Prenatal alcohol exposure alters expression of neurogenesis-related genes in an ex vivo cell culture model

Abstract

Prenatal alcohol exposure can lead to long-lasting changes in functional and genetic programs of the brain, which may underlie behavioral alterations seen in Fetal Alcohol Spectrum Disorder (FASD). Aberrant fetal programming during gestational alcohol exposure is a possible mechanism by which alcohol imparts teratogenic effects on the brain; however, current methods used to investigate the effects of alcohol on development often rely on either direct application of alcohol in vitro or acute high doses in vivo. In this study, we used our established moderate prenatal alcohol exposure (PAE) model, resulting in maternal blood alcohol content of approximately 20 mM, and subsequent ex vivo cell culture to assess expression of genes related to neurogenesis. Proliferating and differentiating neural progenitor cell culture conditions were established from telencephalic tissue derived from embryonic day (E) 15-17 tissue exposed to alcohol via maternal drinking throughout pregnancy. Gene expression analysis on mRNA derived in vitro was performed using a microarray, and quantitative PCR was conducted for genes to validate the microarray. Student's t tests were performed for statistical comparison of each exposure under each culture condition using a 95% confidence interval. Eleven percent of genes on the array had significantly altered mRNA expression in the prenatal alcohol-exposed neural progenitor culture under proliferating conditions. These include reduced expression of Adora2a, Cxcl1, Dlg4, Hes1, Nptx1, and Vegfa and increased expression of Fgf13, Ndn, and Sox3; bioinformatics analysis indicated that these genes are involved in cell growth and proliferation. Decreased levels of Dnmt1 and Dnmt3a were also found under proliferating conditions. Under differentiating conditions, 7.3% of genes had decreased mRNA expression; these include Cdk5rap3, Gdnf, Hey2, Heyl, Pard6b, and Ptn, which are associated with survival and differentiation as indicated by bioinformatics analysis. This study is the first to use chronic low to moderate PAE, to more accurately reflect maternal alcohol consumption, and subsequent neural progenitor cell culture to demonstrate that PAE throughout gestation alters expression of genes involved in neural development and embryonic neurogenesis.

Keywords: Alcohol; Cell culture; Chronic; DNA methylation; Development; Epigenetic; Gene expression; Neurogenesis; Prenatal.

Figure 1. Prenatal alcohol exposure paradigm and ex vivo cell culture

Female C57BL/6 mice, aged 2 months, were acclimated to drinking 10% w/v alcohol with 0.066% (w/v) saccharin or 0.066% (w/v) saccharin only for 4 days. Females continued to drink for 1 week and were then mated for 2 days; 13–15 days after the last day of mating, embryonic tissue (E15–E17) was extracted from pregnant females and the telencephalons from all embryos from one dam were cultured under proliferating conditions with growth factors. After 10 days in vitro (DIV) and 3 passages, RNA was collected from the proliferating NPC cultures. Other NPC cultures had the growth factors removed from their media and were permitted to differentiate for approximately 7–10 DIV for a total of 20 DIV and 3 passages. RNA was collected from the differentiated NPCs at 20 DIV for analysis.

Figure 2. Representative images of cell culture conditions

Neural progenitor cells were collected from E15–E17 tissue without exposure to alcohol and expanded as adherent monolayer cultures in the presence of growth factors (EGF, FGF-2) for immunohistochemistry. Fig. 2A is a 20X representative image of the proliferating NPC culture conditions; Nestin, which marks neural progenitor cells, is in green with a DAPI nuclear stain in blue. Fig. 2B is a 20X representative image of the differentiated NPC culture conditions; GFAP, which marks astrocytes, is in green with a DAPI nuclear stain in blue and the immature neuronal marker doublecortin (DCX) in red.

Figure 3. Exposure to alcohol in utero induces alterations in gene expression in proliferating NPC in vitro

A) A microarray containing probes for genes related to neurogenesis and neural stem cell development was used to assess RNA expression under proliferating conditions. Fold change and p values are provided for each gene significantly altered by PAE. Data presented were acquired from control proliferating NPCs and prenatal alcohol-exposed (PAE) proliferating NPCs (n = 6 dams for each condition). B) Relative gene expression of the most significant changes in gene expression from the array data using RNA derived from the proliferating NPC culture conditions. *p < .05,**p < .01

Figure 4. Exposure to alcohol in utero induces alterations in gene expression in differentiated NPC in vitro

A) A microarray containing probes for genes related to neurogenesis and neural stem cell development was used to assess RNA expression in both culture conditions. Data presented here were acquired from control differentiated NPCs and PAE differentiated NPCs (n = 6 dams for each condition). Relative gene expression of the most significant changes in gene expression from the array data using RNA derived from the differentiated NPC cell culture conditions. *p < .05, **p < .01