In vivo analysis of signaling dynamics in the Notch interaction network

摘要：Notch信号通路在多细胞动物的多细胞命运决定事件中起着关键作用，并且最近已经涉及人类癌症的病理学。遗传和生物化学证据已经揭示了一个大的调节剂网络，其参与建立精确水平的Notch信号传导和确定途径激活的组织特异性结果，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。尽管这些知识大部分是以果蝇为模式生物获得的，但由于果蝇和哺乳动物之间Notch信号传导的基本步骤的显著保守性，因此与理解Notch在人类中的功能有关。然而，一个主要的障碍是无法研究内源性信号事件。在本研究中， 我们建议通过利用重组工程和靶向转基因插入中的遗传学进展，用内源性Notch和Delta基因的荧光和亲和标记的同种型取代内源性Notch和Delta基因，来深入了解Notch信号传导事件。这些转基因将用于研究Notch信号复合物的组成，并用于体内Notch激活过程的成像。完成这些实验 将建立Notch相互作用网络的体内图谱，并揭示活细胞中信号传导的动力学。考虑到上述情况，我们将追求以下具体目标：1）产生含有基因组标记的Notch和Delta的果蝇系，2）分析体内Notch相互作用网络，以及3）研究体内Notch信号传导的动力学。此外，这种合作将为Veraksa博士的职业生涯的持续专业发展带来多重好处。虽然这两个主要的调查人员有一个富有成效的合作历史。Veraksa博士作为一名独立调查员的职业生涯尚早，但Veraksa和Artavanis-Tsakonas都为拟议的项目带来了互补的专业知识。这种合作也将从培训核心中受益匪浅。

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.