Methods Development for CVM Cell Migration Study in Drosophila

描述（申请人提供）：细胞迁移是胚胎发育过程中一个非常重要的过程，它导致细胞从胚胎的一个区域重排到另一个区域，有效地控制细胞间相互作用，驱动细胞分化和器官发生。大多数复杂器官系统的形状都是由内聚细胞群的定向迁移引起的。因此，细胞迁移必须在时间和空间上得到调控，才能使生物体正常发育。我们的研究目标的首要目标是深入了解迁移群体中的细胞如何感知其环境以及这如何有助于他们的集体运动。为了实现这一目标，我们必须首先开发方法来促进果蝇尾端内脏中胚层（CVM）细胞迁移的研究。我们认为CVM细胞迁移是研究细胞迁移如何影响肠道形成的一个很好的模型系统。CVM细胞表现出定向的细胞迁移，从胚胎最后方的位置向前移动，作为身体两侧的两个不同的群体。这些细胞在果蝇的胚胎发生过程中经历了最长的迁移，但人们对它们是如何沿着这条路线迁移的知之甚少。它们的迁移对于沿着肠道的整个长度定位细胞是必要的，因为这些细胞形成纵向肌肉，必须完全包裹肠道。我们的目标是定义能够研究CVM细胞迁移的方法，并将其发展为研究细胞迁移如何影响器官发生的系统。我们的第一个目标是定义支持CVM细胞核活体成像的方法，以提供细胞跟踪数据，以及CVM细胞膜成像，以深入了解细胞如何与环境相互作用。在第二个目标中，我们将定义定量方法来分析细胞运动和细胞投影，以提供细胞迁移的定量描述。细胞跟踪数据和细胞突出的数量和方向可以提供野生型胚胎细胞迁移过程的重要信息，并可作为解释突变表型的参考。此外，果蝇提供了一个很好的系统来测定一个特定的生物过程中基因的功能表型损失。因此，在我们的第三个目标中，我们将优化RNAi策略，利用短发夹RNA转基因和组织特异性Gal4构建物，当它们一起存在于基因组中时，支持RNAi有效地减少CVM中感兴趣基因的表达。在我们的最后一个目标中，我们提出了一种利用光激活重组酶在发育中的果蝇胚胎中创建突变克隆的新方法。这种方法将使我们能够在迁移细胞群中测定空间和时间上的基因功能。这种方法将是强有力的；一般适用于细胞迁移的研究，对整个发育生物学社区都很有用。动物胃肠道的发育非常相似，

DESCRIPTION (provided by applicant): Cell migration is a very influential process during embryonic development as it results in rearrangement of cells from one area of the embryo to another, effectively controlling cell-cell interactions to drive cell differentiation and organogenesis. The shape of most complex organ systems arises from the directed migration of cohesive cell groups. Therefore, cell migration must be regulated temporally and spatially for organisms to develop properly. The overlying goal of our research objective is to provide insight into how cells within a migrating group sense their environment and how this contributes to their collective movement. To accomplish this goal, we must first develop methodology to facilitate study of caudal visceral mesoderm (CVM) cell migration in Drosophila. We contend that CVM cell migration serves as an excellent model system to study how cell migration shapes gut formation. CVM cells exhibit directed cell migration from the posterior-most position of the embryo toward the anterior as two distinct groups on either side of the body. These cells undergo the longest migration in all of Drosophila embryogenesis, but little is understood about how they are directed along their course. Their migration is necessary to position cells along the entire length of the gut, as these cells form longitudinal muscles that must completely ensheath the gut. Our goal is to define methodology that will enable study of the CVM cell migration, to develop it as a system for studying how cell migration influences organogenesis. Our first aim will be to define methods that support live in vivo imaging of CVM cell nuclei to provide cell tracking data, as well as imaging of CVM cell membranes to provide insight into how cells interact with their environment. In the second aim, we will define quantitative methods to analyze cell movements and cellular projections to provide a quantitative description of cells' migration. Cell tracking data and cell protrusion number and orientation can provide important information about the cell migration process in wildtype embryos, and can be used as a reference to interpret mutant phenotype. In addition, Drosophila presents an excellent system to assay loss of function phenotypes for genes with respect to a particular biological process. Therefore, in our third aim, we will optimize an RNAi strategy utilizing short hairpin RNA transgenes and tissue- specific Gal4 constructs which, when present together in the genome, support RNAi to effectively reduce expression of genes of interest within the CVM. In our last aim, we present a novel approach for creating mutant clones within developing Drosophila embryos using a light-activated recombinase. This method will allow us to assay gene function both spatially and temporally within the migrating cell collective. Such methodology would be powerful; generally applicable toward the study of cell migration and useful to the developmental biology community at large. Development of the gastrointestinal tract of animals is highly similar, so studies of how CVM cell migration influences gut formation in Drosophila have the potential to have far-reaching insight into human development. PUBLIC HEALTH RELEVANCE: Uncontrolled cell migration can lead to congenital defects in vertebrates affecting the heart, vasculature, and gastrointestinal tract. It is possible that congenital defects identified in muscles of the stomach, causing neonatal spontaneous gastric perforation which proceeds rapidly and has a high mortality rate, result from aberrant cell migration earlier in development. As development of the gastrointestinal tract of animals is highly similar, studies of how CVM cell migration influences gut formation in Drosophila have the potential for far- reaching implications.