Zhou et al. BMC Genomics 2011, 12:124 http://www.biomedcentral.com/1471-2164/12/124 reduction of many histone variants would alter chroma- tin organization, affecting transcription at a global level [58,59]; this may be an important effect of the alcohol that leads to the reduction of total RNA and induced growth retardation. Modification of epigenetic processes is a potential mechanism by which alcohol may alter gene expression during development, and may be an important candidate mechanism for the pathophysiology of fetal alcohol syndrome. E. Alcohol delayed or induced gene expression Other genes that were present in the control group but absent in the alcohol-treated group (Table 3) likely reflect a delay in onset or a strong inhibition of normal expression at this stage of development. Among them, four hematopoiesis genes [glycophorin A (Gypa), addu- cin 2 (Add2), beta-2 microglobulin (B2m), and cerulo- plasmin (Cp)] associated with blood cell formation were absent in the alcohol-treated groups; these genes are key components in the pathway of white and red blood cell formation [36,38,60-62]. The absence of these genes is in agreement with the low circulating blood cells seen in alcohol treated embryos (Figure 2). The expression of aldehyde dehydrogenase 1B1 (Aldh1b1) was induced in both of our experiments by alcohol treatment during this period of early neurulation (Table 2 last row). Because Aldh1b1 encodes an efficient enzyme for break- down of acetaldehyde formed during metabolism of ethanol, this up-regulation is likely a detoxification response to the high level of ethanol in the environ- ment. However, the metabolism of other substrates of this enzyme (e.g., retinoic acid, corticosteroids, biogenic amines, neurotransmitters, and lipids) that are required for normal development may be adversely affected by this increase in Aldh1b1 expression [63,64]. Conclusion In summary, alcohol exposure during the period of early neurulation at ~E8-E10, is predominantly inhibitory to gene expression, particularly the neural developmental genes. We found major reductions in gene sets involved in neurospecification, neural growth factors, cell growth and hematopoiesis. These effects on gene expression parallel the growth delay and developmental abnormal- ities including brain, neural tube, eye, heart, blood cells, and embryonic vascularization which are major targets in FASD. Our study, in conjunction with others that use different developmental periods of alcohol exposure, provides an important portfolio of alcohol-induced changes in gene expression associated with altered development. Together, these gene profiles should con- tribute to the generation of testable new hypotheses concerning the mechanistic path from gene expression changes to embryonic structural deficits, and for causal mechanisms of alcohol-induced teratogenesis (e.g., brain Page 12 of 17 growth retardation, neural tube midline deficit, craniofa- cial dysmorphology) in fetal alcohol spectrum disorder. Two such hypotheses emerge from the current study. The first is that alcohol causes a delay in development of the nervous system by inhibiting specific sets of genes involved in neural development (Ngn, Nhlh, Sox, Igf, Ntf, and Egf). The second is that neural tube defects are mediated by the inhibition of genes in the epidermal growth factor signaling pathway and genes encoding his- tone variants. Methods Embryonic Culture All experimental procedures were approved by the Insti- tutional Animal Care and Use Committee of the Indiana University School of Medicine (Indianapolis, IN) and are in accordance with the guidelines of the Institutional Animal Care and Use Committee of the National Insti- tute on Drug Abuse, National Institutes of Health, and the Guide for the Care and Use of Laboratory Animals [65]. Two-month-old C57BL/6 mice (~20 g) were pur- chased from Harlan, Inc. (Indianapolis, IN). Upon arri- val, breeder mice were individually housed and acclimated for at least one week before mating began. The mice were maintained on a reverse 12 h light-dark cycle (lights on: 19:00 - 07:00) and provided with labora- tory chow and water ad libitum. Two females were placed with one male for two hours between 08:00 and 10:00. When a vaginal plug was detected after the mat- ing period, it was designated as embryonic day 0 (E0). On E8.25 at 15:00, dams were sacrificed using CO2 gas. The embryos were treated at this stage, which is the beginning of neurulation. The window of 46 hrs treat- ment covered the stages of the formation of the major organs, neural specification and patterning. These stages are known to be vulnerable to alcohol [66]. The technique for whole embryo culture was based on the methods described by New [31]. The gravid uterus was removed and placed in sterile PBS (0.1 M phos- phate buffer containing saline) at 37°C. The embryo in the visceral yolk sac along with a small piece of the ectoplacental cone (hereafter called embryo, unless otherwise stated) was carefully removed from the decid- uas tissues and the Reichert’s membrane in PBS con- taining 4% fetal bovine serum (Sigma, St Louise. MO). After removal, three embryos bearing 3-5 somites (E8.25) were incubated in a culture bottle in 20 mL of medium which consisted of 70% immediately centri- fuged heat-inactivated rat serum (Harlan Sprague- Dawley, Inc, Indianapolis, IN) and 30% phosphate buf- fered saline (137 mM NaCl, 2.7 mM KCl, 0.5 mM MgCl2, 8 mM Na2HPO4, 1.47 mM KH2PO4, 0.9 mM CaCl2, 5.6 mM glucose, 0.33 mM sodium pyruvate, pH7.4) supplemented with 20 units/ml penicillin and