Search for the Molecular Causes of Diabetic Embryopathy

描述(申请人提供)：怀孕前的母亲糖尿病会显著增加先天畸形的风险。畸形发生在妊娠早期，也就是器官发生的初期。为了了解母体糖尿病干扰胚胎发育的分子机制，首席调查者实验室之前的研究表明，在糖尿病妊娠的小鼠模型中，胚胎过度的葡萄糖代谢抑制了Pax3的表达，Pax3基因编码一种转录因子，神经上皮和神经脊的发育所需。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，形成神经管或神经脊的细胞经历了依赖于P53的凋亡，因此，这些结构无法正常形成。最近使用胚胎干细胞(ESC)和癌细胞的研究表明，自我更新和多潜能与高糖酵解率有关。随着胚胎干细胞开始分化，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。Pax3表达的细胞是祖细胞，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，直到细胞停止增殖和终末分化的发育阶段。有待检验的总体假设是，Pax3在神经上皮细胞和神经脊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，从而过早地导致衰老、分化和高速率的氧化代谢。利用小鼠胚胎干细胞获得的证据表明，Pax3抑制了P53的稳定性，并且Pax3和P53存在于彼此的复合体中。然而，目前尚不清楚Pax3如何破坏P53的稳定性，也不知道Pax3对Pax3的反应是否能阻止胚胎细胞开始分化时干细胞特性的丧失。这项建议的具体目的是：(1)确定Pax3诱导P53降解的机制。(2)测试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)测试Pax3对P53的下调是否可以防止无氧平衡的丧失：有氧葡萄糖代谢是未分化细胞的特征。这些实验将深入了解糖尿病妊娠期间畸形发生的机制，以及当Pax3表达受损时的其他情况。此外，它们还将为糖酵解和氧化能量代谢调节早期胚胎和干细胞的命运提供新的信息。 公共卫生相关性：怀孕前存在的糖尿病显著增加了出生缺陷的风险；糖尿病妇女的后代出生缺陷的发生率是非糖尿病孕妇的2-5倍。这种糖尿病并发症被称为糖尿病胚胎病变。出生缺陷发生在胚胎发育的最早阶段，当时器官系统刚刚开始形成。这个项目将研究糖尿病妊娠引起的早期胚胎发育所需的基因表达改变如何导致出生缺陷。

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.