Dynamic analysis of apical constriction activity during neural tube closure

神经管闭合过程中心尖收缩活动的动态分析

• 批准号: 10267657
• 负责人: Austin Baldwin
• 金额: $ 3.96万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-04-01 至 2021-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10267657
• 关键词: Actin-Binding Protein; Actins; Actomyosin; Adherens Junction; Adhesions; Adhesives; Adopted; Affect; Anencephaly; Animals; Apical; Area; Behavior; CRISPR/Cas technology; Cell Shape; Cell model; Cells; Cellular biology; Child; Clustered Regularly Interspaced Short Palindromic Repeats; Columnar Epithelium; Complex; Congenital Abnormality; Contracts; Data; Defect; Development; Embryonic Development; Epithelial Cells; Failure; Future; Genes; Human; Image; Intercellular Junctions; Invertebrates; Label; Link; Location; Maps; Mechanics; Modeling; Molecular; Morphogenesis; Mosaicism; Mus; Mutation; Myosin ATPase; N-Cadherin; Nature; Neural Tube Closure; Neural Tube Defects; Neural tube; Neuraxis; Paper; Pattern; Pregnancy; Process; Proteins; Role; Shapes; Spatial Behavior; Surface; System; Time; Tissues; Transgenes; United States; Variant; Vertebrates; Work; base; cell behavior; constriction; epidemiology study; experimental study; fly; gene function; genetic manipulation; human model; in vivo; knock-down; live cell imaging; mutant; neural plate; protein complex; relating to nervous system; spatiotemporal; tool; tool development

Project Summary/Abstract: Neural tube closure is a necessary stage of vertebrate embryonic development during which the central nervous system is formed. Failures of neural tube closure, termed neural tube closure defects, are common and serious birth defects that can be either debilitating or fatal. Despite the importance of neural tube closure to vertebrate development, little is known about the morphogenetic cell behaviors underlying this process. One important process in neural tube closure is apical constriction, where the epithelial cells shrink their apical surfaces through actomyosin contractions, facilitating tissue bending and proper closure of the neural tube. Failure of apical constriction results in lethal neural tube defects such as anencephaly. Several genes regulating apical constriction in the neural plate have been identified, but it is unknown how various modes of actomyosin contractions contribute to apical constriction and how apical constriction genes contribute to those modes of actomyosin contractions. This proposal contains experiments that will quantitatively describe actomyosin contractions and apical constriction across the neural plate during neural tube closure. This will be achieved thorough live-imaging of cell shape and cell behavior via cell junctions, and actomyosin dynamics via actin and myosin. Data on cell behavior and actomyosin will then be used to map apical constriction behaviors spatially and temporally to the neural plate in order to understand how various modes of actomyosin contraction contribute to neural tube closure. In addition, it is proposed to examine how the neural tube closure genes Shroom3 (necessary for apical constriction) and N-cadherin (necessary for adhesion) contribute to actomyosin contractions during neural tube closure. Gene function will be assessed by using CRISPR/Cas9 mutatgenesis to generate both mosaic and tissue-wide mutants for both Shroom3 and N-cadherin. CRISPR/Cas9 will also be used to generate fluorescent-labeled endogenous N-cadherin in order to mark cell junctions and examine N-cadherin dynamics in live cells. Overall, this proposal aims to both quantitatively analyze a little-studied cell behavior which is necessary to neural tube closure and implicated in serious birth defects. In addition, molecular tools and strategies will be developed to aid study of neural tube closure in proposed experiments and into the future.

项目摘要/摘要： 神经管闭合是脊椎动物胚胎发育的必要阶段，在此阶段，中枢神经管闭合 神经系统形成。神经管闭合失败（称为神经管闭合缺陷）很常见 以及可能使人衰弱或致命的严重出生缺陷。尽管神经管闭合很重要 对于脊椎动物的发育，人们对这一过程背后的形态发生细胞行为知之甚少。一 神经管闭合的重要过程是顶端收缩，即上皮细胞收缩其顶端 通过肌动球蛋白收缩表面，促进组织弯曲和神经管的正确闭合。 顶端收缩失败会导致致命的神经管缺陷，例如无脑畸形。几个基因 调节神经板顶端收缩的机制已被确定，但尚不清楚各种模式是如何调节的 肌动球蛋白收缩有助于顶端收缩以及顶端收缩基因如何促进这些收缩 肌动球蛋白收缩的模式。 该提案包含定量描述肌动球蛋白收缩和心尖收缩的实验 神经管闭合期间神经板的收缩。这将实现彻底的实时成像 通过细胞连接了解细胞形状和细胞行为，通过肌动蛋白和肌球蛋白了解肌动球蛋白动力学。细胞上的数据 然后，行为和肌动球蛋白将用于将心尖收缩行为在空间和时间上映射到 神经板，以了解肌动球蛋白收缩的各种模式如何影响神经管 关闭。 此外，建议检查神经管闭合基因Shroom3（心尖所必需的）如何 收缩）和 N-钙粘蛋白（粘附所必需）有助于神经管期间肌动球蛋白的收缩 关闭。将通过使用 CRISPR/Cas9 突变生成嵌合体和嵌合体来评估基因功能 Shroom3 和 N-钙粘蛋白的组织范围突变体。 CRISPR/Cas9也将用于生成 荧光标记的内源性 N-钙粘蛋白，用于标记细胞连接并检查 N-钙粘蛋白动态 在活细胞中。 总体而言，该提案旨在定量分析很少研究的细胞行为，这对于 神经管闭合并与严重的出生缺陷有关。此外，分子工具和策略将 旨在帮助研究拟议的实验和未来的神经管闭合。