Global investigation of cell trajectory and lineage relationships in the vertebrate brain with single-cell transcriptomics

利用单细胞转录组学对脊椎动物大脑中的细胞轨迹和谱系关系进行整体研究

• 批准号: 10021448
• 负责人: Bushra Raj
• 金额: $ 12.86万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-19 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10021448
• 关键词: Address; Advisory Committees; Algorithms; Animal Model; Award; Bar Codes; Behavior; Bioinformatics; Brain; Brain region; CRISPR/Cas technology; Cell Lineage; Cell division; Cells; Chromatin; Code; Competence; Core Facility; Data; Data Set; Development; Developmental Biology; Disease model; Embryo; Enhancers; Feedback; Gene Expression; Gene Expression Profile; Genetic; Genetic Transcription; Genomics; Goals; Grant; Investigation; Maps; Memory; Mentors; Mentorship; Methods; Modeling; Molecular; Molecular and Cellular Biology; Natural regeneration; Neurobiology; Neurodevelopmental Disorder; Neuroglia; Neurons; Neurotransmitters; Organoids; Pathway interactions; Population; Process; Proliferating; Radial; Recording of previous events; Regulator Genes; Regulatory Element; Research; Resolution; Resources; Role; Running; Specific qualifier value; Study models; Technology; Time; To specify; Training; Trees; Type I Epithelial Receptor Cell; Vision; Writing; Zebrafish; analytical method; base; career; cell fate specification; cell type; experience; genome resource; genome-wide; graduate student; imprint; insight; mathematical model; meetings; member; nerve stem cell; neurodevelopment; neurogenesis; progenitor; programs; reconstruction; relating to nervous system; segregation; single cell analysis; single-cell RNA sequencing; tool; transcriptome; transcriptomics; translational approach; zebrafish development

PROJECT SUMMARY/ABSTRACT A key goal in developmental biology is to understand how the brain is specified and organized regionally, cellularly and molecularly. Central to this vision is determining the origins and fates of cells during development, and thus map the progressive steps of cell specification and lineage divergences. Focused efforts have provided insight into specific cell types and lineages, however global views of these processes have been lacking. Recent technological breakthroughs in single-cell transcriptomics and lineage tracing using CRISPR-Cas9 tools are now enabling the realization of this vision. The long-term goal of this project is to obtain global views of cellular relationships and molecular changes during neural development and cell type diversification in the vertebrate brain. These include generating large-scale, single-cell resolution cell specification trajectories that describe molecular cascades underlying cell fate specification (Aim 1), and lineage trees that describe the history of cell divisions (Aim 2). These trees represent many key aspects of developmental decisions and can be used to determine gene expression cascades during cell specification and regulatory factors involved in progenitor priming and neuron identity (Aim 1). Furthermore, they can address how often transcriptional and lineage identities are related (Aim 2). These studies will generate resources for genome-wide and single-cell analysis of brain development and reveal cellular and molecular mechanisms for generating neuronal cell diversity. My career goal is to run an academic lab aimed at investigating cellular and molecular features underlying brain development, neurogenesis and neural stem cell activity using global and focused approaches. The proposed research draws on my previous experience with characterizing gene regulatory networks in neurogenesis and extends it to a new model organism, zebrafish, while exposing me to new experimental and analytical methods. I have developed a detailed training plan with my co-mentors, Drs. Len Zon, Alex Schier, and Josh Sanes, who have combined expertise in development, behavior, neurobiology, single-cell analysis and disease modeling. To help me transition to independence, we will meet regularly to discuss research progress, brainstorm ideas, and obtain guidance on grant writing, mentorship and lab management. My K99 advisory committee consists of Drs. Allon Klein, Sean Megason and Gord Fishell, whose collective expertise in single-cell genomics, zebrafish development, and neurobiology will provide me with technical and conceptual feedback in executing my research plan. I will continue to mentor a research technician/graduate student, will present my research at two meetings per year, take courses on bioinformatic analysis, mathematical modeling and grant writing, and attend seminars to broaden my scientific training. As a member of the Harvard Department of Molecular and Cellular Biology, I will have access to leaders in developmental biology, neurobiology and genetics, as well as cutting-edge core facilities. The Pathway to Independence Award will provide me with resources to initiate an ambitious research program and obtain additional training to maximize my chances of a successful transition to independence.

项目摘要/摘要 发育生物学的一个关键目标是了解大脑是如何被指定和组织的， 在细胞和分子上。这一愿景的核心是决定细胞在发育过程中的起源和命运， 从而绘制出细胞规格和谱系分化的渐进步骤。有重点的努力提供了 对特定的细胞类型和谱系的洞察，然而，对这些过程的全球视角一直缺乏。近期 使用CRISPR-Cas9工具在单细胞转录和谱系追踪方面的技术突破现在 使这一愿景得以实现。该项目的长期目标是获得蜂窝网络的全球视角 脊椎动物神经发育和细胞类型多样化过程中的关系和分子变化 大脑。其中包括生成大规模的单元格分辨率像元规范轨迹，该轨迹描述 基于细胞命运规范的分子级联(目标1)，以及描述细胞历史的谱系树 分部(目标2)。这些树代表了发展决策的许多关键方面，可用于 确定细胞分化过程中基因表达的级联和祖细胞所涉及的调控因子 启动和神经元识别(目标1)。此外，它们还可以处理转录和谱系的频率 身份是相关的(目标2)。这些研究将为全基因组和单细胞分析提供资源 并揭示产生神经细胞多样性的细胞和分子机制。 我的职业目标是管理一个学术实验室，旨在研究大脑潜在的细胞和分子特征 使用全球和重点方法的发育、神经发生和神经干细胞活动。建议数 这项研究借鉴了我以前在神经发生和基因调控网络特征方面的经验 将它扩展到一种新的模式生物斑马鱼，同时让我接触到新的实验和分析方法。 我已经与我的共同导师Len Zon博士、Alex Schier博士和Josh Sanes博士制定了详细的培训计划，他们 拥有开发、行为学、神经生物学、单细胞分析和疾病建模方面的专业知识。至 帮助我过渡到独立，我们将定期会面，讨论研究进展，集思广益，并 获得有关拨款撰写、指导和实验室管理方面的指导。我的K99顾问委员会由K99博士组成。 Allon Klein，Sean Megason和Gord Fishell，他们在单细胞基因组学方面的集体专长，斑马鱼 发展，神经生物学将为我提供执行研究的技术和概念反馈 计划。我将继续指导一名研究技术员/研究生，并将在两次会议上介绍我的研究 每年，参加生物信息学分析、数学建模和拨款申请的课程，并参加研讨会 以扩大我的科学训练。作为哈佛大学分子和细胞生物学系的一名成员，我 将有机会接触到发育生物学、神经生物学和遗传学的领军人物，以及尖端核心 设施。独立之路奖将为我提供资源，以启动一项雄心勃勃的研究 计划和获得额外的培训，以最大限度地增加我成功过渡到独立的机会。