Mapping vertebrate differentiation hierarchies with high-throughput single cell transcriptomics

利用高通量单细胞转录组学绘制脊椎动物分化层次结构

• 批准号: 10087987
• 负责人: Daniel E Wagner
• 金额: $ 24.9万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10087987
• 关键词: Address; Adult; Algorithms; Anatomy; Animals; Architecture; Biological; Biological Process; Biology; Biomedical Research; Cell Differentiation process; Cell Fate Control; Cell Lineage; Cell Therapy; Cells; Communities; Comparative Study; Complex; Computing Methodologies; Coupled; Crush Injury; Custom; Data; Data Set; Deposition; Development; Developmental Biology; Diffuse; Disease; Dissection; Dorsal; Educational workshop; Embryo; Embryonic Development; Environment; Exposure to; Eye; Feedback; Fluorescence-Activated Cell Sorting; Foundations; Future; Gene Expression; Generations; Genes; Genetic; Genetic Screening; Genomics; Goals; Grant; Harvest; Human; Image; In Vitro; Individual; Injury; International; Interview; Knowledge; Laboratories; Learning; Logic; Malignant Neoplasms; Maps; Mentors; Methodology; Methods; Microfluidics; Microscopy; Modeling; Molecular; Molecular Genetics; Natural regeneration; Neural tube; Noise; Occupations; Outcome; Patients; Pattern; Persons; Phase; Positioning Attribute; Postdoctoral Fellow; Process; Protocols documentation; Publications; Regulator Genes; Replacement Therapy; Research; Resolution; Resources; Sampling; Scientist; Signal Transduction; Source; Specific qualifier value; Spinal Cord; System; Systems Biology; Testing; Time; Tissues; Training; Transgenic Organisms; Transitional Cell; Trees; Vertebrates; Work; Writing; Zebrafish; base; biological systems; career; career development; cell type; comparative; cost; embryo tissue; experience; experimental study; hatching; human disease; improved; insight; meetings; microfluidic technology; model organisms databases; nerve stem cell; new technology; novel; progenitor; programs; quantitative imaging; reconstruction; regenerative; single-cell RNA sequencing; skills; spinal cord regeneration; stem cells; student mentoring; success; tissue regeneration; tissue/cell culture; transcriptome; transcriptome sequencing; transcriptomics; zebrafish development; Address; Adult; Algorithms; Anatomy; Animals; Architecture; Biological; Biological Process; Biology; Biomedical Research; Cell Differentiation process; Cell Fate Control; Cell Lineage; Cell Therapy; Cells; Communities; Comparative Study; Complex; Computing Methodologies; Coupled; Crush Injury; Custom; Data; Data Set; Deposition; Development; Developmental Biology; Diffuse; Disease; Dissection; Dorsal; Educational workshop; Embryo; Embryonic Development; Environment; Exposure to; Eye; Feedback; Fluorescence-Activated Cell Sorting; Foundations; Future; Gene Expression; Generations; Genes; Genetic; Genetic Screening; Genomics; Goals; Grant; Harvest; Human; Image; In Vitro; Individual; Injury; International; Interview; Knowledge; Laboratories; Learning; Logic; Malignant Neoplasms; Maps; Mentors; Methodology; Methods; Microfluidics; Microscopy; Modeling; Molecular; Molecular Genetics; Natural regeneration; Neural tube; Noise; Occupations; Outcome; Patients; Pattern; Persons; Phase; Positioning Attribute; Postdoctoral Fellow; Process; Protocols documentation; Publications; Regulator Genes; Replacement Therapy; Research; Resolution; Resources; Sampling; Scientist; Signal Transduction; Source; Specific qualifier value; Spinal Cord; System; Systems Biology; Testing; Time; Tissues; Training; Transgenic Organisms; Transitional Cell; Trees; Vertebrates; Work; Writing; Zebrafish; base; biological systems; career; career development; cell type; comparative; cost; embryo tissue; experience; experimental study; hatching; human disease; improved; insight; meetings; microfluidic technology; model organisms databases; nerve stem cell; new technology; novel; progenitor; programs; quantitative imaging; reconstruction; regenerative; single-cell RNA sequencing; skills; spinal cord regeneration; stem cells; student mentoring; success; tissue regeneration; tissue/cell culture; transcriptome; transcriptome sequencing; transcriptomics; zebrafish development

PROJECT SUMMARY A detailed understanding of how cell fates are specified and differentiate in vertebrate tissues is fundamental to developmental and regenerative biology, and has important implications for understanding, modeling, and treating human disease. To date, we have had great success in identifying key molecular components implicated in cell fate regulation through the use of genetic screens, perturbations and fate mapping. However, a precise understanding of how cells acquire their final identities requires much deeper and unbiased examinations of the transitional cell states during differentiation. The general aim of this proposal is to combine zebrafish genetics with powerful new methods for single cell transcriptomic profiling to (Aim 1) deliver a high- resolution molecular fate map of cell differentiation during embryogenesis, (Aim 2) test hypotheses for how cell differentiation is coupled to tissue patterning in the developing spinal cord, and (Aim 3, the R00 phase) to undertake comparative molecular studies of differentiation in two distinct biological contexts: embryonic development and tissue regeneration. It is anticipated that these studies will reveal both universal and regeneration-specific mechanisms for cell differentiation, will provide significant resources to the zebrafish and larger biomedical research communities, and will help transform our approach to developmental biology, from a tradition that relied heavily on marker genes, microscopy, and qualitative observations, to an unbiased and systematic effort that interrogates the entire transcriptome at single-cell resolution. The proposed research, together with an aggressive plan for my own career development, draws on my extensive scientific background in regenerative biology and also incorporates opportunities to learn a new biological system (zebrafish) and new experimental methods, to gain exposure to a new scientific environment (the HMS Systems Biology department), and to be co-mentored by three fantastic scientists. During a K99- mentored phase, I will receive training from Drs. Sean Megason, Allon Klein, and Alex Schier, whose combined expertise in zebrafish genetics, quantitative imaging, molecular genetics, and droplet microfluidics will be invaluable as I work to build my own independent research program. As I describe below, my plan for transitioning to independence will be facilitated by annual meetings with my co-mentors to evaluate progress, and will include attendance of several workshops (including a 2- week course at Cold Spring Harbor laboratories) to develop my skills in genomics and quantitative biology. I will gain experience presenting research results in talks at international meetings and in 1-2 high-impact publications. I will also gain experience mentoring students, and will hone my lab management and grant writing skills. During my transition to independence, I will benefit from the past experiences of my co-mentors, who have all served on academic search committees and can provide practical advice as I prepare for job interviews. In addition, Dr. Alex Schier has guided 18 of his former postdocs to successful independent research positions, and his insights and feedback will be particularly valuable. My long-term career goal is to direct an independent research program aimed at understanding molecular features of cell differentiation in the contexts of embryonic development, adult tissue regeneration, and (ultimately) cell replacement therapies. So far I have achieved significant progress towards this goal in the form of research experience, successful publications, and many years of engagement with international scientific communities. I firmly believe, however, that a K99 mentored phase will help maximize my chances for success by providing access to key persons and training that would be otherwise lacking from my postdoctoral experience.

项目摘要 对如何指定细胞命运和在脊椎动物组织中分化的详细理解是基本的 发育和再生生物学，对理解，建模和 治疗人类疾病。迄今为止，我们在识别关键分子组件方面取得了巨大成功 通过使用遗传筛选，扰动和命运图与细胞命运调节有关。然而， 对细胞如何获得最终身份的精确理解需要更深入和公正 分化过程中过渡细胞状态的检查。该提议的总体目的是结合 斑马鱼遗传学具有强大的新方法，用于单细胞转录组分析（AIM 1）提供高 分辨率分子命运图在胚胎发生过程中的细胞分化，（AIM 2）检验假设细胞如何 分化与发育中的脊髓中的组织模式耦合，并且（AIM 3，R00相）至 在两个不同的生物学环境下进行分化的比较分子研究：胚胎 开发和组织再生。可以预料，这些研究将揭示普遍和 细胞分化的再生特定机制，将为斑马鱼和 更大的生物医学研究社区，并将有助于改变我们的发展生物学方法 这种传统在很大程度上依赖标记基因，显微镜和定性观察，并依赖于公正和 系统的努力以单细胞分辨率询问整个转录组。 拟议的研究以及对我自己的职业发展的积极计划，借鉴了我​​的 再生生物学的广泛科学背景，还结合了学习新的机会 生物系统（斑马鱼）和新的实验方法，以获取新的科学环境 （HMS系统生物学系），并由三位出色的科学家共同授予。在K99- 指导阶段，我将获得DRS的培训。 Sean Megason，Allon Klein和Alex Schier的结合 斑马鱼遗传学，定量成像，分子遗传学和液滴微流体的专业知识将是 当我努力构建自己的独立研究计划时，无价。 如下所述，我的年度会议将促进我过渡到独立的计划 与我的副官员一起评估进度，并将包括参加几个研讨会（包括2- 在冷泉港实验室举行的周课程），以发展我在基因组学和定量生物学方面的技能。我 将获得在国际会议和1-2个高影响力的谈判中呈现研究结果的经验 出版物。我还将获得指导学生的经验，并将磨练我的实验室管理和授予 写作技巧。在过渡到独立的过程中，我将从我的联合会经历中受益， 所有人都在学术搜索委员会任职，可以在我准备工作时提供实用建议 访谈。此外，Alex Schier博士还指导了他的18个以前的博士后，成功独立 研究职位以及他的见解和反馈将特别有价值。 我的长期职业目标是指导旨在理解的独立研究计划 在胚胎发育，成人组织再生的背景下，细胞分化的分子特征， 和（最终）细胞替代疗法。到目前为止，我已经取得了重大进展 研究经验的形式，成功的出版物以及与国际的多年参与 科学社区。但是，我坚信，K99的指导阶段将有助于最大化我的机会 通过提供对关键人物和培训的访问，成功的成功 经验。