Embryonic Gene Expression During Diabetic Embryopathy

描述（由申请人提供）：患有妊娠前糖尿病的女性的后代中先天性畸形显著增加。在胚胎发育早期，由胚胎过度葡萄糖代谢引起的氧化应激是造成这些畸形的原因。我们已经表明，氧化应激抑制胚胎中控制基本发育过程的基因表达。特别是，神经管和神经嵴发育所需的Pax3的表达在糖尿病小鼠的胚胎中显著降低，抗氧化剂阻断Pax3的抑制以及相关的神经管和神经嵴缺陷。虽然已经确定了导致糖尿病母亲胚胎中氧化应激的几个生化过程，但尚不清楚氧化应激如何破坏胚胎基因表达。在这个建议的中心假设是，PAX3的表观遗传调节，这是与增加有氧代谢在早期胚胎发育过程中被废除由母体高血糖引起的氧化应激。这一假设是基于这样的认识，即早期胚胎的燃料代谢是高度糖酵解的，并且随着细胞分化而变得更加氧化，并且在早期胚胎中表达的基因依赖于染色质修饰。有三个具体目标：（1）测试氧化应激是否阻断分化诱导的基因表达所必需的Pax3转录调节元件的表观遗传修饰。(2)测试是否介质的染色质修饰，这是响应于氧化或细胞氧化还原状态的变化，随着分化而变化，它们是否被氧化应激抑制，以及它们是否调节Pax3的表达。(3)测试AMP活化蛋白激酶（AMPK）是否介导氧化应激的作用，以阻断分化诱导的染色质修饰，AMPK在胚胎和胚胎干细胞（ESC）中响应缺氧和氧化应激而活化。我们的方法是研究ESC中组蛋白甲基化和乙酰化以及DNA甲基化，以及小鼠胚胎和ESC中Pax3的表达。ESC将被诱导分化为神经元前体，并且氧化应激将在ESC或胚胎中被诱导，并且将检查刺激或抑制响应于氧化应激而可能修饰染色质的途径的作用。这项研究意义重大，因为它将确定母体糖尿病引起的异常燃料代谢破坏胚胎基因表达激活从而导致畸形的机制。这些知识可能会导致新的策略，以正常化胚胎基因表达在糖尿病妊娠。这一建议是创新的，因为早期胚胎基因表达是由燃料代谢产生的信号，改变染色质结构的发育调控的概念以前没有被调查。

DESCRIPTION (provided by applicant): Congenital malformations are significantly increased in the offspring of women with pregestational diabetes. Oxidative stress, resulting from excessive glucose metabolism by the embryo, early during embryonic development, is responsible for these malformations. We have shown that oxidative stress inhibits expression of genes in the embryo that control essential developmental processes. In particular, expression of Pax3, which is required for neural tube and neural crest development, is significantly reduced in embryos of diabetic mice, and antioxidants block the inhibition of Pax3 as well as the associated neural tube and neural crest defects. While several of the biochemical processes leading to oxidative stress in embryos of diabetic mothers have been determined, it is not known how oxidative stress disrupts embryo gene expression. The central hypothesis in this proposal is that epigenetic regulation of Pax3 that is associated with increasing aerobic metabolism during early embryonic development is abrogated by oxidative stress resulting from maternal hyperglycemia. This hypothesis is based upon the recognition that fuel metabolism by the early embryo is highly glycolytic and becomes more oxidative as cells differentiate, and that genes that are expressed in the early embryo are dependent upon chromatin modifications. There are three Specific Aims: (1) Test whether oxidative stress blocks epigenetic modifications of the Pax3 transcription regulatory element that are necessary for differentiation-induced gene expression. (2) Test whether mediators of chromatin modifications, which are responsive to changes in oxidation or cellular redox status, change with differentiation, whether they are inhibited by oxidative stress, and whether they regulate Pax3 expression. (3) Test whether AMP-activated protein kinase (AMPK), which is activated in embryos and embryonic stem cells (ESC) in response to hypoxia and oxidative stress, mediates the effects of oxidative stress to block differentiation- induced chromatin modifications. Our approach is to study histone methylation and acetylation and DNA methylation in ESC, and Pax3 expression in mouse embryos and in ESC. ESC will be induced to differentiate into neuronal precursors, and oxidative stress will be induced in ESC or embryos, and the effects of stimulating or inhibiting pathways that potentially modify chromatin in response to oxidative stress will be examined. The proposed study is significant because it will determine the mechanisms by which abnormal fuel metabolism caused by maternal diabetes disrupts the activation of embryonic gene expression, thereby leading to malformations. This knowledge may lead to new strategies to normalize embryo gene expression during diabetic pregnancy. This proposal is innovative because the concept that early embryonic gene expression is developmentally regulated by signals generated by fuel metabolism that alter chromatin structure has not previously been investigated.