Suppression and Epigenetic Regulation of MiR-9 Contributes to Ethanol Teratology: Evidence from Zebrafish and Murine Fetal Neural Stem Cell Models
Article first published online: 25 JUN 2013
Copyright © 2013 by the Research Society on Alcoholism
Alcoholism: Clinical and Experimental Research
Volume 37, Issue 10, pages 1657–1667, October 2013
How to Cite
Pappalardo-Carter, D. L., Balaraman, S., Sathyan, P., Carter, E. S., Chen, W.-J. A. and Miranda, R. C. (2013), Suppression and Epigenetic Regulation of MiR-9 Contributes to Ethanol Teratology: Evidence from Zebrafish and Murine Fetal Neural Stem Cell Models. Alcoholism: Clinical and Experimental Research, 37: 1657–1667. doi: 10.1111/acer.12139
- Issue published online: 3 OCT 2013
- Article first published online: 25 JUN 2013
- Manuscript Accepted: 14 FEB 2013
- Manuscript Received: 16 APR 2012
- NIH. Grant Number: AA013440
- Texas Brain and Spine Institute
- Embryonic Development;
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.
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.
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.
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.