Determining the cell fate programs of mammalian retina development

项目概要/摘要 该提案的首要目标是了解视网膜的典型结构是如何形成的 在胚胎发育过程中。为了实现这一目标，该提案旨在开发新型分子记录 允许基于端点测量重建谱系历史的技术。这个方法 利用基因组编辑技术在合成条形码中随机创建可遗传的突变 随着时间的推移累积编辑的数组。因为这些数组的读出与空间兼容 转录组学技术，这些方法结合起来将允许同时捕获 视网膜组织内单个细胞的转录细胞状态、谱系关系和空间位置。的 然后，将使用一种名为“谱系基序”的新型统计工具对所得谱系树数据集进行分析 分析，一种计算方法，用于识别所有显着过度或不足的细胞模式。这个 方法系统地列举了在逐渐更大的范围内观察到的命运的所有可能安排 子树，然后将它们的频率与基于不相关单元的空模型中预期的频率进行比较 细胞分裂之间的命运。谱系树将揭示细胞类型的出生顺序如何在克隆上受到调节 水平和谱系图案将揭示视网膜中的谱系随机或预编程的程度。 此外，谱系图案代表了对控制视网膜如何形成的“规则”的直接洞察。 发展，并提供一种方法来描述这些“规则”在不同情况下如何变化，例如疾病或 药理干扰。这里提出的数据集和分析将为新的开发提供信息 再生医学治疗导致失明的视网膜疾病的治疗策略。 这里概述的培训计划将为我提供必要的工具和知识，以便 (1) 执行 该提案的目的并获得新颖的生物学理解，以及（2）推动我实现我的职业目标 领导一个研究小组专注于研究胚胎的基本原理 发育和疾病进展。我将与空间转录组学先驱蔡龙博士合作， 学习成像和图像处理技术，并向神经生物学专家Carlos Lois博士学习 小鼠操作和外科手术。 Elowitz 博士和我将定期会面讨论我的研究 进展、撰写论文出版物和资助计划、教学/指导学生以及机会 在加州理工学院和全国会议上展示我的研究。作为加州理工学院的博士生，我将有机会获得 领导者精通应用定量方法来研究发育生物学，最先进的核心 设施以及生物学和统计学方面的尖端课程。通过资助我其余的博士研究， 这项研究金将使我能够揭示视网膜发育和设定的基本原理 当我在后来的职业生涯中转向成为一名独立调查员时，我准备独立。

Project Summary/Abstract The overarching goal of this proposal is to understand how the stereotypical structure of retina forms during embryonic development. Towards this goal, this proposal seeks to develop novel molecular recording technologies that allow the reconstruction of lineage history based on endpoint measurements. This method leverages genome editing techniques to stochastically create heritable mutations within synthetic barcode arrays that accumulate edits over time. Because readout of these arrays is compatible with spatial transcriptomics technologies, these methods when combined will allow the simultaneous capturing of transcriptional cell state, lineage relationships, and spatial position of single cells within retina tissue. The resulting lineage tree datasets will then be analyzed using a novel statistical tool termed Lineage Motif Analysis, a computational approach to identify all significantly over- or under-represented cellular patterns. This approach systematically enumerates all possible arrangements of observed fates on progressively larger subtrees and then compares their frequencies to those expected in a null model based on uncorrelated cell fate between cell divisions. Lineage trees will reveal how birth-order of cell types is regulated on the clonal level and lineage motifs will reveal the extent to which lineage in the retina is stochastic or preprogrammed. Furthermore, lineage motifs represent direct insight into the “rules” that govern how the retina forms during development, and provide a way to describe how such “rules” change in different contexts like disease or pharmacological perturbation. The datasets and analysis proposed here will inform the development of new therapeutic strategies for regenerative medicine to treat retinal diseases that lead to blindness. My training program outlined here will equip me with the necessary tools and knowledge to (1) carry out the aims of this proposal and gain novel biological understanding, and (2) advance me towards my career goal of leading a research team focused on studying the fundamental principles that underlie embryonic development and disease progression. I will work with Dr. Long Cai, a pioneer in spatial transcriptomics, to learn imaging and image processing techniques, as well as Dr. Carlos Lois, an expert in neurobiology, to learn mouse manipulation and surgical procedures. Dr. Elowitz and I will meet regularly to discuss my research progress, writing plans for paper publications and grants, teaching/mentoring students, and opportunities to present my research at Caltech and national conferences. As a PhD student at Caltech, I will have access to leaders well-versed in applying quantitative approaches to study developmental biology, state-of-the-art core facilities, and cutting-edge coursework in both biology and statistics. By funding the rest of my PhD research, this fellowship will enable me to uncover the fundamental principles that underlie retina development and set me up for independence as I transition towards becoming an independent investigator in my later career.