Transcriptional control of epithelial behaviors that drive mammalian neural tube closure

 描述(由申请人提供)：神经管关闭失败是一种毁灭性的出生缺陷。尼斯旺德实验室的研究为NT闭合的分子和细胞调控提供了重要的见解。我们建立并研究了神经管缺陷(NTDS)的小鼠模型，以阐明NT闭合的分子基础。此外，我们还创造了强大而新颖的技术来可视化活体哺乳动物胚胎中NT的闭合。我们的动态成像和关键的基因突变将注意力集中在非神经性外胚层(NNE)在NT闭合中所起的重要作用上，这方面的研究很少，但却很关键。此外，我们还开发了专门分离NNE细胞的方法，为研究NNE细胞的生物学提供了一个完善和强大的平台 不会的。在这里，我们将建立在我们独特的视角之上，并将我们的注意力转向脊柱NTDS，以提供对人类最常见的NTD类型的洞察，以及与小鼠和人类脊柱裂相关的两条遗传途径。目的1验证两个密切相关的GRHL转录因子GRHL2和GRHL3通过靶基因的独特和差异激活而不同地控制颅骨和脊髓NT闭合的假说，部分是通过与激活AP1(cJUN/CFOS)转录因子的JNK信号通路相互作用而介导的。目的2将扩展我们的实时平台，以验证GRHL调控的NNE转录程序通过控制细胞黏附、膜成分的循环、细胞形状变化和/或肌动蛋白动力学来驱动NT关闭的假说。目的3将把我们全面的分子和细胞洞察力与对数百个NTD样本的新的未发表的分析相结合，以检验GRHL3和JNK途径中发现的突变是人类脊髓NTDS的原因这一假设。研究与公共卫生的相关性：拟议的实验将导致对尾部NTDS的原因的新的细胞和分子的见解，尾部NTDS是最常见的NTD类型，它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的一般机制提供新的见解，包括面部和身体壁。在这里获得的洞察力可能导致普遍应用于治疗胚胎组织闭合缺陷的治疗，这些缺陷加在一起占人类出生缺陷的很大比例。建议中使用的缩写：CDH1钙粘附素1或E-钙粘蛋白EMT上皮到间充质转化GRHL Grainyhead-like(GRH是苍蝇同源物)KD击倒mt/镁膜番茄/膜GFP荧光报告，GFP表达由CRE NNE非神经或表面外胚层NT神经管NTD神经管缺陷GRH12-Null：我们将使用我们在ENU筛选中分离的GRH12Nisw等位基因，该等位基因与其他Grhl2零等位基因具有相同的表型。Grhl3-Cre：我们将使用Grhl3-Cre，它会产生一个空等位基因(从S.Coughlin获得；Grh13tm1(Cre)CGH)。

 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).