Mechanism of vertebrate neural tube morphogenesis

描述（由申请人提供）：我们研究的长期目标是了解脊椎动物胚胎神经管闭合的分子机制和细胞过程。我们的重点是一个基本的细胞形状变化称为顶端收缩。根尖收缩，柱状细胞转化为楔形细胞，促进上皮片的弯曲，对神经管闭合起重要作用。我们之前已经证明，一个单独的蛋白Shroom3是诱导上皮细胞顶端收缩的必要和充分条件。Shroom3对于神经管闭合也是必不可少的，因此对于青蛙、老鼠和小鸡的大脑和脊髓的器官形成也是必不可少的。最近的数据表明，Shroom3在眼睛和肠道的上皮形态形成中有额外的需求。对人类的研究表明，蘑菇3与肾脏疾病和高血压有关。尽管如此，这种蛋白质的功能机制仍然不清楚。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控制水平上研究Shroom3的功能。最近的研究表明，顶端质膜内吞作用是细胞形状改变和神经板弯曲的必要条件，但这一过程是如何调节的仍是完全未知的。在本应用的目的1中，我们提出了实验来确定Shroom3在触发根尖内吞作用中的作用，并将阐明表达Shroom3的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是由一层扁平的细胞组成的，这些细胞会卷起并闭合，形成中空的神经管。已经确定了几种潜在的产生力的机制，有助于神经管关闭，包括顶端收缩，趋同延伸，以及邻近表皮细胞产生的不明确的推力。尽管取得了这一进展，但我们仍然没有全面了解这些不同的产生力的“引擎”如何协同作用以影响神经管闭合。在Aim II中，我们将在大量野生型胚胎和缺乏Shroom3功能的胚胎中量化神经管闭合过程中神经和非神经上皮细胞的行为。Shroom3在神经管、晶状体和肠道发育过程中对上皮细胞的形状改变至关重要。由于Shroom3足以诱导剧烈的细胞形状变化，因此了解该基因的转录控制对于全面了解脊椎动物胚胎上皮片弯曲至关重要。Aim III的实验将鉴定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