Zhou et al. BMC Genomics 2011, 12:124 http://www.biomedcentral.com/1471-2164/12/124 and Fabp4; [20]), nucleotide excision repair gene, (Ercc6l) [23], stress-related genes (e.g. heat shock pro- tein 47 [24]), and differentiation and apoptosis genes such as Timp4, Bmp15, Rnf25, Akt1, Tulp4, Dexras1 [25]. These altered genes suggest potential mechanisms for the abnormal development in FASD. However, the wide-ranging developmental abnormalities in FASD are likely a consequence of the interaction of multiple genes. Examination of global gene expression provides a holistic view of genes that potentially interact and colla- boratively contribute to the abnormal development. Alcohol exposure induced changes in a group of cellular adhesion genes (e.g. L1cam and integrin) in neuroblas- toma cells [26]. A brief ethanol exposure (3 h) at gesta- tion day 8 (E8) in mouse embryos altered expression of genes of metabolic, cell programming and cytoskeletal signaling pathways [27]. An earlier alcohol exposure at E6-E8 also altered a set of genes related to PLUNC, neurofilament, and pale ear [28]. In animal models of prenatal alcohol exposure, sources of variability include the pattern, concentration, amount, and developmental stage of alcohol exposure, maternal stress, embryonic growth and maturation of embryos between litters and even within a given litter and within inbred strains of mice [29]. Control of all these variables in rapidly developing embryos is virtually unattainable in vivo. To limit these variables, a whole embryonic culture [30,31] was adopted, including stage alignment based on somite number, in which the pat- tern, amount and concentration of alcohol and embryo- nic staging were controlled. Inbred C57BL/6 mice, with known susceptibility to ethanol teratogenesis [32,33], were used for this study. Differences in the dose and timing of alcohol exposure are known contributors to variation in the phenotypic spectrum in FASD. Understanding the pattern of gene alterations that co-vary with different outcomes pro- duced by different alcohol doses or developmental tim- ing of exposure would provide valuable insights into mechanisms underlying this phenotypic variability. As development is highly dynamic throughout gestation, we asked how alcohol exposure might affect genome-wide gene expression at the critical stage of neurulation (E8- 10), when the nervous system (and other major organs) are actively forming in mouse. We have shown that at this key stage, neural tube formation was highly sensi- tive to the alcohol insult [29]. DNA methylation was altered, with the degree of change commensurate with severity of neural tube defect [34]. In the current study, in an initial experiment, cluster analysis indicated dis- tinct differences in gene expression not only between control- and alcohol-treated embryos, but also between two phenotypic subsets of alcohol-treated embryos dis- cernable at the end of alcohol treatment, one group Page 2 of 17 which had a closed neural tube (ALC-NTC) and the other group with an open neural tube (ALC-NTO). A second study with a larger set of arrays was then per- formed in which alcohol-treated embryos of both neural tube phenotypes were specifically compared. We report here the correlation of alcohol-induced embryonic growth retardation and neural tube abnormalities with changes in expression in networks of genes known to regulate embryonic growth, organ development, and neural specification processes. Results Embryonic Growth Retardation/Abnormalities As was seen in our previous report [29], the size and somite number varied (from 1-6) among embryos within a litter at the time of harvesting from the mother. We selected embryos of similar developmental stages (3-5 somites) and randomly assigned them to the two treat- ment groups (alcohol or control). The alcohol concen- tration profile of the culture media over the 46 hours was similar to that in our previous report [29]. The con- centration of ethanol in the medium was ~88 mM at the start of each day (when first added to the media) and declined to ~44 mM by the end of each day. Among all cultured embryos, more than 95% maintained active heartbeats and blood circulation over this time, and only those were used for analysis. Development of the heart, caudal neural tube, brain vesicles, optic sys- tem, and limb buds in the embryos were significantly compromised in the alcohol treated group (Table 1). Brain vesicle development was retarded and the brain vesicles were smaller in size in the alcohol group. The significant effects in multiple organs and regions and in total scores (Table 1) demonstrated that alcohol treat- ment resulted in retardation of the overall growth and interfered with development of several specific struc- tures, including brain, heart, and limb development, in this embryonic culture model. The overall growth retardation was accompanied by varying degrees of abnormality in organ system develop- ment (Figure 1). These abnormalities included an increased size of the heart and ventricular chambers, reduced size of lung buds, flattened forebrain, small/ slanted eyes, abnormal tail morphology, abnormal limb web, and unfinished turning of neural axis. A reduced blood/vascular system was also evident by less vasculari- zation in yolk sac (Table 1), and lower red coloration apparent in many blood vessels of yolk sacs and embryos in the alcohol-treated than the control embryos (Figure 2). Among 127 samples of alcohol-treated embryos, 34 (27%) had various degrees of incomplete neural tube closing (Figure 1); this compares to 3 (2%) out of the 139 controls. These openings in the neural tube mostly