Transcriptional control of epithelial behaviors that drive mammalian neural tubeclosure

驱动哺乳动物神经管闭合的上皮行为的转录控制

• 批准号: 9660106
• 负责人: Lee A. Niswander
• 金额: $ 9.37万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-01 至 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9660106
• 关键词: Transcriptional; control; epithelial; behaviors; drive

 DESCRIPTION (provided by applicant): Failure of neural tube closure is a devastating birth defect. Research in the Niswander lab has provided significant insights into the molecular and cellular regulation of NT closure. We have created and studied mouse models with neural tube defects (NTDs) to elucidate the molecular foundations of NT closure. Moreover, we have created robust and novel technology to visualize NT closure in a living mammalian embryo. Our dynamic imaging and key genetic mutants have focused attention on the little studied but critical role for the non-neural ectoderm (NNE) in NT closure. In addition we developed methods to specifically isolate NNE cells to provide a refined and robust platform to study the biology of the NNE. Here we will build upon our unique perspective and turn our attention to spinal NTDs, to provide insight into the most common type of NTD in humans, and to two genetic pathways that are associated with spina bifida in mice and humans. Aim 1 will test the hypothesis that the two closely related Grainyhead-like (GRHL) transcription factors, GRHL2 and GRHL3, differentially control cranial and spinal NT closure through unique and differential activation of target genes, in part mediated by interaction with the JNK signaling pathway that activates the AP1 (cJUN/cFOS) transcription factor. Aim 2 will extend our live platform to test the hypothesis that GRHL-regulated NNE transcriptional programs drive NT closure by controlling cell adhesion, recycling of membrane components, cell shape changes, and/or actin dynamics. Aim 3 will combine our comprehensive molecular and cellular insights with novel unpublished analyses of hundreds of NTD samples to test the hypothesis that mutations identified in GRHL3 and the JNK pathway are causative for spinal NTDs in humans. Relevance of research to public health: The proposed experiments will lead to new cellular and molecular insights into the causes of caudal NTDs, the most common type of NTD and which leads to a profoundly important and frequently disrupted aspect of mammalian embryogenesis. Moreover, our studies will impart novel insights into the general mechanisms of embryonic tissue fusion including the face and body wall. The insights gained here may lead to therapies of general application for treatment of embryonic tissue closure defects that together represent a significant percentage of human birth defects. Abbreviations used in proposal: CDH1 Cadherin1 or E-cadherin EMT Epithelial-to-mesenchymal transition GRHL Grainyhead-like (GRH is the fly homolog) KD Knock-down mT/mG Membrane tomato/membrane GFP fluorescent reporter, GFP expression is activated by Cre NNE Non-neural or surface ectoderm NT Neural tube NTD Neural tube defect Grhl2-null: We will use Grhl21Nisw allele that we isolated in our ENU-screen and which has the same phenotype as other Grhl2 null alleles. Grhl3-Cre: We will use Grhl3-Cre which generates a null allele (obtained from S. Coughlin; Grhl3tm1(cre)Cgh).

 描述（由申请人提供）：神经管闭合失败是一种毁灭性的出生缺陷。 Niswander 实验室的研究为 NT 闭合的分子和细胞调节提供了重要的见解。我们创建并研究了神经管缺陷 (NTD) 小鼠模型，以阐明 NT 闭合的分子基础。此外，我们还创造了强大且新颖的技术来可视化活体哺乳动物胚胎中的 NT 闭合。我们的动态成像和关键基因突变体将注意力集中在非神经外胚层 (NNE) 在 NT 闭合中研究很少但至关重要的作用。此外，我们开发了专门分离 NNE 细胞的方法，为研究 NNE 细胞的生物学提供了一个精致而强大的平台。 北东北。 在这里，我们将基于我们独特的视角，将注意力转向脊柱 NTD，以深入了解人类最常见的 NTD 类型，以及与小鼠和人类脊柱裂相关的两种遗传途径。目标 1 将检验以下假设：两个密切相关的 Grainyhead 样 (GRHL) 转录因子 GRHL2 和 GRHL3 通过靶基因的独特和差异激活来差异控制颅骨和脊髓 NT 闭合，部分是通过与激活 AP1 (cJUN/cFOS) 转录因子的 JNK 信号通路相互作用介导的。目标 2 将扩展我们的实时平台，以测试 GRHL 调节的 NNE 转录程序通过控制细胞粘附、膜成分回收、细胞形状变化和/或肌动蛋白动力学来驱动 NT 闭合的假设。目标 3 将把我们全面的分子和细胞见解与数百个 NTD 样本的未​​发表的新颖分析相结合，以检验 GRHL3 和 JNK 通路中发现的突变是人类脊髓 NTD 病因的假设。研究与公共卫生的相关性：拟议的实验将对尾部 NTD 的原因产生新的细胞和分子见解，尾部 NTD 是最常见的 NTD 类型，并且导致哺乳动物胚胎发生的一个极其重要且经常被破坏的方面。此外，我们的研究将为胚胎组织融合（包括面部和体壁）的一般机制提供新的见解。这里获得的见解可能会导致治疗胚胎组织闭合缺陷的普遍应用的疗法，这些缺陷共同代表了人类出生缺陷的很大一部分。提案中使用的缩写： CDH1 Cadherin1 或 E-cadherin EMT 上皮间质转化 GRHL Grainyhead-like（GRH 是果蝇同源物） KD Knock-down mT/mG 膜番茄/膜 GFP 荧光报告基因，GFP 表达由 Cre 激活 NNE 非神经或表面外胚层 NT 神经管 NTD 神经 管缺陷 Grhl2-null：我们将使用我们在 ENU 筛选中分离的 Grhl21Nisw 等位基因，该等位基因与其他 Grhl2 null 等位基因具有相同的表型。 Grhl3-Cre：我们将使用 Grhl3-Cre，它生成无效等位基因（从 S. Coughlin 获得；Grhl3tm1(cre)Cgh）。