MECHANISMS OF ETHANOL INDUCED TERATOGENICITY

描述(由申请者提供)：本研究项目旨在增加我们的 对酒精致伤机制及分子病理学的认识 畸形发育。它跟进了我们之前确定的大脑、耳部和视神经原始区域作为乙醇诱导的细胞凋亡和随后的出生缺陷的目标，我们正在进行的微阵列分析的结果，以及其他人对这一领域的重大贡献。聚焦于小鼠的发育阶段，对应于人类胚胎发生的3-6周，并采用原位杂交，我们将检验这一假设，即在接触乙醇后的几个小时内，图案基因在胚胎的大脑、眼睛和内耳中发生异常表达；这些变化预示着随后的畸形发生。为了阐明致病序列，可以检测到改变的图案的时间将与最初可以识别细胞凋亡的时间进行比较。利用急性酒精暴露范例、全胚胎培养和激光共聚焦成像，将有助于分析小鼠胚胎发生的时间和区域特异性变化。认识到酒精暴露会干扰维甲酸(RA)的代谢，维甲酸(RA)调节基因表达；我们还将检验这一假说，即减少RA依赖的基因信号是乙醇致畸的基础。在这项工作中，我们将比较基因表达模式 以及维甲酸缺乏(BMS493处理)和乙醇暴露的小鼠胚胎发育中的脑、眼和内耳的细胞凋亡模式，并测试RA的改善潜力。此外，我们建议进行体外和体内研究，以检验改变遗传(特别是。Sonic hedgehog)信号转导源于乙醇诱导的RA缺乏症，将加剧胆固醇降低后信号转导减弱。这些实验将利用药理学(AY9944)和遗传(7脱氢胆甾醇还原酶基因修饰)诱导的胆固醇缺乏，两者都会产生与乙醇引起的畸形一致的畸形。这项工作有望提供与可能影响人类人群对乙醇致畸胎敏感性的因素相关的重要新数据。它还将为在其他发育阶段和脆弱组织的后续行动中使用类似的分析奠定基础。

DESCRIPTION (provided by applicant): This research project is designed to increase our understanding of the mechanisms and molecular pathology underlying alcohol-induced dysmorphogenesis. It follows up on our previous identification of selected regions of the brain, otic, and optic primordial as targets of ethanol-induced apoptosis and subsequent birth defects, results of our ongoing microarray analyses, and significant contributions to this field by others. Focusing on Developmental stages in mice that correspond to weeks 3-6 of human embryogenesis and employing in situ hybridization, we will test the hypothesis that within hours following exposure to ethanol, abnormal expression of patterning genes occurs in the embryonic brain, eye and inner ear; changes that presage subsequent dysmorphogenesis. To elucidate pathogenic sequences, the time by which altered patterning is detectable will be compared to that for which apoptosis can initially be identified. Analyses of temporally and regionally-specific alterations in mouse embryogenesis, will be facilitated by utilization of an acute ethanol exposure paradigm, whole embryo culture, and laser confocal imaging. Recognizing that ethanol exposure can interfere with retinoid metabolism and that retinoic acid (RA) regulates gene expression; we will also examine the hypothesis that diminishing RA-dependent gene signaling underlies ethanol's teratogenicity. For this work, we will compare gene expression patterns and patterns of apoptosis in the developing brain, eye and innerear of retinoid-deficient (BMS493-treated)and ethanol-exposed mouse embryos and test RA's ameliorative potential. Additionally, we propose to conduct in vitro and in vivo investigations to test the hypothesis that altered genetic (esp. sonic hedgehog) signaling stemming from ethanol-induced RA deficiency will exacerbate diminished signaling subsequent to cholesterol reduction. These experiments will utilize both pharmacologically (AY9944) and genetically (7 dehydrocholestrol reductase gene modification) - induced cholesterol deficiency, both of which yield malformations consistent with those caused by ethanol. This work is expected to provide important new data relative to factors that may influence sensitivity to ethanol-induced teratogenesis in human populations. It will also establish a foundation for similar analyses to be utilized in following up on other developmental stages and vulnerable tissues.