Mechanism of vertebrate neural tube morphogenesis

描述(申请人提供)：我们研究的长期目标是了解脊椎动物胚胎神经管形态发生的分子和细胞机制。这项拟议的实验意义重大，因为神经管形态发生障碍是人类第二常见的出生缺陷。我们的重点是一种称为心尖收缩的细胞形状变化，即柱状细胞转变为楔形细胞，这对神经管闭合起重要作用。顶端收缩涉及极化上皮细胞顶端表面肌动蛋白细丝的聚集。这一过程背后的分子机制尚不清楚，但我们已经证明，单一的蛋白质Shroom足以诱导顶端肌动蛋白组装和顶端收缩。蘑菇对于神经管的关闭是必不可少的。这项建议旨在了解蘑菇介导的肌动蛋白细胞骨架重组如何影响神经管关闭所必需的细胞形状变化。我们建议确定蘑菇介导的肌动蛋白组装的分子机制。我们假设这一过程需要肌动蛋白调节剂Arp2/3、Forin和Mena。我们将使用蘑菇活性和肌动蛋白调节蛋白抑制剂的功能增益试验来测试这一点。我们认为，与根尖收缩相关的细胞行为是相互关联的，并受Sroom控制。我们将通过非洲爪哇胚胎的功能丧失实验和共聚焦成像来解决这个问题。最后，我们认为一种名为APXL的蘑菇相关基因控制着缺乏蘑菇表达的细胞的顶端收缩。我们将同时使用增益型和损失型策略来检验这一假设。这项工作将有助于阐明控制细胞形状变化的调控网络，特别是神经管的关闭。此外，这项工作有可能揭示人类神经管缺陷的基础。

DESCRIPTION (provided by applicant): The long-term goal of our research is to understand the molecular and cellular mechanisms governing morphogenesis of the neural tube in vertebrate embryos. The proposed experiments are significant because failure of neural tube morphogenesis is the second most common human birth defect. Our focus is on a cell shape change called apical constriction, a conversion of columnar cells into wedge-shaped cells, that contributes importantly to neural tube closure. Apical constriction involves accumulation of actin filaments at the apical surface of polarized epithelial cells. The molecular mechanisms underlying this process are unknown, but we have shown that a single protein, Shroom, is sufficient to induce both apical actin assembly and apical constriction. Shroom is essential for neural tube closure. This proposal aims to understand how Shroom-mediated reorganization of the actin cytoskeleton influences cell shape changes that are necessary for neural tube closure. We propose to determine the molecular mechanisms of Shroom-mediated actin assembly. We hypothesize that the actin regulators, Arp2/3, formin, and Mena are required for this process. We will test this using a gain-of-function assay for Shroom activity and inhibitors of the actin regulatory proteins. We suggest that cell behaviors associated with apical constriction are inter-related and controlled by Shroom. We will address this with loss-of-function experiments in Xenopus embryos and confocal imaging. Finally, we suggest that a Shroom-related gene, called APXL, controls apical constrictions in cells that lack Shroom expression. We will use both gain- and loss-of-function strategies to test this hypothesis. This work will help to elucidate the regulatory networks that govern cell shape-change in general and neural tube closure in particular. Moreover, this work has the potential to shed light on the underpinnings of human neural tube defects.