Zhou et al. BMC Genomics 2011, 12:124 http://www.biomedcentral.com/1471-2164/12/124 Page 4 of 17 Figure 1 Alcohol causes dysmorphology of growing embryos. Control embryos (a), Alcohol-treated (b-f). There are many dysmorphologies including microencephaly of forebrain (b, c, f), failure of closure of midbrain (mb; c) or hindbrain (hb; f), dysmorphic optical vesicle (optic; d), flex tail (ft; e) in caudal neural tube, delay formation of heart (H) chamber (b) and occasional detachment of epicardium (epic; b and e), neural tube opening at midbrain (mb; c, arrowheads) and hindbrain (hb; f, arrowheads) in the alcohol group. Majority of the brain vesicles in alcohol-treated group were closed (ALC-NTC; b, e). Approximately 30% of the embryos were found with a neural tube opening (ALC-NTO), usually in the head fold. Scale bars: a, b, c, e, f = 0.05mm; d = 0.25 mm. open neural tubes (ALC-NTO) (Table 3 last row). No gene was found to be absent in Control but present in ALC-NTC. Another retinol regulating gene, cellular reti- nol binding protein 1 (Crbp1), was reduced by alcohol exposure (Table 2). Gene Set Enrichment Analysis (GSEA) Analyses Four GSEA analyses were conducted within each experi- ment: control versus all alcohol-treated (ALC), control versus ALC-NTC, control versus ALC-NTO, and ALC- NTC versus ALC-NTO. As 415 GO gene sets and 191 stem cell related gene set were pre-selected, there were totally 4 × (415+191) = 2424 GSEA tests. We found 15 gene sets that were significant at 5% and shared the same enrichment direction in both experiments. By chance, one would expect only 2424 × (0.05 × 0.05 × 0.5) = 3; therefore, the FDR is 3/15 = 20%. The signifi- cant gene sets common to the two experiments are out- lined below. a. Early Developmental Biology Gene Sets GSEA analysis using the GO biological function cate- gories selected as being related to development (Addi- tional files 1 and 2.) identified 20 enriched sets in Experiment 2. Of these 20 sets, 9 were also identified by Experiment 1 (Table 4). Included in these shared gene sets are multiple GO categories related to growth, eye and heart development, and epigenetics. When comparing the control embryos to all alcohol treated embryos, there were 7 GO categories that were enriched in the control groups (i.e., down-regulated in the alcohol-treated