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。我们的重点是一个重要的细胞形状变化称为顶端收缩。顶端收缩，柱状细胞转化为楔形细胞，促进上皮片的弯曲，并有助于重要的神经管关闭。我们以前已经表明，一个单一的蛋白质，Shroom3，是必要的和足够的诱导上皮细胞的顶端收缩。Shroom3也是神经管闭合所必需的，因此对于青蛙、小鼠和小鸡的大脑和脊髓的器官形成也是必需的。最近的数据表明，在眼睛和肠道的上皮形态发生中对Shroom3有额外的要求。人类研究将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不清楚。在这里，我们提出了一个综合的方法，将调查Shroom3功能的水平上的基本细胞生物学，组织形态发生，和基因表达的转录控制。 最近的研究表明，顶端质膜内吞和顶端内吞是必不可少的细胞形状的变化和弯曲的神经板，但如何调节这一过程仍然是完全未知的。在本申请的目的I中，我们提出了将确定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的实验。脊椎动物的中枢神经系统最初发育为一片扁平的细胞，这些细胞会卷起并封闭起来形成中空的神经管。已经确定了几个潜在的力产生机制，有助于神经管关闭，包括顶端收缩，收敛延伸，和一个不明确的推力所产生的相邻的表皮细胞。尽管取得了这些进展，我们仍然没有全面了解这些不同的力产生“引擎”如何协同作用，以影响神经管关闭。在目标II中，我们将量化大量野生型胚胎和缺乏Shroom3功能的胚胎中神经管闭合期间神经和非神经上皮细胞的行为。 Shroom3在神经管、透镜和肠的发育过程中对上皮细胞的形状变化至关重要。由于Shroom3足以诱导细胞形状的巨大变化，因此了解该基因的转录控制对于全面了解脊椎动物胚胎中的上皮片层弯曲至关重要。目的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 公共卫生相关性：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和细胞机制。人类中枢神经系统最初在早期胚胎中发育为一片扁平的细胞，随后卷起并封闭形成一个管。这种滚动和密封过程的失败导致脊柱裂和其他称为神经管缺陷的出生缺陷。该申请提出了集中在三个领域的实验。1)我们将研究神经系统发育中细胞形状变化的基本细胞生物学过程。2)我们将研究单个细胞形状的变化如何影响相邻细胞的形状，以及这如何影响组织的滚动。3)我们将确定细胞形状变化的遗传调节因子。总之，这些实验将为神经管出生缺陷的病因学提供新的见解。

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 PUBLIC HEALTH RELEVANCE: The long-term goal of our research is to understand the molecular and cellular mechanisms governing neural tube closure in vertebrate embryos. The human central nervous system develops initially as a flat sheet of cells in the early embryo and subsequently rolls up and seals shut to form a tube. Failure of this rolling and sealing process leads to spina bifida and other birth defects called Neural Tube Defects. The application proposes experiments centered in three areas. 1) We will examine the basic cell biological processes underlying cell shape change in the developing nervous system. 2) We will examine how changes in the shape of an individual cells impacts shape in neighboring cells and how this impacts the rolling of the tissue generally. 3) We will identify the genetic regulators of cell shape change. Together, these experiments will provide new insights into the etiology of neural tube birth defects.