Search for the Molecular Causes of Diabetic Embryopathy

描述（由申请人提供）：神经管是脊椎动物胚胎发育过程中最早形成的结构之一;它形成大脑和脊髓发育的支架，因此，神经管的正确建立对于中枢神经系统的正常发育至关重要。Pax 3编码一种DNA结合转录因子，其在神经管形成早期表达，并且对于神经管在吻侧和尾侧神经孔处的闭合以及对于背板的适当背侧化是必需的。我们发现母体糖尿病抑制糖尿病小鼠胚胎中Pax 3的表达，并增加神经管缺陷（NTD）。NTD是女性糖尿病患者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Pax 3的表达，以及Pax 3的生产不足如何导致NTD，对于了解这些缺陷如何发生非常重要。最近，我们发现，p53缺陷拯救胚胎与非功能性Pax 3等位基因从NTD，和稳态p53蛋白水平降低，在胚胎表达野生型Pax 3相比，胚胎表达非功能性Pax 3。基于这些发现，我们推测Pax 3通过抑制p53的合成或稳定性来抑制p53依赖的凋亡，从而指导神经管发育。我们将通过确定Pax 3是否在神经管融合前和融合后神经管背侧化期间阻止神经褶皱中的细胞周期停滞和随后的p53依赖性凋亡来测试该假设（目的1），以及确定p53合成或稳定性是否被Pax 3抑制，并研究p53合成或稳定性受损的发生机制（目的2）。目标1将使用敲除小鼠系进行，其中Pax 3基因已用报告基因标记，使得Pax 3表达细胞可在神经管形成期间进行命运作图，所述敲除小鼠系已与p53敲除小鼠杂交。目的2将使用表达或不表达Pax 3和p53的细胞系来研究p53的合成或稳定性。这项研究将大大推进我们的知识如何关闭和神经管的背侧化的调节，并提供新的信息的分子机制，糖尿病妊娠导致NTD。

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.