In vivo analysis of signaling dynamics in the Notch interaction network

摘要：Notch信号通路在后生动物的多个细胞命运决定事件中起着关键作用，最近被认为与人类癌症的病理有关。遗传和生化证据表明，从Notch受体与其配体Delta和Serrate的相互作用到Notch靶基因的转录调节，一个庞大的调节剂网络参与了Notch信号的精确水平的建立，并决定了通路激活的组织特异性结果。虽然这些知识大部分是用果蝇作为模式生物获得的，但由于果蝇和哺乳动物之间Notch信号传导的基本步骤显着保持，因此它与理解人类Notch功能有关。然而，一个主要的障碍是无法研究内源性信号事件。在这项研究中，

Abstract: The Notch signaling pathway plays a critical role in multiple cell fate decision events in metazoans, and recentiy has been implicated in the pathology of cancer in humans. Genetic and biochemical evidence has revealed a large network of modulators that are involved in establishing a precise level of Notch signaling and determining tissue-specific outcomes of pathway activation, from the interaction ofthe Notch receptor with its ligands Delta and Serrate to transcriptional regulation of the Notch target genes. Though much of this knowledge has been obtained using Drosophila as a model organism, it is relevant for understanding Notch function in humans due to a remarkable conservation ofthe fundamental steps in Notch signaling between flies and mammals. Yet, a major obstacle has been an inability to study endogenous signaling events. In this study, we propose to gain insight into the Notch signaling events by using the genetic advances in recombineering and targeted transgene insertions to replace the endogenous Notch and Delta genes with their fluorescent and affinity tagged isoforms. These transgenes will be used for studying the composition of Notch containing signaling complexes and for imaging the process of Notch activation in vivo. Completion of these experiments will establish an in vivo map ofthe Notch interaction network and reveal the dynamics of signaling in living cells. With the above in mind, we will pursue the following specific aims: 1) To generate Drosophila lines containing genomically tagged Notch and Delta, 2) To analyze the Notch interaction network in vivo, and 3) To study the dynamics of Notch signaling in vivo. Moreover, this collaboration will have multiple benefits for the continued professional development of Dr. Veraksa's career. While the two principle investigators have a history of productive collaboration. Dr. Veraksa is eariy in his career as an independent investigator; yet both Veraksa and Artavanis-Tsakonas bring complementary expertise to the proposed project. This collaboration will also significantiy benefit from the Training Core.