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关闭的分子机制和细胞过程。我们的重点是一种基本的细胞形状变化，称为根尖收缩。根尖狭窄是柱状细胞转化为楔形细胞的过程，有助于上皮片的弯曲，并对神经管闭合起重要作用。我们之前已经证明，单一的蛋白质Shroom3是诱导上皮细胞心尖收缩的必要条件和充分条件。Shroom3对神经管的闭合以及青蛙、小鼠和小鸡的大脑和脊髓的器官发生也是必不可少的。最近的数据表明，Shroom3在眼睛和肠道的上皮形态发生中需要额外的需求。在人类身上的研究将Shroom3与肾脏疾病和高血压联系起来。尽管如此，这种蛋白质的功能机制仍然没有得到很好的解释。在这里，我们提出了一种综合的方法，将在基础细胞生物学、组织形态发生和基因表达的转录调控水平上研究Shroom3的功能。最近的研究表明，顶端质膜是内吞的，顶端内吞作用对于细胞形状的改变和神经板的弯曲是必不可少的，但这一过程是如何调控的仍是完全未知的。在这一应用的第一个目标中，我们提出了一些实验，以确定Shroom3在触发顶端内吞作用中的作用，并将阐明Shroom3表达细胞中驱动内吞作用的蛋白质机制。脊椎动物的中枢神经系统最初发育为一层扁平的细胞，这些细胞会卷曲并关闭，形成中空的神经管。已经确定了几种潜在的力量产生机制，包括顶端收缩、收敛伸展和邻近表皮细胞产生的模糊推力。尽管取得了这些进展，但我们仍然没有全面了解这些不同的产生力量的“引擎”是如何协同工作来影响神经管关闭的。在AIM II中，我们将量化大量野生型胚胎和缺乏Shroom3功能的胚胎在神经管关闭过程中神经和非神经上皮细胞的行为。Shroom3在神经管、晶状体和肠道的发育过程中对上皮细胞的形状变化是必不可少的。由于Shroom3足以引起细胞形状的戏剧性变化，了解该基因的转录控制对于全面理解脊椎动物胚胎中的上皮片弯曲将是至关重要的。AIM III的实验将确定Shroom3表达的转录激活因子，并为脊椎动物上皮形态发生的基因调控网络奠定基础。第5页

DESCRIPTION (provided by applicant): The long-term goal of our research is to understand the molecular mechanisms and cellular processes governing neural tube closure in vertebrate embryos. Our focus is on an essential cell shape change called apical constriction. Apical constriction, a conversion of columnar cells into wedge-shaped cells, facilitates the bending of epithelial sheets and contributes importantly to neural tube closure. We have previously shown that a single protein, Shroom3, is both necessary and sufficient to induce apical constriction in epithelial cells. Shroom3 is also essential for neural tube closure and thus for organogenesis of the brain and spinal cord in frogs, mice and chicks. Recent data demonstrate additional requirements for Shroom3 in epithelial morphogenesis of the eye and the gut. Studies in humans link Shroom3 to renal disease and hypertension. Nonetheless, the mechanisms by which this protein functions remain poorly defined. Here, we propose an integrated approach that will investigate Shroom3 function at the level of fundamental cell biology, tissue morphogenesis, and transcriptional control of gene expression. Recent studies demonstrate that apical plasma membrane is endocytosed and that apical endocytosis is essential for cell shape change and for bending of the neural plate, but how this process is regulated remains entirely unknown. In Aim I of this application, we propose experiments that will determine the role of Shroom3 in triggering apical endocytosis and will elucidate the protein machinery driving endocytosis in Shroom3 expressing cells. The central nervous system of vertebrates develops initially as a flat sheet of cells that will roll up and seal shut to form the hollow neural tube. Several potentially force-generating mechanisms have been identified that contribute to neural tube closure, including apical constriction, convergent extension, and a poorly-defined pushing force generated by the neighboring epidermal cells. Despite this progress, we still have no comprehensive understanding of how these different force-generating "engines" function cooperatively to effect neural tube closure. In Aim II, we will quantify the behavior of both neural and non-neural epithelial cells during neural tube closure in large numbers of wild-type embryos and in embryos lacking Shroom3 function. Shroom3 is essential for epithelial cell shape change during development of the neural tube, the lens and the gut. Because Shroom3 is sufficient to induce dramatic cell shape changes, understanding the transcriptional control of this gene will be essential to any comprehensive understanding of epithelial sheet- bending in vertebrate embryos. Experiments in Aim III will identify transcriptional activators of Shroom3 expression and lay the foundation for a gene regulatory network governing epithelial morphogenesis in vertebrates. Page 5