Genetic analysis of apoptotic cell clearance in flies

描述（由申请人提供）：凋亡细胞在体内被吞噬作用迅速清除。凋亡细胞吞噬作用的缺陷与几种人类病理学如系统性红斑狼疮有关。在没有吞噬的情况下，剩余的组织可能因暴露于垂死细胞的内容物而受损。 在果蝇中，凋亡的尸体迅速被循环的吞噬细胞吞噬。我们已经表明，croquemort基因是必需的凋亡细胞在胚胎中的有效吞噬。我们建议识别和表征其他基因参与吞噬，果蝇遗传学，组织培养试验和差异表达筛选的组合。 基因组缺失的筛选确定了胚胎中凋亡细胞有效吞噬所需的许多区域。在我们的屏幕中确定的基因组区域之一含有磷脂酰丝氨酸受体的果蝇同源物。这种受体在哺乳动物组织培养试验中参与了细胞凋亡尸体的吞噬作用。虽然我们没有检测到这种受体在果蝇胚胎吞噬中的主要作用，但我们确实发现它的功能是抑制细胞凋亡。我们将使用这种表型来筛选由受体激活的信号通路。此外，我们将进一步探讨这种受体在吞噬中的需求。这些研究将为了解蛋白质的一般功能提供帮助。 我们还确定了其他基因组区域，包含吞噬所需的基因。我们将使用遗传和RNA干扰技术来区分负责基因。已经开发了基于组织培养的凋亡细胞吞噬测定以促进RNAi技术。这将使我们能够检查吞噬细胞和凋亡靶细胞中所需基因的作用。 在我们最初的研究中，我们发现在凋亡细胞存在的情况下，克罗克莫特水平上调。 为了鉴定吞噬细胞中暴露于凋亡细胞时表达变化的其他基因，我们启动了差异表达筛选。将测试响应于凋亡细胞的存在而显示表达变化的基因在组织培养物和体内的吞噬中的作用。这种策略的组合将使我们能够识别和表征许多参与凋亡细胞吞噬的基因，并了解它们在这一重要过程中的功能。

DESCRIPTION (provided by applicant): Apoptotic cells are rapidly cleared by phagocytosis in vivo. Defects in apoptotic cell phagocytosis have been implicated in several human pathologies such as Systemic Lupus Erythematosus. In the absence of engulfment, the remaining tissue may be damaged by exposure to the contents of the dying cells. In Drosophila, apoptotic corpses are rapidly engulfed by circulating phagocytic cells. We have shown that the croquemort gene is required for the efficient engulfment of apoptotic cells in the embryo. We propose to identify and characterize other genes involved in engulfment, using a combination of Drosophila genetics, tissue culture assays and differential expression screens. A screen of genomic deletions identified a number of regions required for efficient engulfment of apoptotic cells in the embryo. One of the genomic regions identified in our screen contains the Drosophila homologue of the phosphatidylserine receptor. This receptor has been implicated in the phagocytosis of apoptotic corpses in mammalian tissue culture assays. Although we do not detect a major role for this receptor in engulfment in the Drosophila embryo, we do find that it functions to inhibit apoptosis. We will use this phenotype to screen for signaling pathways activated by the receptor. In addition, we will further explore the requirement for this receptor in engulfment. These studies will provide insight into the general function of the protein. We have also identified other genomic regions that contain genes required for engulfment. We will distinguish the responsible genes using genetic and RNA interference techniques. A tissue culture based apoptotic cell engulfment assay has been developed to facilitate RNAi techniques. This will allow us to examine the role of genes required in both the phagocyte and in the apoptotic target cell. In our initial studies, we found that croquemort levels were upregulated in the presence of apoptotic cells. To identify other genes in the phagocyte that show changes in expression on exposure to apoptotic cells, we have initiated a differential expression screen. Genes that show expression changes in response to the presence of apoptotic cells will be tested for a role in engulfment in tissue culture and in vivo. This combination of strategies will allow us to identify and characterize many genes involved in apoptotic cell engulfment, and to understand how they function in this important process.