Zhou et al. BMC Genomics 2011, 12:124 http://www.biomedcentral.com/1471-2164/12/124 Page 3 of 17 Table 1 Embryonic dysmorphology after alcohol exposure, scored according to Maele-Fabry et al,1992 Region Allantois Branchial bars Brain: Forebrain Brain: Midbrain Brain: Hindbrain Caudal Neural Tube Flexion Heart Limb: Forelimb Limb: Hindlimb Mandibular process Maxillary process Olfactory system Optic system Otic system Somites Total score Control 3 ± 0 2.77 ± 0.09 4.76 ± 0.10 4.52 ± 0.11 4.71 ± 0.10 4.76 ± 0.12 4.80 ± 0.09 4.80 ± 0.10 2.01 ± 0.06 0.53 ± 0.10 2.08 ± 0.11 2.41 ± 0.14 0.47 ± 0.08 3.59 ± 0.14 3.95 ± 0.12 4.81 ± 0.09 53.97 ± 0.66 Alcohol 2.80 ± 0.08 2.15 ± 0.21 3.81 ± 0.27* 3.71 ± 0.27 3.86 ± 0.24* 4.11 ± 0.19* 4.33 ± 0.19 4.10 ± 0.16** 1.51 ± 0.13** 0.20 ± 0.08* 1.99 ± 0.09 2.06 ± 0.16 0.26 ± 0.08 2.87 ± 0.14** 3.68 ± 0.11 4.38 ± 0.16 45.83 ± 1.54** ALC-NTC 2.86 ± 0.10 2.17 ± 0.26 4.57 ± 0.14 4.50 ± 0.14 4.50 ± 0.14 4.09 ± 0.26* 4.59 ± 0.19 4.15 ± 0.19* 1.48 ± 0.18* 0.21 ± 0.09 2.12 ± 0.08 2.21 ± 0.18 0.29 ± 0.11 3.02 ± 0.17* 3.88 ± 0.10 4.50 ± 0.17 49.14 ± 1.54** ALC-NTO 2.70 ± 0.14 2.11 ± 0.39 2.29 ± 0.29** ^^ 2.14 ± 0.14** ^^ 2.57 ± 0.30** ^^ 4.14 ± 0.26* 3.81 ± 0.36* 4.00 ± 0.31* 1.57 ± 0.20 0.19 ± 0.14 1.71 ± 0.18 1.76 ± 0.30 0.20 ± 0.13 2.57 ± 0.20** 3.29 ± 0.18* ^ 4.14 ± 0.34 39.23 ± 1.72** ^^ * P-Value <0.05, ** <0.01; compared to control. ^ P-Value <0.05, ^^ <0.01; compared to NTC. Control n = 21; Alcohol (all alcohol-treated, n = 21; ALC-NTC, n = 14; ALC-NTO, n = 7). occurred in the head fold, although delayed or incom- plete neural tube closure in midbrain and hindbrain was also seen. The abnormalities and developmental delays are clearly more severe in ALC-NTO than in ALC-NTC subgroups, particularly in development of the neural axis including hindbrain, midbrain, forebrain, otic vesicle. Differences in Gene Expression At the end of the culture period, the total RNA extracted from alcohol-treated embryos was approxi- mately half that of controls: controls = 2.8 ± 0.5 (n = 13), ALC-NTC = 1.6 ± 0.5 (n = 13, P < 0.05 com- pared with control), ALC-NTO = 1.2 ± 0.5 (n = 8, P < 0.05 compared with control). In Experiment 1, 14,243 out of 22,690 probe sets (62.7%) were present in at least half of the samples in either control or alcohol treated groups. Hierarchical clustering by arrays (Figure 3. Exp 1) clearly separated the samples into three groups, control, ALC/NTC, and ALC/NTO, rather than just two (ALC vs. control). In Experiment 2, 26,674 out of 45101 probe sets (59.1%) were present in at least half of the samples in either control or alcohol treated group. Again, the hierarchical cluster analysis (Figure 3. Exp 2) separated the samples into the same three groups, con- trol, ALC/NTC, and ALC/NTO. In Experiment 1, 850 probe sets (6% of the probe sets that were present) were differentially expressed in alco- hol-treated embryos as a group (p ≤ 0.05). In Experi- ment 2, which had more power due to the larger number of arrays and also examined twice as many probe sets, 2519 probe sets (9.4% of the probe sets that were present) were differentially expressed in alcohol- treated embryos considered as a group (p ≤ 0.05). These relaxed stringencies were employed to reduce false nega- tives when comparing genes across the two experiments. The probe sets on the Mouse Genome 430A GeneChip were a subset of those on the Mouse Genome 430 2.0 GeneChip. Comparing this common subset across the two experiments, 87 probe sets were significant in both experiments and consistent in direction; because there are 13810 genes present in both experiments, the expectation is that only 17 genes would be expected to be in common with the same direction of change. 49 probe sets were lower in alcohol-treated embryos and 38 were higher (Table 2). Among these were genes for alcohol metabolism, epigenetics (histone and histone variants), hematopoiesis, neurotrophic factors, retinol metabolism, cell cycle, cell adhesion, homeobox genes, and oncogenes. Furthermore, in Experiment 2 (which had more power to detect differences), a number of genes in addition to the above list were present in the controls but were absent in the alcohol treated samples (Table 3). Notably, glycophorin A (Gypa) and beta-2 microglobulin (B2m) genes were absent in ALC-NTO, and ceruloplasmin (Cp), adducin 2 (Add2), B2 m, and ceruloplasmin (Cp) genes were absent in ALC-NTC. All of these are critical in hematopoiesis and/or red blood cell function [35-39]. In contrast, the aldehyde dehydrogenase 1 family, B1 (Aldh1b1), which catalyzes oxidation of retinaldehyde, was present only in the alcohol-treated embryos with