamel formation, isolated prisms appear among the crystals continuous with the prism-free zone crystals. These initial prisms are in general inclined incisally, corresponding to the prisms in the outer enamel layer in the fully established enamel. Inner enamel with the characteristic decussation pattern is added somewhat further apically and increases in thickness with increasing enamel thickness. Based on the structure of the enamel that they produce, the ameloblasts producing the initial, thin, prism-free enamel at the incisal tip of the unworn mouse incisor are probably differently configured (e.g. lacking Tomes' processes), probably differently organized (e.g. not organized in transverse rows moving sideways in opposite directions), and probably have a shorter life-span than the more apically situated ameloblasts which produce thicker enamel with the full four-layered configuration of mouse incisor enamel. For this reason it would be of interest to compare the gene expression profile of the incisal tip segment where prism-free enamel is being formed with the profile of the immediately adjacent segment where enamel with all four layers is being formed. As long as the incisor is unworn enamel formation and gene expression in these segments will likely reflect mechanistic differences between these segments as regards enamel biosynthesis. MicroRNAs (miRNA) are a class of non-coding RNAs that regulate gene expression at a post-transcriptional level, and are considered as important regulatory molecules during foetal development. By binding to target mRNA, miRNA induce mRNA decay or translation repression. Recent bioinformatic predictions of miRNA targets in vertebrates indicate that hundreds of miRNAs are responsible for regulating the expression of up to 30% of the human protein-coding genes, and miRNAs have recently also been shown to regulate expression of hundreds of mRNAs in cultured cells. Recently, miRNA expression profiles of the developing murine molar tooth germ and submandibular salivary gland have been established. We here report expression profiling of miRNAs present in the two adjacent segments of the incisor tooth germ on which we also have carried out mRNA expression profiling. The results suggest that also miRNAs are abundantly, and differentially, expressed during development of the incisor tooth germ in mouse. By analogy with the mRNA data also miRNA expression is dynamic, with respect to both the two incisor segments investigated and to the developmental stage of the incisor.

Dataset Information

Enamel organ mRNA expression in the limiited bedding and nesting (LBN) mouse model

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