Mapping vertebrate differentiation hierarchies with high-throughput single cell transcriptomics

项目摘要 对脊椎动物组织中细胞命运如何被指定和分化的详细理解是至关重要的 发展和再生生物学，并具有重要的意义，理解，建模， 治疗人类疾病到目前为止，我们已经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、扰动和命运作图，参与细胞命运调控。然而，在这方面， 要精确理解细胞如何获得它们的最终身份， 在分化期间检查过渡细胞状态。本提案的总体目标是将联合收割机 斑马鱼遗传学与强大的单细胞转录组学分析的新方法（目的1）提供高， 胚胎发生过程中细胞分化的分子命运图，（目的2）测试细胞如何分化的假设 分化与发育中的脊髓中的组织图案化偶联，并且（目标3，R00期） 在两种不同的生物学背景下进行分化的比较分子研究：胚胎分化 发育和组织再生。预计这些研究将揭示普遍和 细胞分化的再生特异性机制，将为斑马鱼提供重要的资源， 更大的生物医学研究社区，并将有助于改变我们的方法发育生物学，从 一个传统，严重依赖于标记基因，显微镜，定性观察，以一个公正的， 以单细胞分辨率询问整个转录组的系统性努力。 拟议的研究，连同一个积极的计划，为我自己的职业发展，借鉴了我的 在再生生物学广泛的科学背景，也包括学习新的 生物系统（斑马鱼）和新的实验方法，以接触新的科学环境 (the HMS系统生物学系），并由三位出色的科学家共同指导。在K99- 在指导阶段，我将接受肖恩·梅加松博士、阿隆·克莱因博士和亚历克斯·希耶博士的培训，他们的组合 斑马鱼遗传学、定量成像、分子遗传学和液滴微流体学方面的专业知识将被 在我致力于建立自己的独立研究项目时，这是非常宝贵的。 如我下文所述，我的向独立过渡计划将通过年度会议得到促进 与我的共同导师评估进展，并将包括出席几个研讨会（包括2- 在冷泉港实验室的一周课程），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个高影响力会议上发表研究成果的经验 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资助 写作技巧。在我向独立过渡的过程中，我将受益于我的共同导师过去的经验， 他们都曾在学术搜索委员会任职，在我准备工作时可以提供实用的建议 采访此外，亚历克斯希耶博士已指导他的前博士后18成功的独立 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独立的研究项目， 在胚胎发育，成体组织再生， 以及（最终）细胞替代疗法。到目前为止，我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，并与国际多年的参与 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， 通过提供接触关键人员和培训的机会，我的博士后将获得成功， 体验.

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.