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

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.

项目概要 详细了解脊椎动物组织中细胞命运如何指定和分化是基础 对发育和再生生物学具有重要意义，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成分方面取得了巨大成功 通过使用遗传筛选、扰动和命运图谱参与细胞命运调控。然而， 准确理解细胞如何获得其最终身份需要更深入和公正的研究 检查分化过程中的过渡细胞状态。该提案的总体目标是结合 斑马鱼遗传学具有强大的单细胞转录组分析新方法，以（目标 1）提供高 解析胚胎发生过程中细胞分化的分子命运图，（目标 2）检验细胞如何分化的假设 分化与发育中的脊髓中的组织模式相关，并且（目标 3，R00 阶段） 在两种不同的生物背景下进行分化的比较分子研究：胚胎 发育和组织再生。预计这些研究将揭示普遍性和 细胞分化的再生特异性机制将为斑马鱼和 更大的生物医学研究社区，并将有助于改变我们的发育生物学方法，从 这一传统严重依赖标记基因、显微镜和定性观察 以单细胞分辨率询问整个转录组的系统性工作。 拟议的研究以及我自己职业发展的积极计划借鉴了我的经验 再生生物学方面广泛的科学背景，并且还包含学习新知识的机会 生物系统（斑马鱼）和新的实验方法，接触新的科学环境 （HMS 系统生物学系），并由三位出色的科学家共同指导。在 K99- 指导阶段，我将接受博士的培训。肖恩·梅加森 (Sean Megason)、阿隆·克莱因 (Allon Klein) 和亚历克斯·席尔 (Alex Schier) 斑马鱼遗传学、定量成像、分子遗传学和液滴微流控方面的专业知识 当我致力于建立自己的独立研究计划时，这是无价的。 正如我在下面所描述的，我向独立过渡的计划将通过年度会议来促进 与我的共同导师一起评估进展情况，并将包括参加多个研讨会（包括 2- 冷泉港实验室的周课程）以培养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 个高影响力的演讲中展示研究成果的经验 出版物。我还将获得指导学生的经验，并磨练我的实验室管理和资助 写作技巧。在我向独立过渡的过程中，我将从我的共同导师过去的经验中受益， 他们都曾在学术搜索委员会任职，可以在我准备工作时提供实用的建议 采访。此外，Alex Schier 博士还指导他的 18 名前博士后成功独立 研究职位，他的见解和反馈将特别有价值。 我的长期职业目标是指导一个旨在理解的独立研究项目 胚胎发育、成体组织再生中细胞分化的分子特征， 和（最终）细胞替代疗法。到目前为止，我已经在实现这一目标方面取得了重大进展 形式的研究经验、成功的出版物以及多年与国际的接触 科学界。然而，我坚信 K99 指导阶段将有助于最大限度地提高我的机会 通过提供与关键人员的接触和培训来取得成功，而这些是我的博士后所缺乏的 经验。