Effects of Early Life Adversity on Tooth Enamel Formation

Abstract

In a systemic effort to survive environmental stress, organ systems fluctuate and adapt to overcome external pressures. The evolutionary drive back toward 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 the enamel organ gene expression and enamel matrix mineralization. On average, postnatal day 12 (P12) ELA mice weighed significantly less than the controls. When adjusted for animal weight, ELA molar enamel volume was reduced as compared with the controls, and the relative mineral density of molar enamel was significantly increased. There were no obvious changes in enamel matrix crystal morphology or structure in ELA as compared with the control mouse enamel. RNAseq showed extracellular matrix organization to be the most significantly affected GO and reactome pathways, whereas butanote metabolism was the most significantly altered KEGG pathway. Transcripts expressing the enamel matrix proteins amelogenin (Amelx) and enamelin (Enam) were among the top 4 most differentially expressed genes. When evaluating molecular mechanisms for the changes in gene expression in ELA enamel organs, we found significantly increased expression of Dlx3, while transcripts for clock genes Per1 and Nrd1 were downregulated. These findings support the possibility that the developing enamel organ is sensitive to the pressures of early life adversity and produces molecular and structural biomarkers reflecting these challenges.

Keywords: RNAseq; ameloblasts; early life adversity (ELA); enamel; limited bedding and nesting (LBN); mineralization.

FIGURE 1 |

First molar relative enamel mineral density and enamel volume is associated with animal weight. Black triangles = control, white triangles = ELA.

FIGURE 2 |

Mineralized enamel volume and relative density in molars from P12 mice, normalized for body weight. (A) Enamel volume was significantly less in ELA (n = 10) as compared to controls (n = 10). (B) Enamel density was significantly higher in ELA mice as compared to controls *p < 0.05. Error bars: SD.

FIGURE 3 |

Backscattered electron SEM images of enamel in (A,B) control and (C,D) ELA mice. Qualitatively, there are no differences in the structural organization of rod (R)-interrod (IR) enamel, and (B,D) also no apparent difference in appearance of crystal profiles. IE, inner enamel; OE, outer enamel.

FIGURE 4 |

Venn diagram showing the number of genes that are uniquely expressed within each group yellow = ELA mice, purple = weight-matched controls.

FIGURE 5 |

Log-fold changes of transcripts amplified by qPCR. Blue boxes show upregulated transcripts and red boxes show downregulated transcripts expressed in ELA enamel organs as compared to controls (gray boxes). *p <= 0.05.