Zhou et al. BMC Genomics 2011, 12:124 http://www.biomedcentral.com/1471-2164/12/124 Validation by Quantitative RT-PCR Quantitative RT-PCR (qRT-PCR) was used to verify some of the genes that were significantly affected by alcohol, including a sample of genes from the functional gene sets for neural specification and trophic factors identified in GSEA (Tables 6 and 7). These studies used independent embryos subjected to identical ethanol exposure. The qRT-PCR verified that all 11 down- regulated neural specification genes (Table 6) and neurotrophic/growth factor genes (Table 7) tested dif- fered in the same direction. One gene (Mylc2) that did not differ in the microarray experiments was also tested and the lack of difference was confirmed. Discussion 1. Developmental Deficits and Correlation with Gene Expression Profiles The abnormal embryonic development resulting from the alcohol treatment at this specific stage of develop- ment (Figure 1; Table 1) was consistent with our pre- vious report [29] and those of others [41,42]. Two different facets of abnormal development could be iden- tified: growth delay and frank teratogenesis. Delays in growth were also evident by the significant reductions in the total RNA per embryo and in the delayed morpholo- gical staging (Table 1). The affected structures were derived from each of the three germ layers, i.e., neural tube and brain vesicles (ectoderm), somites and cardio- vascular system (mesoderm/endoderm), and involved a wide range of tissues and organs (e.g., heart, head, limbs). Alterations in all of these have been observed in FAS cases. The teratogenic consequences were evident as dysmorphology of various organs (central nervous system, eye, and heart) that involved pathogenic effects beyond just the observed delay of the normal course of development. Examples include enlarged heart primor- dium and abnormally enlarged ventricular chambers, detached pericardial sac, small forebrain, flat telencepha- lic vesicle, failure in neural tube closure, and small and irregularly shaped eyes. Neural tube defect We observed in Experiment 1 that gene expression pro- files from alcohol treatment of embryos in this controlled Page 10 of 17 culture system yielded two distinguishable patterns; com- parison to the morphological data revealed that these were correlated with two different phenotypes: open (ALC- NTO) and closed neural tubes (ALC-NTC). The pheno- types and correlated gene expression differences were reproduced in Experiment 2. The embryos with open neural tubes (ALC-NTO) had more severe delays in brain and otic development than those with closed neural tubes (ALC-NTC) (Table 1). These different phenotypes are consistent with our previous in vivo observation in a liquid diet model of prenatal alcohol exposure in C57BL/6 mice, which resulted in partial penetration of incomplete neural tube closure (as late as embryonic day 15) and a cascade of deficits in midline structural development [43]. Finding this difference in development in experimentally con- trolled culture conditions indicates either a stochastic event or that an extremely sensitive gene-environment interaction is involved, e.g. different outcomes based on small differences in developmental stage at the time of exposure or small differences in tissue concentrations of alcohol across embryos. We have recently found greater DNA hypermethylation in ALC-NTO than in ALC-NTC embryos, particularly in genes on chromosomes 7, 10, and X. Remarkably, there was a >10 fold increase in the num- ber of hypermethlyated genes on chromosomes 10 and X in ALC-NTO than ALC-NTC [34]. Both the ALC-NTC and the ALC-NTO embryos demonstrated lower expression of genes in sets related to cell growth, growth factors, heart (angiogenesis), and eye (in NTC vs. Control) (Table 4; Table 7). The ALC-NTC and ALC-NTO embryos also differed in other sets of func- tionally related genes. The histone gene set was selectively reduced in ALC-NTO compared to controls. The epider- mal growth factor signaling pathway genes were lower in ALC-NTO than ALC-NTC (Table 4). At the single gene analysis level, Experiment 2 showed a greater number of neurotrophic/growth factor genes were down-regulated in ALC-NTO than in ALC-NTC groups, particularly in the TGFb, NTF3, S100, and EGF families. These differences in gene expression between the ALC-NTO and ALC-NTC embryos appear to be correlated with the more severe ter- atogenic trajectory of the ALC-NTO group, but causal relationships have yet to be established. Gene Bhlhb5 Ngn2 Ngn1 Sox5 Mylc2 GenBank NM_021560 BC055743 NM_010896 AI528773 NM_023402 *Control versus alcohol-treated. Table 6 RT-PCR confirmation of differences in gene expression: Neural specification genes from Experiment 1 Microarray Fold Change* qRT-PCR Fold Change -1.6 -2 -1.3 -1.4 -1 p-value* 0.0021 0.0015 0.047 0.015 0.8 -2.3 -2 -1.7 -1.7 -1 p-value Description 0 0 0.021 0.005 0.19 basic helix-loop-helix domain, class B5 neurogenin 2 neurogenin 1 SRY-box containing gene 5 myosin light chain