Engulfment of Dying Cells in Drosophila Embryos

描述（由申请人提供）：程序性细胞死亡是一个必要的过程，通过这个过程，不需要的或有缺陷的细胞以有序的方式被移除。因此，对垂死细胞的吞噬必须迅速而彻底，而不释放垂死细胞的内容物。细胞死亡失败导致畸形和癌症。未能吞噬死亡细胞也可能导致形态发生缺陷、模式错误、继发性坏死和炎症。细胞吞噬的研究主要集中在吞噬细胞上。已经确定了几个果蝇和秀丽隐杆线虫吞噬所需的基因。令人惊讶的是，没有一种是垂死细胞所独有的。我们对垂死细胞内促进其吞噬的细胞变化知之甚少。我们最近在了解果蝇胚胎发生过程中死亡细胞的吞噬方面取得了重要进展，通过开发一种使用荧光吞噬底物（我们称之为VGAL）的活胚胎吞噬敏感试验。这种吞噬实验表明，吞噬的模式忠实地反映了细胞死亡的模式。令人惊讶的是，在无法激活caspase的胚胎中，细胞吞噬的模式没有受到干扰，这表明细胞吞噬的信号独立于caspase激活级联。不依赖半胱天冬酶的细胞吞噬是一种新的和潜在的重要现象，我们所知甚少。caspase独立的吞噬挑战了caspase级联控制吞噬过程的教条。这个探索性的R21拨款提案旨在创建可视化和操纵体内吞噬的新工具。VGAL的一个重要限制是，它必须注射以检测吞噬，这限制了它在遗传筛查中的应用。第一个目标是开发一种基于gfp的细胞吞噬报告基因，这将为吞噬缺陷提供一种筛选方法，并进一步证实不依赖于caspase的吞噬。这种基于gfp的报告基因也可用于研究自噬。第二个目标将测试一个新的假设，即死亡上皮细胞内的细胞骨架变化允许/促进其健康邻居对死亡细胞的吞噬。这个模型试图解释为什么所有已知的吞噬突变都需要在吞噬细胞中发生，而不是在死亡细胞中发生。成功完成这两个目标将对发育调节的细胞死亡和吞噬的研究产生重大影响。

DESCRIPTION (provided by applicant): Programmed cell death is an essential process by which unwanted or defective cells are removed in an orderly fashion. The engulfment of dying cells, therefore, must be rapid and complete, without releasing the contents of the dying cells. Failures in cell death lead to deformities and cancer. Failure to engulf dying cells may also result in morphogenetic defects, patterning errors, secondary necrosis and inflammation. The study of cell engulfment has primarily focused on the phagocytic cells. Several Drosophila and C. elegans genes required for engulfment have been identified. Surprisingly, none are required exclusively in the dying cell. We have very little understanding of the cellular changes within the dying cell that promote its engulfment. We recently made an important advance to understanding the engulfment of dying cells during Drosophila embryogenesis by developing a sensitive assay for engulfment in living embryos using a fluorogenic, engulfment substrate, which we call VGAL. This engulfment assay showed that the pattern of engulfment faithfully mirrors the pattern of cell death. Surprisingly, the pattern of cell engulfment was unperturbed in embryos that were unable to activate their caspases, indicating that the signal for cell engulfment is independent of the caspase activation cascade. Caspase-independent cell engulfment is a new and potentially important phenomenon that we know very little about. Caspase-independent engulfment challenges the dogma that the caspase cascade controls the engulfment process. This exploratory, R21 grant proposal is intended to create new tools for visualizing and manipulating engulfment in vivo. A significant limitation of VGAL is that it must be injected in order to detect engulfment, which limits its use for genetic screening. The first aim is to develop a GFP-based cell engulfment reporter that will provide a screening method for engulfment defects and further confirm caspase- independent engulfment. This GFP-based reporter can also be used to study autophagy. The second aim will test a novel hypothesis that cytoskeletal changes within dying, epithelial cells permit/promote the engulfment of dying cells by their healthy neighbors. This model attempts to explain why all known engulfment mutations are required in the engulfing cells, but not the dying cells. Successful completion of both aims will have a significant impact on the study of developmentally-regulated cell death and engulfment.