Genetic analysis of apoptotic cell clearance in flies

描述(申请人提供)：凋亡细胞在体内通过吞噬作用迅速清除。凋亡细胞吞噬功能缺陷与系统性红斑狼疮等多种人类疾病有关。在没有被吞噬的情况下，剩余的组织可能会因为暴露在死亡细胞的内容物中而受损。在果蝇中，凋亡的身体会迅速被循环中的吞噬细胞吞噬。我们已经证明Croquemort基因是有效吞噬胚胎中的凋亡细胞所必需的。我们建议利用果蝇遗传学、组织培养分析和差异表达筛选相结合的方法，识别和表征与吞噬有关的其他基因。基因组缺失筛查确定了有效吞噬胚胎中的凋亡细胞所需的一些区域。在我们的筛选中发现的基因组区域之一包含果蝇磷脂酰丝氨酸受体的同源物。在哺乳动物组织培养试验中，这种受体与吞噬凋亡身体有关。虽然我们没有检测到这种受体在果蝇胚胎吞噬过程中的主要作用，但我们确实发现它具有抑制细胞凋亡的功能。我们将利用这种表型来筛选由受体激活的信号通路。此外，我们还将进一步探讨吞噬过程中对该受体的要求。这些研究将提供对该蛋白质的一般功能的洞察。我们还确定了包含吞噬所需基因的其他基因组区域。我们将使用遗传和RNA干扰技术来区分负责的基因。为了促进RNAi技术的发展，建立了一种基于组织培养的凋亡细胞吞噬试验。这将使我们能够研究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，我们发现在存在凋亡细胞的情况下，Croquemort水平被上调。为了确定吞噬细胞中的其他基因在暴露于凋亡细胞时表现出表达的变化，我们启动了差异表达筛选。在组织培养和活体中，将测试对存在凋亡细胞而表现出表达变化的基因在吞噬过程中的作用。这种策略的结合将使我们能够识别和表征许多与细胞凋亡吞噬有关的基因，并了解它们在这一重要过程中如何发挥作用。

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.