C57BL/6J (B6) mice are susceptible to in utero growth retardation and a number of morphological malformations following prenatal alcohol exposure, while DBA/2J (D2) mice are relatively resistant. We have previously shown that genomic imprinting may play a role in differential sensitivity between B6 and D2 (Downing and Gilliam 1999). The best characterized mechanism mediating genomic imprinting is differential DNA methylation. In the present study we examined DNA methylation and gene expression, in both embryonic and placental tissue, at the mouse Igf2 locus following in utero ethanol exposure. We also examined the effects of a methyl-supplemented diet on methylation and ethanol teratogenesis. In embryos from susceptible B6 mice, we found small decreases in DNA methylation at four CpG sites in one of the differentially methylated regions of the Igf2 locus; only one of the four sites showed a statistically significant decrease. We observed no significant decreases in methylation in placentae. All Igf2 transcripts showed approximately 1.5 fold decreases following intrauterine alcohol exposure. Placing dams on a methyl-supplemented diet before pregnancy and throughout gestation brought methylation back up to control levels. Methyl-supplementation also resulted in lower prenatal mortality, greater prenatal growth, and decreased digit malformations; it dramatically reduced vertebral malformations. Thus, while prenatal alcohol had only small effects on DNA methylation at the Igf2 locus, placing dams on a methyl-supplemented diet partially ameliorated ethanol teratogenesis.
Background We hypothesized that rapid tolerance (one-day tolerance) for the duration of the loss of righting reflex (“sleep time”; ST) was mediated by an increase in AFT. We also hypothesized that increased AFT would correspond to increased drinking. These questions were addressed using the LXS RI panel. Methods Mice were given a pretreatment dose of either saline or 5 g/kg alcohol on day one. On day two, mice were tested for ST (4.1 g/kg) using a method with which it is possible to accurately assess AFT. Genetic correlation analysis was conducted among the ST-related variables and also with “drinking in the dark” (DID) which was previously measured by Saba et al. (2011). Results Saline pretreated mice showed a continuous distribution of ST ranging from ~40 minutes to over 3 hours. Out of the 43 strains tested, 9 showed significantly decreased ST after alcohol pretreatment while in 3 strains, ST was significantly increased. AFT scores ranged from 0 to over 200 mg% in the saline group and in the alcohol group, 8 strains showed a significant increase in AFT and 2 strains showed significant decrease in AFT. In the saline group, AFT was significantly correlated to ST (r = −0.47), but not in the alcohol group (r = −0.22). DID was significantly correlated to only AFT in the alcohol pretreated group (r = 0.64). Conclusions The results suggest that AFT is an important component of the overall ST response, but that the alcohol pretreatment-induced change in AFT does not contribute to rapid ST tolerance. The significant correlation between DID and AFT in the alcohol group suggests that AFT may be a more relevant predictor of drinking behavior than the static measurement of ST. Moreover, pre-exposure to alcohol seems to change AFT in a way that makes it an even stronger predictor of drinking behavior.
Background We previously reported that acute functional tolerance (AFT) to the hypnotic effects of alcohol was significantly correlated to drinking in the dark (DID) in the LXS RI panel, but only in mice that had been pretreated with alcohol. Here we have conducted QTL mapping for AFT. DNA sequencing of the progenitor ILS and ISS strains and microarray analyses were also conducted to identify candidate genes and functional correlates. Methods LXS mice were given either saline or alcohol (5 g/kg) on day one and then tested for loss of righting reflex (LORR) AFT on day two. QTLs were mapped using standard procedures. Two microarray analyses from brain were conducted: 1) naïve LXS mice and 2) an alcohol treatment time course in the ILS and ISS. The full genomes of the ILS and ISS were sequenced to a depth of ~30×. Results A significant QTL for AFT in the alcohol pretreatment group mapped to distal chromosome 4; numerous suggestive QTLs were also mapped. Preference drinking and DID have previously been mapped to the chromosome 4 locus. The credible interval of the significant chromosome 4 QTL spanned 23 Mb and included 716 annotated genes of which 150 had at least one non-synonymous SNP or small indel that differed between the ILS and ISS; expression of 48 of the genes was cis-regulated. Enrichment analysis indicated broad functional categories underlying AFT including proteolysis, transcription regulation, chromatin modification, protein kinase activity, apoptosis, and others. Conclusions The chromosome 4 QTL is a key region containing possibly pleiotropic genes for AFT and drinking behavior. Given that the region contains many viable candidates and a large number of the genes in the interval fall into one or more of the enriched functional categories, we postulate that many genes of varying effect size contribute to the observed QTL effect.
Valproic acid (VPA) is used worldwide to treat epilepsy, migraine headaches, and bipolar disorder. However, VPA is teratogenic and in utero exposure can lead to congenital malformations. Using inbred C57BL/6J (B6) and DBA/2J (D2) mice, we asked whether genetic variation could play a role in susceptibility to VPA teratogenesis. Whereas B6 fetuses were more susceptible than D2 fetuses to digit and vertebral malformations, D2 fetuses were more susceptible to rib malformations. In a reciprocal cross between B6 and D2, genetically identical F1 mice carried in a B6 mother had a greater percentage of vertebral malformations following prenatal VPA exposure than F1 mice carried in a D2 mother. This reciprocal F1 difference is known as a maternal effect and shows that maternal genotype/uterine environment is an important mediator of VPA teratogenecity. VPA is a histone deacetylase inhibitor, and it is possible that the differential teratogenesis in B6 and D2 is because of strain differences in histone acetylation. We observed strain differences in acetylation of histones H3 and H4 in both embryo and placenta following in utero VPA exposure, but additional studies are needed to determine the significance of these changes in mediating teratogenesis. Our results provide additional support that genetic factors, both maternal and fetal, play a role in VPA teratogenesis. Lines of mice derived from B6 and D2 will be a useful model for elucidating the genetic architecture underlying susceptibility to VPA teratogenesis.
Many studies have utilized the Inbred Long Sleep and Inbred Short Sleep mouse strains to model the genetic influence on initial sensitivity to ethanol. The mechanisms underlying this divergent phenotype are still not completely understood. In this study, we attempt to identify genes that are differentially expressed between these two strains and to identify baseline networks of co-expressed genes, which may provide insight regarding their phenotypic differences. We examined the whole brain and striatal transcriptomes of both strains, using next generation RNA sequencing techniques. Many genes were differentially expressed between strains, including several in chromosomal regions previously shown to influence initial sensitivity to ethanol. These results are in concordance with a similar sample of striatal transcriptomes measured using microarrays. In addition to the higher dynamic range, RNA-Seq is not hindered by high background noise or polymorphisms in probesets as with microarray technology, and we are able to analyze exome sequence of abundant genes. Furthermore, utilizing Weighted Gene Co-expression Network Analysis (WGCNA) we identified several modules of co-expressed genes corresponding to strain differences. Several candidate genes were identified, including protein phosphatase 1 regulatory unit 1b (Ppp1r1b), prodynorphin (Pdyn), proenkephalin (Penk), ras association (RalGDS/AF-6) domain family member 2 (Rassf2), myosin 1d (Myo1d), and transthyretin (Ttr). In addition, we propose a role for potassium channel activity as well as map kinase signaling in the observed phenotypic differences between the two strains.
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