Synthetic recording and in situ readout of cell lineage and molecular history in mammalian retina

摘要 这个项目的长期目标是描述哺乳动物视网膜的复杂结构是如何在 发展。为了实现这一目标，本提案的重点是开发新型的分子记录 能够基于原位终点重建细胞谱系和分子历史的技术 测量。这些方法利用基因组编辑技术的最新进展，使目标 合成条形码阵列中的突变，可永久且可继承地记录个体的历史 它们基因组中的细胞。重要的是，这里开发的方法与基于成像的读数兼容 信息，并可与原位转录分析相结合，以提供当前的统一视图 单个单元格的状态、过去历史和空间背景。通过应用这些方法，我将绘制出克隆人 小鼠视网膜的结构，调查局部信号对细胞命运决定的影响程度，揭示 视网膜谱系的随机和预先编程的方面，并重建视网膜的单细胞轨迹 祖细胞在经历不同的能力状态时。总之，这些实验提供了 对哺乳动物视网膜细胞命运指定过程的根本新见解，因此将有助于 未来在再生医学方面的努力，以补救错误指定或视网膜退化的情况 神经元导致失明。 我的职业目标是经营一个学术实验室，研究细胞如何在 发展，使用分子记录和合成生物学方法。拟议中的实验将 为我提供视网膜生物学、多路序列FISH技术和单细胞方面的进一步培训 基因组学是实现这一目标所必需的。我和我的导师迈克尔博士制定了一份详细的培训计划 埃洛维茨，帮助我过渡到独立。我将定期与Elowitz博士会面，讨论研究 进展、助学金撰写策略、学生辅导和实验室管理。去实践我的导师之道 技能，我将在实验室监督一名研究生和一名技术人员的工作。为了扩大我的科学研究 网络，我将在每年2-3次的国际会议上介绍我的工作。要寻求更多的指导，请在 在这个项目成功的关键领域，我将与康妮·切普科博士合作并接受他的指导， 视网膜生物学专家，顺序FISH方法的先驱蔡龙博士和 单细胞基因组学。作为加州理工学院生物和生物工程系的一员，我将拥有 接触合成生物学和发育生物学、尖端课程和培训计划的领军人物， 和最先进的核心设施。独立之路奖将提供时间和资源 需要启动一项雄心勃勃的研究计划，重建 哺乳动物的视网膜。

Abstract The long term goal of this project is to describe how the intricate structure of mammalian retina is made during development. To achieve this goal, this proposal focuses on developing novel molecular recording technologies that enable reconstruction of cell lineage and molecular history based on in situ endpoint measurements. These methods leverage recent advances in genome editing technology to make targeted mutations in synthetic barcode arrays that make a permanent and inheritable record of history of individual cells in their genome. Importantly, the methods developed here are compatible with imaging based readout of information and can be combined with in situ transcriptional profiling to provide a unified view of the current state, past history, and spatial context of individual cells. By applying these methods, I will map the clonal structure of the mouse retina, investigate the extent to which local signals affect cell fate decisions, reveal the stochastic versus pre-programmed aspects of retinal lineage, and reconstruct the single cell trajectory of retinal progenitor cells as they proceed through different competence states. Together, these experiments provide fundamental new insight into cell fate specification process in mammalian retina and, therefore, will aid the future efforts in regenerative medicine to remedy conditions in which misspecification or degeneration of retinal neurons lead to blindness. My career goal is to run an academic lab that studies how cells acquire and maintain different identities during development, using molecular recording and synthetic biology approaches. The proposed experiments will provide me with further training in retina biology, multiplexed sequential FISH technology, and single cell genomics that are required for this goal. I have developed a detailed training plan with my mentor, Dr. Michael Elowitz, to help me transition to independence. I will meet regularly with Dr. Elowitz to discuss research progress, strategies for grant writing, student mentorship and lab management. To practice my mentorship skills, I will oversee the work of a graduate student and a technician in the lab. To broaden my scientific network, I will present my work at 2-3 international meetings per year. To seek out additional mentorship, in areas critical to the success of this project, I will collaborate with and receive guidance from Dr. Connie Cepko, expert in retina biology, Dr. Long Cai, pioneer in sequential FISH methods, and Dr. Jay Shendure, expert in single cell genomics. As a member of Caltech Division of Biology and Biological Engineering, I will have access to leaders in synthetic as well as developmental biology, cutting-edge courses and training programs, and state-of-the-art core facilities. The Pathway to Independence Award will provide the time and resources required to initiate an ambitious research program on the reconstruction of developmental history of mammalian retina.