Prenatal alcohol exposure causes a continuum of birth defects that includes growth deficiency, mental retardation and craniofacial anomalies. The gestational period of fetal exposure determines the nature and severity of the defects. Ethanol exposure during the gastrulation period of embryogenesis results in craniofacial and neural tube disorders in humans and in several vertebrate species. Accumulating evidence indicates that ethanol produces developmental anomalies by altering specific regulatory pathways used by embryonic cells to direct their development. This idea is supported by studies on signal transduction in mouse embryonic cells during the preimplantation stage of development. Preimplantation embryos are not susceptible to the teratogenic effects of ethanol, but are a superior stage to work with experimentally because the embryos may be cultured outside the female reproductive system in simple, serum-free media. It is hypothesized that the proximal intracellular signaling pathways altered by ethanol in preimplantation embryos are affected similarly at other stages of embryogenesis; however, downstream physiological changes that influence subsequent development are stage-specific. Studies are proposed to examine the signaling events perturbed by ethanol in postimplantation embryos during the period of gastrulation, which has been established as an animal model of ethanol-induced teratogenesis. Common intracellular signaling pathways are responsible for inducing cell proliferation, differentiation and programmed cell death (apoptosis). We will determine whether the proximal pathways altered by ethanol are shared by preimplantation and gastrulation-stage embryos, and whether embryonic cells at the two stages are differentially programmed in their physiological response to a common signaling pathway. Accordingly, the effect of ethanol on cell proliferation and apoptosis will be assessed in embryonic cells at these two developmental stages using quantitative in situ techniques. The balance of cell survival genes is a major determining factor in the response of cells to intracellular signals. Preimplantation embryos appear to be programmed for proliferation through autocrine growth factor signaling, while growth factor expression during postimplantation development may be more limited and localized. Experiments will be carried out to determine whether ethanol-induced cell proliferation and apoptosis are alternative outcomes determined by the relative expression levels of cell survival genes. The expression of mRNA for pro-apoptotic and anti-apoptotic genes of the bcl-2 and c-myc proto-oncogene families will be measured in embryos at the two stages, along with mRNA for growth factors that protect cells from entering the apoptosis pathway. Based on the observed effects of ethanol on postimplantation embryonic cells regarding their cellular activities and expression of survival-related genes, experiments are designed to delineate the proximal signaling pathways altered by ethanol during gastrulation. The proposed experiments may identify signal transduction inhibitors or growth factors to ameliorate the downstream effects of ethanol in vitro, and suggest strategies for therapeutic intervention during pregnancy.
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