Search for the Molecular Causes of Diabetic Embryopathy

描述（由申请人提供）：孕妇在怀孕前患有糖尿病会显著增加先天性畸形的风险。畸形发生在妊娠早期，器官发生的初期。为了了解母体糖尿病扰乱胚胎发育的分子机制，先前的研究表明，在糖尿病妊娠小鼠模型中，胚胎过量的葡萄糖代谢抑制了Pax3的表达，Pax3是一种编码神经上皮和神经嵴发育所需转录因子的基因。Pax3的表达减少与神经管和心脏流出道缺陷的增加有关，这是糖尿病妊娠中最常见的两种缺陷。由于Pax3表达不足，形成神经管或神经嵴的细胞发生p53依赖性凋亡，从而导致这些结构不能正常形成。最近对胚胎干细胞（ESC）和癌细胞的研究表明，自我再生性和多能性与高速率的糖酵解有关。随着ESC开始分化，p53被激活，并调节促进分化、衰老和氧化代谢的基因和酶活性的表达。表达pax3的细胞是祖细胞，必须能够增殖并保持可塑性才能发育成多种不同的特化细胞类型，直到细胞停止增殖并最终分化的发育阶段。需要验证的总体假设是，Pax3在神经上皮细胞和神经嵴细胞的早期发育过程中是必需的，因为它必须抑制p53的推力，从而过早导致衰老、分化和高氧化代谢率。在之前的资助期内使用小鼠ESC获得的证据表明，Pax3抑制p53的稳定性，并且Pax3和p53相互存在于一个复合物中。然而，目前尚不清楚Pax3如何破坏p53的稳定性，也不清楚Pax3对p53的降解是否会阻止胚胎细胞开始分化时干细胞特征的丧失。本提案的具体目的是：(1)确定Pax3诱导p53降解的机制。(2)检验Pax3下调p53是否通过支持自我更新和抑制分化所需基因的表达来防止干细胞特性的丧失。(3)检测Pax3下调p53是否能阻止未分化细胞的无氧：好氧糖代谢平衡的丧失。这些实验将深入了解糖尿病妊娠和其他条件下Pax3表达受损时发生畸形的机制。此外，它们将为糖酵解和氧化能代谢对早期胚胎和干细胞命运的调控提供新的信息。

DESCRIPTION (provided by applicant): Maternal diabetes pre-dating pregnancy significantly increases the risk for congenital malformations. The malformations occur very early during gestation, at the beginning of organogenesis. In an effort to understand the molecular mechanisms by which maternal diabetes disturbs embryonic development, previous research from the Principal Investigator's laboratory has shown that, in a mouse model of diabetic pregnancy, excess glucose metabolism by the embryo inhibits expression of Pax3, a gene that encodes a transcription factor required for development of neuroepithelium and neural crest. Reduced expression of Pax3 is associated with increased neural tube and cardiac outflow tract defects, two of the most common defects that occur in diabetic pregnancy. As a result of insufficient Pax3 expression, cells forming the neural tube or neural crest undergo p53-dependent apoptosis, and consequently, these structures fail to form properly. Recent research using embryonic stem cells (ESC) and cancer cells has demonstrated that self-renewability and pluripotentiality are coupled to a high rate of glycolysis. As ESC start to differentiated, p53 becomes activated and regulates expression of genes and enzyme activity that promote differentiation, senescence, and oxidative metabolism. Pax3-expressing cells are progenitors that must be able to proliferate and maintain plasticity to develop into multiple different specialized cell types until the stages of development when cells should stop proliferating and terminally differentiate. The overall hypothesis to be tested is that Pax3 is required during early development of neuroepithelial and neural crest cells because it must inhibit the thrust of p53 to prematurely cause senescence, differentiation, and high rates of oxidative metabolism. Evidence acquired during the previous funding period using mouse ESC indicates that Pax3 inhibits p53 stability, and that Pax3 and p53 are found in a complex with each other. However, it is not known how Pax3 destabilizes p53, nor whether degradation of p53 in response to Pax3 prevents the loss of stem cell characteristics as embryo cells start to differentiate. The Specific Aims of this proposal are to: (1) Determine the mechanism by which Pax3 induces p53 degradation. (2) Test whether down regulation of p53 by Pax3 prevents loss of stem cell characteristics by supporting expression of genes required for self-renewal and inhibition of differentiation. (3) Test whether down regulation of p53 by Pax3 prevents loss of the balance of anaerobic: aerobic glucose metabolism that is characteristic of undifferentiated cells. These experiments will provide insight into the mechanisms by which malformations occur during diabetic pregnancy and other conditions when Pax3 expression is impaired. Furthermore, they will provide new information into the regulation of early embryo and stem cell fate by glycolytic and oxidative energy metabolism. PUBLIC HEALTH RELEVANCE: Diabetes existing before pregnancy significantly increases risk for birth defects; the incidence of birth defects in the offspring of diabetic women is 2-5 times that in nondiabetic pregnancy. This diabetic complication is known as, diabetic embryopathy. The birth defects occur at the very earliest stages of embryonic development, when the organ systems are just starting to form. This project will investigate how altered expression of a gene that is required during early embryo development, resulting from diabetic pregnancy, leads to birth defects.