Suppression and epigenetic regulation of MiR-9 contributes to ethanol teratology: evidence from zebrafish and murine fetal neural stem cell models

Alcohol Clin Exp Res. 2013 Oct;37(10):1657-67. doi: 10.1111/acer.12139. Epub 2013 Jun 25.

Abstract

Background: Fetal alcohol exposure produces multiorgan defects, making it difficult to identify underlying etiological mechanisms. However, recent evidence for ethanol (EtOH) sensitivity of the miRNA miR-9 suggests one mechanism, whereby EtOH broadly influences development. We hypothesized that loss of miR-9 function recapitulates aspects of EtOH teratology.

Methods: Zebrafish embryos were exposed to EtOH during gastrulation, or injected with anti-miR-9 or nonsense control morpholinos during the 2-cell stage of development and collected between 24 and 72 hours postfertilization (hpf). We also assessed the expression of developmentally important, and known miR-9 targets, FGFR-1, FOXP2, and the nontargeted transcript, MECP2. Methylation at CpG islands of mammalian miR-9 genes was assessed in fetal murine neural stem cells (mNSCs) by methylation-specific PCR, and miRNA processing assessed by qRT-PCR for pre-miR-9 transcripts.

Results: EtOH treatment and miR-9 knockdown resulted in similar cranial defects including microcephaly. Additionally, EtOH transiently suppressed miR-9, as well as FGFR-1 and FOXP2, and alterations in miR-9 expression were correlated with severity of EtOH-induced teratology. In mNSCs, EtOH increased CpG dinucleotide methylation at the miR-9-2 locus and accumulation of pre-miR-9-3.

Conclusions: EtOH exerts regulatory control at multiple levels of miR-9 biogenesis. Moreover, early embryonic loss of miR-9 function recapitulated the severe range of teratology associated with developmental EtOH exposure. EtOH also disrupts the relationship between miR-9 and target gene expression, suggesting a nuanced relationship between EtOH and miRNA regulatory networks in the developing embryo. The implications of these data for the expression and function of mature miR-9 warrant further investigation.

Keywords: Embryonic Development; Ethanol; Methylation; MiR-9; Zebrafish.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Epigenesis, Genetic / drug effects
  • Epigenesis, Genetic / physiology*
  • Ethanol / toxicity*
  • Fetal Stem Cells / drug effects
  • Fetal Stem Cells / physiology*
  • Gene Knockdown Techniques / methods
  • Mice
  • Mice, Inbred C57BL
  • MicroRNAs / physiology*
  • Neural Stem Cells / drug effects
  • Neural Stem Cells / physiology*
  • Random Allocation
  • Teratogenesis / drug effects
  • Teratogenesis / physiology*
  • Zebrafish

Substances

  • MIRN9 microRNA, mouse
  • MicroRNAs
  • Ethanol