Chris Downing

Subtle decreases in DNA methylation and gene expression at the mouse Igf2 locus following prenatal alcohol exposure: effects of a methyl-supplemented diet

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. 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, although prenatal alcohol had only small effects on DNA methylation at the Igf2 locus, placing dams on a methyl-supplemented diet partially ameliorated ethanol teratogenesis.

Ethanol Teratogenesis in Five Inbred Strains of Mice

BACKGROUND Previous studies have demonstrated individual differences in susceptibility to the detrimental effects of prenatal ethanol exposure. Many factors, including genetic differences, have been shown to play a role in susceptibility and resistance, but few studies have investigated the range of genetic variation in rodent models. METHODS We examined ethanol teratogenesis in 5 inbred strains of mice: C57BL/6J (B6), Inbred Short-Sleep, C3H/Ibg, A/Ibg, and 129S6/SvEvTac (129). Pregnant dams were intubated with either 5.8 g/kg ethanol (E) or an isocaloric amount of maltose-dextrin (MD) on day 9 of pregnancy. Dams were sacrificed on day 18 and fetuses were weighed, sexed, and examined for gross morphological malformations. Every other fetus within a litter was then either placed in Bouins fixative for subsequent soft-tissue analyses or eviscerated and placed in ethanol for subsequent skeletal analyses. RESULTS B6 mice exposed to ethanol in utero had fetal weight deficits and digit, kidney, brain ventricle, and vertebral malformations. In contrast, 129 mice showed no teratogenesis. The remaining strains showed varying degrees of teratogenesis. CONCLUSIONS Differences among inbred strains demonstrate genetic variation in the teratogenic effects of ethanol. Identifying susceptible and resistant strains allows future studies to elucidate the genetic architecture underlying prenatal alcohol phenotypes.

Gene Expression Changes in C57BL/6J and DBA/2J Mice Following Prenatal Alcohol Exposure

BACKGROUND Prenatal alcohol exposure can result in fetal alcohol spectrum disorders (FASD). Not all women who consume alcohol during pregnancy have children with FASD and studies have shown that genetic factors can play a role in ethanol teratogenesis. We examined gene expression in embryos and placentae from C57BL/6J (B6) and DBA/2J (D2) mice following prenatal alcohol exposure. B6 fetuses are susceptible to morphological malformations following prenatal alcohol exposure while D2 are relatively resistant. METHODS Male and female B6 and D2 mice were mated for 2 hours in the morning, producing 4 embryonic genotypes: true-bred B6B6 and D2D2, and reciprocal B6D2 and D2B6. On gestational day 9, dams were intubated with 5.8 g/kg ethanol, an isocaloric amount of maltose dextrin, or nothing. Four hours later, dams were sacrificed and embryos and placentae were harvested. RNA was extracted, labeled and hybridized to Affymetrix Mouse Genome 430 v2 microarray chips. Data were normalized, subjected to analysis of variance and tested for enrichment of gene ontology molecular function and biological process using the Database for Annotation, Visualization and Integrated Discovery (DAVID). RESULTS Several gene classes were differentially expressed in B6 and D2 regardless of treatment, including genes involved in polysaccharide binding and mitosis. Prenatal alcohol exposure altered expression of a subset of genes, including genes involved in methylation, chromatin remodeling, protein synthesis, and mRNA splicing. Very few genes were differentially expressed between maltose-exposed tissues and tissues that received nothing, so we combined these groups for comparisons with ethanol. While we observed many expression changes specific to B6 following prenatal alcohol exposure, none were specific for D2. Gene classes up- or down-regulated in B6 following prenatal alcohol exposure included genes involved in mRNA splicing, transcription, and translation. CONCLUSIONS Our study identified several classes of genes with altered expression following prenatal alcohol exposure, including many specific for B6, a strain susceptible to ethanol teratogenesis. Lack of strain specific effects in D2 suggests there are few gene expression changes that confer resistance. Future studies will begin to analyze functional significance of the expression changes.

No Effect of Prenatal Alcohol Exposure on Activity in Three Inbred Strains of Mice

AIMS Prenatal exposure to alcohol can have adverse effects on the developing fetus. Two of the hallmarks of children exposed to alcohol prenatally are attention deficits and hyperactivity. While hyperactivity has been observed in rats following prenatal ethanol exposure, few studies have examined these effects in mice. The present study investigated the effects of prenatal ethanol exposure on activity in mice from three inbred strains: C57BL/6 (B6), Inbred Long Sleep (ILS) and Inbred Short Sleep (ISS). METHODS On Days 7 through 18 of gestation, mice were intragastrically intubated twice daily with either 3.0 g/kg ethanol (E) or an isocaloric amount of maltose-dextrin (MD); non-intubated control (NIC) litters were also generated. Offspring activity was monitored at 30, 60, 90 and 150 days of age. RESULTS While results showed no effects of prenatal ethanol exposure on any measures of activity, we did observe differences in baseline activity among the strains. ISS mice were more active than B6 and ILS for all activity measures except stereotypy; B6 mice had higher measures of stereotypy than ILS and ISS. Younger mice were more active than older mice. The only sex effects were on measures of stereotypy, where males had higher scores. CONCLUSIONS Mice are an excellent organism to study genetic influences on many phenotypes. However, our study and others have shown few effects of prenatal ethanol exposure on behavior in mice. It appears as if the prenatal period in mice, corresponding to organogenesis, is not a sensitive period for producing behavioral deficits following ethanol exposure. It is likely that the first 2 weeks postnatally, corresponding to the brain growth spurt, are more sensitive for producing behavioral effects.

Genetic and Maternal Effects on Valproic Acid Teratogenesis in C57BL/6J and DBA/2J Mice

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.

The metabotropic glutamate receptor subtype 5 mediates sensitivity to the sedative properties of ethanol.

Objective Inbred long-sleep and short-sleep mice (ILS and ISS) were selectively bred for differential sensitivity to the sedative effects of ethanol. Lines of mice derived from these progenitors have been used to identify several quantitative trait loci (QTLs) mediating loss of the righting reflex due to ethanol (LORE). This study investigated the metabotropic glutamate receptor subtype 5 (mGluR5) as a candidate gene underlying Lore7, a QTL mediating differential LORE sensitivity. Methods We used knockout mice, a quantitative complementation test, pharmacological antagonism of mGluR5, real-time quantitative PCR, radioligand binding, DNA sequencing, and bioinformatics to examine the role of mGluR5 in ethanol-induced sedation. Results mGluR5 knockout mice had a significantly longer LORE duration than wildtype controls. Administration of the mGluR5 antagonist 2-methyl-6-(phenylethyl)-pyridine (MPEP) had differential effects on LORE in ILS and ISS mice. A quantitative complementation test also supported mGluR5 mediating LORE. Two intronic single-nucleotide polymorphisms in mGluR5 were highly correlated with LORE in recombinant inbred mice derived from a cross between ILS and ISS (LXS RIs). Differences in mGluR5 mRNA level and receptor density were observed between ILS and ISS in distinct brain regions. Finally, data from WebQTL showed that mGluR5 expression was highly correlated with several LORE phenotypes in the LXS RIs. Conclusion Altogether, this data provides convincing evidence that mGluR5 mediates differential sensitivity to the sedative effects of ethanol. Studies from the human literature have also identified mGluR5 as a potential candidate gene for ethanol sensitivity.

Mapping quantitative trait loci mediating sensitivity to etomidate.

Long- and Short-Sleep (LS and SS) mice were selectively bred for differences in ethanol-induced loss of the righting reflex (LORR) and have been found to differ in LORR induced by various anesthetic agents. We used a two-stage mapping strategy to identify quantitative trait loci (QTLs) affecting duration of LORR caused by the general anesthetic etomidate and brain levels of etomidate (BEL) following regain of the righting reflex. Analysis of recombinant-inbred strains derived from a cross between LS and SS mice (LSXSS) yielded a heritability estimate of 0.23 for etomidate-induced LORR and identified one marker that showed suggestive linkage for a QTL, on mouse Chromosome (chr) 12. Mapping in an F2 population derived from a cross between inbred LS and SS (ILS and ISS) revealed a significant QTL for etomidate-induced LORR on Chr 12, and two significant QTLs mediating BEL on Chrs 6 and 12. Several QTLs showing suggestive linkage for etomidate-induced LORR and BEL were also identified in the F2 population. Brain levels of etomidate in the RI and F2 mice suggested that differences in LORR were due to differential central nervous system sensitivity, rather than differential etomidate metabolism. Interestingly, the region on Chr 7 has also been identified as a region influencing ethanol-induced LORR, suggesting the possibility of a common genetic mechanism mediating etomidate and ethanol sensitivity. These QTL regions need to be further narrowed before the testing of candidate genes is feasible.

Quantitative Trait Locus Mapping for Ethanol Teratogenesis in BXD Recombinant Inbred Mice

BACKGROUND Individual differences in susceptibility to the detrimental effects of prenatal ethanol (EtOH) exposure have been demonstrated. Many factors, including genetics, play a role in susceptibility and resistance. We have previously shown that C57BL/6J (B6) mice display a number of morphological malformations following an acute dose of EtOH in utero, while DBA/2J (D2) mice are relatively resistant. Here, we present the results of quantitative trait locus (QTL) mapping for EtOH teratogenesis in recombinant inbred strains derived from a cross between B6 and D2 (BXD RIs). METHODS Pregnant dams were intubated with either maltose-dextrin or 5.8 g/kg EtOH on day 9 of gestation (GD9). On GD 18, dams were sacrificed and fetuses and placentae were removed. Placentae and fetuses were weighed; fetuses were sexed and examined for gross morphological malformations. Fetuses were then either placed in Bouins fixative for subsequent soft-tissue analyses or eviscerated and placed in EtOH for subsequent skeletal examinations. QTL mapping for maternal weight gain (MWG), prenatal mortality, fetal weight (FW) at c-section, placental weight (PW), and several morphological malformations was performed using WebQTL. RESULTS Heritability for our traits ranged from 0.06 for PW to 0.39 for MWG. We found suggestive QTLs mediating all phenotypes and significant QTLs for FW and digit and rib malformations. While most QTL regions are large, several intriguing candidate genes emerged based on polymorphisms between B6 and D2 and gene function. CONCLUSIONS In this first mapping study for EtOH teratogenesis, several QTLs were identified. Future studies will further characterize these regions. Identification of genes and epigenetic modifications mediating susceptibility to the teratogenic effects of alcohol in mice will provide targets to examine in human populations.

Maternal effects on ethanol teratogenesis in a cross between A/J and C57BL/6J mice

Genetic factors influence adverse pregnancy outcome in both humans and animal models. Animal research reveals that both the maternal and fetal genetic profiles are important for determining the risk of physical birth defects and prenatal mortality. Using a reciprocal-cross breeding design, we investigated whether the mothers genes may be more important than fetal genes in determining risk for ethanol teratogenesis. Examination of possible synergistic genetic effects on ethanol teratogenesis was made possible by using two mouse strains known to be susceptible to specific malformations. Inbred A/J (A) and C57BL/6J (B6) mice were mated to produce four fetal genotype groups: the true-bred AċA and B6ċB6 genotypes and the genetically identical AċB6 and B6ċA genotypes (the F(1) genotype). Dams were administered either 5.8 g/kg ethanol or an isocaloric amount of maltose-dextrin on day 9 of pregnancy. Fetuses were removed by laparotomy on gestation day 18, weighed, and assessed for digit, vertebral, and kidney malformations. Digit malformations in the genetically identical F(1) ethanol-exposed litters showed a pattern consistent with a maternal genetic effect (AċB6 [2%] and B6ċA [30%]). In contrast, vertebral malformations were similar in all ethanol-exposed litters (AċA [26%], AċB6 [18%], B6ċA [22%], and B6ċB6 [33%]). The percentage of malformations did not differ between male and female fetuses, indicating sex-linked factors are not responsible for the maternal effect. Ethanol exposure decreased litter weights but did not affect litter mortality compared with maltose-exposed controls. This study supports the idea that genes influence malformation risk following in utero alcohol exposure. Specifically, maternal genes influence risk more than fetal genes for some teratogenic outcomes. No evidence supported synergistic genetic effects on ethanol teratogenesis. This research supports the conclusion that uterine environment contributes to determining risk of Fetal Alcohol Spectrum Disorder.

Murine Models of Alcoholism

Most behavioral responses to alcohol are known to be influenced by genetic factors. Human twin and adoption studies consistently show that susceptibility to alcohol abuse is heritable (1). The mode of inheritance is unknown, but is certainly polygenic and multifactorial, with a substantial environmental effect (1,2). Despite much research, the genes and causal pathways determining susceptibility to alcohol abuse and dependence remain relatively unknown. Identifying genes that mediate alcoholism will improve strategies for diagnosis, treatment, and ultimately prevention.