Innovative reporters to characterize heterogeneous states among cells

 描述：这项建议是对RFA-RM-13-021“用于单细胞分析的特殊创新工具和技术(R21)”的资助机会公告的回应。我们的提案涉及研究目标中提出的目标：“开发新一代工具，区分原位细胞之间的异质状态”，特别是“在单个细胞内和表面上相似的原位细胞之间，在分子/结构/活动的灵敏度、选择性或时空分辨率方面提供重大进展的工具”。基因激活模式的复杂性差异很大。有些是简单和静态的，比如许多看家基因的模式，这些基因在生命中的所有细胞中都有表达。其他的则异常复杂，表达随时间或细胞波动 转到牢房。基因表达的变化反映了细胞转录格局的变化，在许多情况下，这种变化是对细胞外信号的反应。因此，表征基因表达的时空变化是细胞和发育生物学中的一个关键问题。此外，除了特定细胞内的时间动力学外，细胞-细胞连接和交流的空间架构也是至关重要的。在多细胞生物体中，细胞间的直接接触在几乎所有的生物过程中都发挥着至关重要的作用，从细胞增殖、分化和迁移等基本功能，到免疫系统激活、身体运动和记忆形成等高级功能。为了解决当前技术的一些局限性，我们建议开发一系列工具，这些工具将在分析体内细胞异质性和细胞接触方面取得重大进展。具体地说，我们建议开发：目的1.敏感的基因表达传感器，以可视化组织内细胞组之间的异质性。驱动荧光蛋白(FP)表达的转录报告通常被可视化地用于分析通路的激活；然而，这些报告的稳定性使得不可能获得分析动态基因表达变化所需的时间分辨率。我们已经开始开发一系列新的荧光记者(动态荧光记者)，它们极大地改善了基因活动的时间可视化，以解决这些基本问题。我们建议进一步优化这些工具，并展示它们在体内的应用：1.研究表面上均一的细胞群体之间的异质性；2.检查单个细胞对形态原分子的转录反应。目的2.检测细胞间接触的转录传感器。我们已经开发出强大的体外合成配体-受体系统，可以在细胞与细胞直接接触后激活转录报告或效应器。我们建议在体内建立这些系统，并评估它们在以下方面的应用：1.研究细胞线，即在想象的盘状上皮中与其他细胞进行远程接触的细胞投射；2.研究神经元的连接性。在AIMS 1和2中开发的工具将有许多额外的应用，并使研究信号通路之间的串扰；血细胞归巢；通过产生分泌的配体与其细胞靶标的结合 Ligand-GFP分子；以及分离不同发育/功能状态的特定细胞类型，用于RNAseq转录组分析。最后，虽然R21中描述的实验将在果蝇身上进行，但一旦建立起来，这些工具将很容易转移到其他系统，特别是脊椎动物。

 DESCRIPTION: This proposal is in response to the Funding Opportunity Announcement RFA-RM-13-021 "Exceptionally Innovative Tools and Technologies for Single Cell Analysis (R21)." Our proposal addresses the goals stated in the Research Objectives: "to develop next-generation tools that distinguish heterogeneous states among cells in situ", in particular "Tools that provide significant advances in sensitivity, selectivity or spatiotemporal resolution of molecules/structures/activities within single cells and between ostensibly similar cells in situ". Gene activation patterns vary widely in complexity. Some are straightforward and static, such as the pattern of many housekeeping genes, that are expressed in all cells at all times in life. Others are extraordinarily intricate with expression fluctuating from minute to minute or from cell to cell. Changes in gene expression reflect changes in the transcriptional landscape of the cell, which in many cases occur in response to extracellular signals. Characterizing the spatiotemporal changes in gene expression is therefore a critical question in cell and developmental biology. In addition, besides temporal dynamics within particular cells, the spatial architecture of cell-cell connection and communication are also critical. In multicellular organisms, direct intercellular contacts play vital roles in almost every biological process, from basic functions like cell proliferation, differentiation, and migration; to higher functions like immune system activation, body movement, and memory formation. To address some of the limitations with current technologies, we propose to develop a series of tools that will provide significant advances in the analysis of cellular heterogeneity and cell contact in vivo. Specifically, we propose to develop: Aim 1. Sensitive sensors of gene expression to visualize heterogeneity among groups of cells within tissues. Transcriptional reporters that drive the expression of fluorescent proteins (FPs) are commonly visualized to analyze the activation of pathways; however, the stability of these reporters makes it impossible to achieve the temporal resolution needed to dissect dynamic gene expression changes. We have begun to develop a series of new fluorescence reporters (Dynamic Fluorescence Reporters) that improve dramatically the temporal visualization of gene activities to address these fundamental questions. We propose to further optimize these tools and demonstrate their in vivo applications to: 1. study heterogeneity among apparently homogeneous cell populations; and 2. examine the transcriptional response of individual cells to a morphogen molecule. Aim 2. Transcriptional sensors to detect contacts between cells. We have developed robust in vitro synthetic ligand-receptor systems that activate a transcriptional reporter or effector following direct cell-cell contact. We propose to establish these systems in vivo and evaluate their use for: 1. the study of cytonemes, cellular projections making long-range contact with other cells, in imaginal disc epithelia; and 2. the study of neuronal connectivity. The tools developed in Aims 1 and 2 will have many additional applications and enable studies of cross-talk between signaling pathways; blood cell homing; the binding of secreted ligands to their cellular targets by generating secreted ligand-GFP molecules; as well as isolation of specific cell types at different developmental/functional states for RNAseq transcriptome analyses. Finally, although the experiments described in this R21 will be performed in Drosophila, the tools once established will be easily transferrable to other systems, especially vertebrates.