Search for the Molecular Causes of Diabetic Embryopathy

描述(申请人提供)：神经管是脊椎动物胚胎发育过程中最早形成的结构之一，它形成了大脑和脊髓发育的支架，因此，神经管的正确建立对中枢神经系统的正常发育至关重要。Pax3编码一种DNA结合的转录因子，在神经管形成早期表达，对神经管在吻端和尾端神经孔的闭合以及翼板的适当背侧是必不可少的。我们发现，母体糖尿病抑制了糖尿病小鼠胚胎中Pax3的表达，并增加了神经管缺陷(NTD)。NTD是糖尿病女性后代中最常见的先天性畸形之一。因此，了解母体糖尿病如何干扰Pax3的表达，以及Pax3的产生不足如何导致NTD，对于了解这些缺陷是如何发生的非常重要。最近，我们发现P53缺失可以挽救NTD中携带非功能性Pax3等位基因的胚胎，并且表达野生型Pax3的胚胎与表达非功能性Pax3的胚胎相比，稳定状态下的P53蛋白水平降低。基于这些发现，我们假设Pax3通过抑制P53的合成或稳定性来抑制依赖于P53的细胞凋亡，从而指导神经管的发育。我们将通过确定Pax3是否在神经管融合前和融合后神经管背侧期间阻止细胞周期停滞和随后的P53依赖的细胞凋亡来验证这一假说(目标1)，并确定Pax3是否抑制P53的合成或稳定性，并研究发生P53合成或稳定性受损的机制(目标2)。目的1利用Pax3基因被标记为报告基因的基因敲除小鼠品系，以便表达Pax3的细胞在神经管形成过程中能够被命运定位，并与P53基因敲除小鼠杂交。目的2将使用表达Pax3或不表达Pax3的细胞系和P53来研究P53的合成或稳定性。这项研究将极大地提高我们对神经管闭合和背侧是如何调节的认识，并为糖尿病妊娠导致NTD的分子机制提供新的信息。

DESCRIPTION (provided by applicant): The neural tube is one of the earliest structures to form during vertebrate embryogenesis; it forms the scaffolding upon which the brain and spinal cord will develop, therefore, proper establishment of the neural tube is critical for normal development of the central nervous system. Pax3 encodes a DNA-binding transcription factor that is expressed early during neural tube formation and is essential for neural tube closure at the rostral and caudal neuropores, and for proper dorsalization of the alar plate. We have found that maternal diabetes inhibits expression of Pax3 in embryos of diabetic mice and increases neural tube defects (NTD). NTD are among the most common of the congenital malformations in the offspring of women with diabetes. Therefore, understanding how maternal diabetes disturbs the expression of Pax3, and how insufficient production of Pax3 leads to NTD, is important to understand how these defects occur. Recently, we showed that p53 deficiency rescues embryos with nonfunctional Pax3 alleles from NTD, and that steady state p53 protein levels are reduced in embryos expressing wild type Pax3 compared to embryos expressing nonfunctional Pax3. Based on these findings we hypothesize that Pax3 directs neural tube development by suppressing p53-dependent apoptosis by inhibiting p53 synthesis or stability. We will test this hypothesis in this proposal by determining whether Pax3 prevents cell cycle arrest and subsequent p53-dependent apoptosis in the neural folds prior to neural tube fusion, and during dorsalization of the neural tube following fusion (Aim 1), and determining whether p53 synthesis or stability is inhibited by Pax3 and investigating the mechanisms by which impaired synthesis or stability of p53 occurs (Aim 2). Aim 1 will be performed using knockout mouse lines, in which the Pax3 gene has been tagged with a reporter gene so that Pax3- expressing cells can be fate mapped during neural tube formation, that have been crossed with p53 knockout mice. Aim 2 will be performed using cells lines that express Pax3 or not, and p53 to study p53 synthesis or stability. This research will significantly advance our knowledge of how closure and dorsalization of the neural tube is regulated, and provide new information on the molecular mechanisms by which diabetic pregnancy causes NTD.