Combining dynamics of ligand presentation with dynamics of hESC response in colonies with defined architecture

项目摘要 在早期胚胎发生过程中，多能细胞通过整合多个信号来指定它们的命运。 不同的时间，不同的地点，不同的动力。在小鼠胚胎中， BMP 4、Wnt和Nodal通路启动胚胎胚层的诱导和形成。然而，在这方面， 这些途径与人类发展的相关性尚不清楚。此外，多个动态 整合信号通路以确定细胞命运的情况尚不清楚。在这里，我们建议使用人类胚胎 干细胞（hESC），以了解单个细胞如何处理这些动态信号， 命运为了解决这个问题，我们开发了两项创新技术。首先，微流体技术可以精确地控制 配体应用的时机，并遵循信号转导器SMAD 4的行为，我们 表明TGFβ信号是适应性的。第二，控制hESC集落大小的微图案， 为了显示响应于BMP 4，在圆形集落中培养的hESC自组织成放射状， 分散的胚胎胚层的对称模式。这显著地概括了近端-远端轴， 原肠胚形成中的小鼠胚胎。在这一竞争性更新中，我们将联合收割机的两种技术优势结合起来， 通过微流体将配体呈递的不同动力学传递到在培养的CRISPR编辑的hESC系中， 微型菌落四个独立的信号报告人胚胎干细胞系，荧光标记SMAD 1， SMAD 2、SMAD 4和β-连环蛋白将用于可视化信号传导，一个三重标记的fate报告基因将用于可视化信号传导。 CRISPR编辑的荧光标记SOX 2，BRACHYURY和SOX 17的细胞系将用于监测命运 采集我们的CRISPR报告细胞系将用于测量信号动力学和命运获取， 单细胞分辨率和实时视频显微镜，当细胞与BMP 4，Wnt 3A， 激活素/Nodal，作为一个定义浓度的持续步骤，或作为一个定义持续时间的步骤。我们提出 三个具体目标。在aim 1中，这三种配体将被呈递给我们的信号报告CRISPR系， 跟踪四个标记的信号换能器的行为并确定每个信号换能器的动态行为 通路在aim 2中，使用相同的方法，我们将通过测量 三种配体的转录报告基因，并使用我们的三重命运报告基因CRISPR系来跟踪命运。 采集这两种方法将建立信号动力学、转录和转录之间的定量联系。 输出和命运决定。在aim 3中，从aims 1和aims 2获得的大型数据集将用于对 信号转导的动力学，并提供一个数学范式来解释hESC自组织。的 解决我们的三个目标将对我们理解的动态整合产生重大影响， 作为人类细胞命运特化基础的信号通路与基本理解， 和临床应用。

Project Summary During early embryogenesis, pluripotent cells specify their fates by integrating multiple signals delivered at different times, places, and with different dynamics. In the mouse embryo, interplay between 3 signaling pathways - BMP4, Wnt, and Nodal - initiate the induction and patterning of embryonic germ layers. However, the relevance of these pathways to human development is not understood. Furthermore, how multiple dynamic signaling pathways are integrated to define cellular fate is unclear. Here we propose to use human embryonic stem cells (hESCs) to understand how individual cells process these dynamic signals to generate discrete fates. To address this, we developed 2 innovative technologies. First, microfluidics to precisely control the timing of ligand application and follow the behavior of the signal transducer SMAD4, with which we demonstrated that TGFβ signaling was adaptive. Second, micropatterns to control hESC colony size and geometry, to show that in response to BMP4, hESCs cultured in circular colonies self-organize into radially symmetric patterns of discrete embryonic germ layers. This remarkably recapitulates the proximal-distal axis of the gastrulating mouse embryo. In this competitive renewal, we combine the strengths of both technologies to deliver distinct dynamics of ligand presentation by microfluidics to CRISPR-edited hESC lines cultured in micropatterned colonies. Four independent signaling-reporter hESC lines that fluorescently tag SMAD1, SMAD2, SMAD4, and β-CATENIN will be used to visualize signaling, and one triple-tagged, fate-reporter CRISPR-edited line that fluorescently tags SOX2, BRACHYURY, and SOX17, will be used to monitor fate acquisition. Our CRISPR-reporter lines will be used to measure signaling dynamics and fate acquisition, with single cell resolution and in real-time by video-microscopy, when cells are presented with BMP4, Wnt3A, and Activin/Nodal either as a persistent step of defined concentration, or as one of defined duration. We propose three specific aims. In aim1, the three ligands will be presented to our signaling-reporter CRISPR lines, to follow the behavior of the four tagged signal transducers and to determine the dynamic behavior of each pathway. In aim2, using the same approach, we will evaluate pathway output by measuring the activity of transcriptional reporters for the three ligands, and use our triple fate-reporter CRISPR line to follow fate acquisition. These two approaches will establish a quantitative link between signaling dynamics, transcriptional output, and fate determination. In aim3, large datasets obtained from aims1 and 2, will be used to model the kinetics of signal transduction, and provide a mathematical paradigm to explain hESC self-organization. The resolution of our three aims will have a strong impact on our understanding of the dynamic integration of signaling pathways underlying human cell fate specification with direct relevance to both basic understanding, and clinical applications of hESCs.