Methods Development for CVM Cell Migration Study in Drosophila

描述（由申请人提供）：细胞迁移是胚胎发育期间非常有影响力的过程，因为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-细胞相互作用以驱动细胞分化和器官发生。大多数复杂器官系统的形状是由粘附细胞群的定向迁移形成的。因此，细胞迁移必须在时间和空间上进行调节，以使生物体正常发育。我们的研究目标的首要目标是深入了解迁移组内的细胞如何感知它们的环境，以及这如何有助于它们的集体运动。为了实现这一目标，我们必须首先发展的方法，以促进研究尾部内脏中胚层（CVM）细胞迁移的果蝇。我们认为，CVM细胞迁移作为一个很好的模型系统来研究细胞迁移如何塑造肠道形成。CVM细胞表现出从胚胎的最后位置向身体两侧的两个不同群体的前部定向细胞迁移。这些细胞在果蝇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迁移的却知之甚少。它们的迁移是沿着肠道的整个长度定位细胞所必需的，因为这些细胞形成必须完全包裹肠道的纵向肌肉。我们的目标是定义方法，使研究的CVM细胞迁移，开发它作为一个系统，研究细胞迁移如何影响器官发生。我们的第一个目标将是定义支持CVM细胞核的活体体内成像的方法，以提供细胞跟踪数据，以及CVM细胞膜的成像，以深入了解细胞如何与其环境相互作用。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起的数量和方向可以提供有关野生型胚胎中细胞迁移过程的重要信息，并可用作解释突变表型的参考。此外，果蝇提出了一个很好的系统，以测定功能表型的基因损失相对于一个特定的生物过程。因此，在我们的第三个目标中，我们将利用短发夹RNA转基因和组织特异性Gal 4构建体优化RNAi策略，当它们一起存在于基因组中时，支持RNAi以有效地减少CVM内感兴趣的基因的表达。在我们的最后一个目标中，我们提出了一种新的方法，用于创建突变体克隆内开发果蝇胚胎使用光激活重组酶。这种方法将使我们能够测定迁移细胞集合中基因的空间和时间功能。这样的方法将是强大的，一般适用于细胞迁移的研究和有用的发育生物学社区。动物胃肠道的发育是高度相似的， 因此，对CVM细胞迁移如何影响果蝇肠道形成的研究有可能对人类发育产生深远的影响。 公共卫生相关性：不受控制的细胞迁移可导致脊椎动物的先天性缺陷，影响心脏、脉管系统和胃肠道。在胃部肌肉中发现的先天性缺陷可能是由于发育早期的异常细胞迁移造成的，这种缺陷导致新生儿自发性胃穿孔，其进展迅速，死亡率高。由于动物胃肠道的发育是高度相似的，研究CVM细胞迁移如何影响果蝇肠道的形成具有深远的意义。

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.