Page 11 of 17 -2.0 -1.8 -2.0 -1.9 -1.2 Not tested Not tested p-value* 0.048 0.010 0.045 0.038 0.053 Zhou et al. BMC Genomics 2011, 12:124 http://www.biomedcentral.com/1471-2164/12/124 Gene symbol Ctgf Edil3 Efemp1 Igf1 Igfbp2 Ntf3 Tieg1 GenBank NM_010217 NM146015 NM_146015 NM_184052 NM_008342 NM_008742 NM_013692 Table 7 RT-PCR confirmation of differences in gene expression: Growth/neurotrophic factor genes from Experiment 2 ALC-NTC ALC-NTO Microarray Fold Change* p-value* qRT-PCR Fold Change* p-value* Microarray Fold p-value* qRT-PCR Fold Change* -1.7 -1.6 -1.5 -1.4 -2.0 -1.2 -1.2 0.01 0.00 0.01 0.02 0.02 0.30 0.36 -1.2 -1.4 -2.3 -2.6 -1.6 -1.7 -1.3 0.300 0.019 0.002 0.001 0.084 0.060 0.015 -2.2 -1.9 -2.3 -1.3 -1.5 -1.8 -1.5 0.004 0.025 0.001 0.060 0.035 0.030 0.010 *Control versus alcohol-treated. Fold change: positive = ratio of alcohol-treated to control, negative = ratio of control to alcohol-treated. p-value: t-test of control vs. alcohol treated. The neural tube abnormality may either be a delay in neural tube closure or a neural tube defect. In either case, a delay in closing of the neural tube is associated with defi- cits in midline brain development due to disruption of the timing of critical events of early brain development. At more mature stages, such midline deficits include cranio- facial abnormalities, corpus callosum, olfactory bulb, cere- bellum, and raphe neuron formation [43-50]. 2. Patterns of Gene Expression A. Temporal patterns Green and colleagues [27] reported that a 3 to 4 h binge- like alcohol exposure, with blood alcohol concentration 300 to 400 mg/dL at E8, produced a major abnormality in craniofacial and eye development in C57BL/6 mice at E15 or E17 (effects in the C57BL/6J substrain were greater than in the C57BL/6N substrain). Alterations of gene expression were reported to occur within hours of alcohol exposure at E8; these genes included metabolic and cellular gene, down-regulated ribosome and protea- some pathways; upregulated glycolysis and pentose phos- phate, tight junction, and Wnt signaling pathways, as well as other cellular profile genes. In another study, a comparable high dose of alcohol exposure at an earlier stage, E6-E8, produced growth retardation, abnormal tail torsion, open neural tube, reduction of somite number, and other malformations [28]. The altered gene expres- sion at E10 included cytoskeletal (Neurofilament), signal transduction (Zinc finger protein, MAP kinase related, Transcription factor Nf2l2), and metabolic genes (lactate dehydrogenase, Aldolase 1). In the current study, a simi- lar dose of alcohol exposure at the stage of neurulation (E8-10) produced a major neural and cardiovascular retardation and other organ system abnormalities. The trends of gene expression are consistent with the observed developmental delay and growth retardation in FASD. Among the genes with reduced expression in the alcohol-treated embryos were those involved in growth retardation, neural development, heart and hematopoiesis, and epigenetics. Among the identified functionally related gene sets, the most notable effect was the down regulation of growth-related genes, which represented the largest group of affected genes (Table 4). These genes provide plausible candidates for mechanistic links to the observed embryonic growth retardation. B. Neural specification genes Expression of neural specification genes (Table 5 and 7) and neurotrophic/growth factor genes (Table 4 and 7) was also reduced by the ethanol exposure. These partici- pate in neuronal specification, neural stem cell differen- tiation, and neural fate determination [51-55]. Suppression of these genes predicts a downstream reduction in the early formation of neural cells. Null neurog 1 (Ngn1) or neurog 2 (Ngn2) leads to sensory abnormality [56,57]). These differential expression of neuronal specification/patterning genes together with neurotrophic genes supports the dysmorphism and developmental delay of neural tube and fore-to mid- brain formation. The Igf1 and EGF genes were also identified by a microarray study with 3 h alcohol treat- ment [27] indicating they are altered early after ethanol exposure. The down-regulation of these neural specifica- tion and neural trophic/growth factor genes may play a major role in the neurodevelopmental deficit observed in the current study and featured in FASD. C. Genes related to other organ defects Although heterogeneity of tissue arising from use of whole embryos might have masked some changes in specific tissues, two functional gene sets, optic vesicle and the heart (Table 4), were identified and specifically linked to our observed developmental delay and abnormalities. Also, the collective down-regulation of key hematopoiesis genes that were either absent (Table 3) or reduced (Table 2) is consistent with the reduced blood circulation observed in the embryos. D. Histone variants Many histone genes related to epigenetic regulation of transcription were affected by ethanol (Table 4). The