EMBRYONIC RETINOID HOMEOSTASIS AND ALCOHOL TERATOGENESIS

胎儿酒精综合征是指由酒精引起的人类 新生儿畸形，主要是中枢神经系统和 神经嵴，这是由于过量的母亲乙醇 消费这种综合症是智力迟钝的主要原因， 证明了详细了解致畸机制的努力是正确的。 尽管有许多研究旨在分析乙醇的胚胎毒性， 人类，啮齿动物和其他脊椎动物，没有单一的潜在机制， 乙醇的致畸作用已被广泛接受。一个假设的 胎儿酒精综合征的机制涉及乙醇诱导的维甲酸 该实验室提出了酸缺乏症，并将在 这个提议。维甲酸是维生素A的活性代谢产物， 在胚胎发育中起着重要的作用，作为基因的调节者， 表达，特别是在其中起关键作用的同源异型盒基因家族， 中枢神经系统的模式。由于视黄醇的转化 已知通过醇脱氢酶将维生素A（醇）转化为视黄酸， 在体外被乙醇抑制，这是非常感兴趣的，以确定是否 乙醇可影响体内视黄酸水平以及 已知由视黄酸调节的基因的表达。 乙醇致畸作用与维甲酸致畸作用的相似性 进一步暗示乙醇是干扰视黄酸水平的试剂。 怀孕期间摄入过量的维甲酸对人体有致畸作用 胎儿，新生儿表现出与观察到的缺陷相似的缺陷 乙醇致畸作用因此，人类胚胎将经历正常的 将少量母体衍生的维生素 A（视黄醇）以组织特异性方式转化为视黄酸，但可能 当对增加或减少的事件作出反应时， 维甲酸水平超出正常范围 本建议的目标如下：（l）研究各种措施的影响 定时剂量的产前乙醇对小鼠胚胎中类维生素A水平的影响 使用HPLC技术分析分离组织中的类维生素A水平 生物化学，并使用lacZ-视黄酸指示细胞系， 生物学监测胚胎组织中的视黄酸水平;（2）分析 通过北方印迹和原位杂交， (and给药时间）对已知视黄酸靶基因表达的影响 在小鼠胚胎中。研究的基因将包括那些编码 同源异型盒家族，视黄酸受体，细胞视黄酸 结合蛋白，细胞视黄醇结合蛋白，以及各种 类维生素A代谢酶，包括乙醇脱氢酶、醛 脱氢酶和细胞色素P450;（3）检查 乙醇脱氢酶（ADH）在维甲酸合成中的作用 转基因小鼠的突变。三个ADH基因已被表征， 小鼠（Adh-1，Adh-2和Adh-3），我们计划制备无效突变 每个基因。将分析这些敲除小鼠以确定是否存在缺失。 一种特殊的ADH导致胚胎发育的变化， 酸合成或视黄酸靶基因表达。

Fetal alcohol syndrome is defined as a collection of ethanol-induced human neonatal malformations, predominantly of the central nervous system and neural crest, that are the result of excessive maternal ethanol consumption. This syndrome is a leading cause of mental retardation, justifying efforts to understand the teratogenic mechanism in detail. Despite numerous studies designed to analyze ethanol embryotoxicity in humans, rodents, and other vertebrates, no single underlying mechanism for the teratogenic action of ethanol has been widely accepted. A hypothetical mechanism for fetal alcohol syndrome involving an ethanol-induced retinoic acid deficiency has been proposed by this laboratory and will be tested in this proposal. Retinoic acid is the active metabolite of vitamin A and plays a central role in embryonic development as a regulator of gene expression, particularly of-he homeobox gene family which plays a key role in patterning the central nervous system. Since the conversion of retinol (vitamin A alcohol) to retinoic acid by alcohol dehydrogenase is known to be inhibited by ethanol in vitro, it is of great interest to determine if ethanol can effect retinoic acid levels in vivo as well as effect expression of genes known to be regulated by retinoic acid. The similarities between ethanol teratogenesis and retinoid teratogenesis further implicate ethanol as an agent which disturbs retinoic acid levels. Excessive retinoic acid taken during pregnancy is teratogenic to the human fetus, with neonates exhibiting defects similar to those observed for ethanol teratogenesis. Thus, human embryos will undergo normal morphogenesis when converting a small amount of maternally-derived vitamin A (retinol) into retinoic acid in a tissue-specific fashion, but may undergo teratogenesis when responding to events which increase or decrease retinoic acid levels out of the normal range. Goals for this proposal are as follows: (l) Study the effect of various timed doses of prenatal ethanol on retinoid levels in the mouse embryo using HPLC technology to analyze retinoid levels in isolated tissues biochemically, and using a lacZ-retinoic acid indicator cell line to monitor retinoic acid levels in embryo tissues biologically; (2) Analyze by Northern blotting and in situ hybridization the effect of ethanol dose (and timing of dose) on the expression of known retinoic acid target genes in the mouse embryo. The genes studied will include those encoding the homeobox family, the retinoic acid receptor, the cellular retinoic acid binding protein, the cellular retinol binding protein, and various retinoid-metabolizing enzymes including alcohol dehydrogenase, aldehyde dehydrogenase, and cytochrome P450; (3) Examine the in vivo role of alcohol dehydrogenase (ADH) in retinoic acid synthesis by producing ADH mutations in transgenic mice. Three ADH genes have been characterized in the mouse (Adh-1, Adh-2, and Adh-3), and we plan to prepare null mutations in each gene. These knockout mice will be analyzed to determine if loss of a particular ADH results in a change in embryonic development, retinoic acid synthesis, or retinoic acid target gene expression.