Mapping the signaling landscape of vertebrate development at single cell resolution

摘要(项目摘要) 发育生物学的一个主要目标是了解所有胚胎细胞的详细分子进程。 血统，从多能性到成年期，以及信号通路如何控制血统选择的每一步 差异化。这样的理解解决了发育生物学中的几个基本问题， 同时对疾病中的细胞重新编程和细胞的体外分化具有实际意义 心理治疗。最近，我们发展了液滴微流控单细胞RNA测序(scRNA-Seq)技术， 它允许以低成本分析数万个单细胞的转录组，而且我们还 发展了一种将液滴scRNA-Seq与谱系追踪相结合的方法，并给出了计算方法 从scRNA-Seq和谱系数据重建分化的时间序列所需的。在前期工作中，我们 应用这些工具生成了斑马鱼发育过程中细胞状态轨迹的综合地图 通过受精后的第一个24小时。在这项提案中，我们将扩展我们的斑马鱼发展地图， 将scRNA-Seq与克隆分析相结合跟踪斑马鱼胚胎发育中的每一种细胞状态 受精后天数。然后我们将使用七个主要信号通路的阶段性、急性扰动， 然后是scRNA-Seq，以定义哪些信号通路控制细胞沿不同轨迹流动 在整个发育过程中，以及它们的上下文相关和不变的基因靶标。深度聚焦于 神经管模式，然后我们将剖析整合信号通路的转录因子网络， 通过CRISPR/Cas9扰动偶联到scRNA-Seq.该提案建立在多年合作的基础上 拥有强大和协同专业知识的两个实验室--Megason实验室使用斑马鱼 发育系统生物学和克莱恩实验室在单细胞基因组学和分析方面的研究。

Abstract (Project Summary) A major goal of developmental biology is to understand the detailed molecular progression of all embryonic cell lineages, from pluripotency to adulthood, and how signalling pathways control lineage choices at every step of differentiation. Such an understanding addresses several fundamental questions in developmental biology, while having practical implications for re-programming cells in disease, and for in vitro differentiation for cell therapy. Recently, we developed droplet microfluidic single cell RNA-Sequencing (scRNA-Seq) technology, which allows profiling the transcriptome of tens of thousands of single cells at low cost, and we additionally developed a method to combine droplet scRNA-Seq with lineage tracing, and the computational methods required to reconstruct time series of differentiation from scRNA-Seq and lineage data. In preliminary work, we applied these tools to generate a comprehensive map of cell state trajectories in zebrafish development through the first 24 hours post fertilization. In this proposal, we will extend our map of zebrafish development, combining scRNA-Seq with clonal analysis to track every cell state in the developing zebrafish embryo up to 7 days post-fertilization. We will then use staged, acute perturbations of seven major signaling pathways, followed by scRNA-Seq, to define which signaling pathways control the flow of cells down different trajectories throughout development, as well as their context dependent and invariant gene targets. Focusing deeply on neural tube patterning, we will then dissect the transcription factor networks that integrate signaling pathways, by CRISPR/Cas9 perturbation coupled to scRNA-Seq. This proposal builds on a multi-year collaboration between two labs with strong and synergistic expertise -- the Megason lab in the use of zebrafish for developmental systems biology and the Klein lab in single cell genomics and analysis.