Embryonic Gene Expression During Diabetic Embryopathy

描述（由申请人提供）：妊娠糖尿病妇女的后代先天性畸形明显增加。在胚胎发育早期，由胚胎过度的葡萄糖代谢引起的氧化应激是造成这些畸形的原因。我们已经证明氧化应激抑制胚胎中控制基本发育过程的基因的表达。特别是，糖尿病小鼠胚胎中神经管和神经嵴发育所需的Pax3的表达显著降低，抗氧化剂阻断了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。该提案的中心假设是，胚胎早期发育过程中与有氧代谢增加相关的Pax3的表观遗传调控被母体高血糖引起的氧化应激所取消。这一假说是基于这样一种认识，即早期胚胎的燃料代谢是高度糖酵解的，随着细胞分化，燃料代谢变得更加氧化，并且早期胚胎中表达的基因依赖于染色质修饰。有三个具体目的：(1)测试氧化应激是否阻断分化诱导基因表达所必需的Pax3转录调控元件的表观遗传修饰。(2)检测响应氧化或细胞氧化还原状态变化的染色质修饰介质是否随分化而变化，是否受到氧化应激的抑制，是否调节Pax3的表达。(3)检测胚胎和胚胎干细胞（ESC）在缺氧和氧化应激下激活的amp活化蛋白激酶（AMPK）是否介导氧化应激阻断分化诱导的染色质修饰的作用。我们的方法是研究ESC中组蛋白甲基化、乙酰化和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，并且在ESC或胚胎中诱导氧化应激，并且刺激或抑制可能改变染色质以响应氧化应激的途径的影响将被检查。这项研究具有重要意义，因为它将确定由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。这一知识可能会导致新的策略，使胚胎基因表达正常化在糖尿病妊娠。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调控的，这种信号改变了染色质结构，这一概念以前没有被研究过。

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.