Genomic tools for massively parallel recording of signaling activity at cellular resolution in a brain-wide manner

项目总结/摘要 信号通路涉及无数的功能，包括介导细胞命运决定， 扩散，迁移和空间模式，以及其他作用。传统上，这些研究在 特定细胞类型，在有限数量的时间点，在少量细胞中。这是三个 局限性：1）我们对这些途径的理解依赖于细胞群的“批量分析”，这稀释了 单个细胞的信号作用。2)实时成像技术保留了细胞分辨率，但缺乏 可扩展性以扩展到整个组织或器官。3)在一个或两个时间点的测量不能捕获 在不同的发育阶段改变这些途径的作用。因此，我们需要从小规模转向 从信号活动的“局部”快照到大规模的“全局”快照。在这里，我描述了一种新颖的技术， 使用CRISPR/Cas工具记录每个细胞基因组中的信号输入，并在高水平上读取这些信号。 单细胞RNA测序（scRNA-seq）。 作为概念验证，我将应用这个工具来研究斑马鱼大脑中的信号事件。几个关键 已经确定了影响神经祖细胞命运和调节神经元空间模式的信号通路。 大脑区域。在这项研究中，我专注于Notch和FGF信号通路，以测试和验证该技术。 Notch信号通路是维持神经前体细胞间平衡的重要机制 增殖和分化成神经元细胞类型。FGF信号在大脑中具有多种功能， 前脑发育，空间模式和调节左右不对称。建议的第一部分 描述了记录斑马鱼信号活动的工具。它有5个核心组件：1）它使用3个Cas orthogonal 蛋白质独立记录信号。2)它对Cas激活有时间诱导作用。3)它使连续 在多个时间点记录信号。4)它便于记录多个信号输入。5)它可以被缩放 整个组织和器官，并保留单个细胞的分辨率。在第二部分中，我描述了 这项技术将为Notch和FGF信号转导提供新的生物学见解。我将调查1）诺奇是否 祖细胞中的信号传导与祖细胞分化成哪种类型的神经元相关; 2）是否 Notch介导的不对称分裂减少了祖细胞生态位内的细胞异质性; 3）如何以及何时 Notch和FGF的活性交叉调节神经发生。 CRISPR/Cas介导的信号条形码化和scRNA-seq的组合提供了一个强大的平台， 快速，可扩展和高分辨率的信号活动在发展过程中的调查。我设想， 技术将为组织工程、疾病和癌症建模的创新提供框架， 研究成体再生

PROJECT SUMMARY/ABSTRACT Signaling pathways have been implicated in a myriad of functions including mediating cell fate decisions, proliferation, migration, and spatial patterning, among other roles. Traditionally, these have been studied in specific cell types, at a limited number of timepoints, and in small numbers of cells. This presents three limitations: 1) Our understanding of these pathways has relied on “bulk analysis” of cell populations, which dilutes the signaling effects of individual cells. 2) Live imaging techniques preserve cellular resolution but lack the scalability to extend to whole tissues or organs. 3) Measurements at one or two timepoints do not capture the changing roles of these pathways at various developmental stages. Thus, we need to shift from small-scale “local” snapshots of signaling activities to large-scale “global” snapshots. Here I describe a novel technology that uses CRISPR/Cas tools to record signaling inputs in each cell’s genome and reads these signals at high throughput with single-cell RNA sequencing (scRNA-seq). As a proof-of-concept, I will apply this tool to investigate signaling events in the zebrafish brain. Several critical signaling pathways have been identified that influence neural progenitor fates and regulate spatial patterning of brain regions. In this study, I focus on the Notch and Fgf signaling pathways to test and validate the technology. The Notch pathway is an important mechanism that maintains the balance between neural progenitor cell proliferation and differentiation into neuronal cell types. Fgf signaling has multiple functions in the brain including forebrain development, spatial patterning and modulating left-right asymmetry. The first part of the proposal describes tools to record signaling activity in the zebrafish. It has 5 core components: 1) It uses 3 Cas orthologous proteins to independently record signals. 2) It has temporal inducibility of Cas activation. 3) It enables continuous signal recording at multiple timepoints. 4) It facilitates recording of multiple signaling inputs. 5) It can be scaled to whole tissues and organs and preserves the resolution of single cells. In the second part, I describe how the technology will provide new biological insights into Notch and Fgf signaling. I will investigate 1) whether Notch signaling in progenitors correlates with which types of neurons the progenitors differentiate into; 2) whether Notch-mediated asymmetric divisions reduced the cell heterogeneity within a progenitor niche; 3) how and when the activities of Notch and Fgf intersect to regulate neurogenesis. The combination of CRISPR/Cas-mediated signal barcoding and scRNA-seq provides a powerful platform for rapid, scalable and high-resolution investigation of signaling activity during development. I envision that this technology will provide a framework for innovation in tissue engineering, modeling disease and cancer, and studying adult regeneration.