Molecular recording to understand the determinants of cell fate transitions in early development

分子记录以了解早期发育中细胞命运转变的决定因素

• 批准号: 10643190
• 负责人: Junhong Choi
• 金额: $ 11.43万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-01 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10643190
• 关键词: Biological Assay; Biological Models; Biology; Caenorhabditis elegans; Cell Lineage; Cell model; Cells; Complex; DNA; Data; Decision Making; Development; Developmental Biology; Developmental Process; Embryo; Event; Foundations; Gene Activation; Genetic Transcription; Genome; Genomics; Goals; Health; Human; Link; Mammalian Cell; Maps; Measurement; Mentors; Mentorship; Methods; Microscopy; Mission; Modality; Modeling; Molecular; Morphogenesis; Mus; National Human Genome Research Institute; Organism; Outcome; Phase; Positioning Attribute; Process; Recording of previous events; Regenerative Medicine; Research; Resolution; Scientific Inquiry; Signal Transduction; Specific qualifier value; Structure; System; Systems Development; Techniques; Technology; Testing; Transcriptional Activation; career; cell type; cellular development; clinical application; design; epigenomic profiling; experimental study; genome editing; human genomics; improved; in vivo; insight; model development; post-doctoral training; single-cell RNA sequencing; temporal measurement; tool; transcriptome; transcriptomic profiling

Project Summary During mammalian development, a single cell gives rise to thousands of diverse and functionally distinct cell-types. Understanding how each cell-type is determined during development is one of the central questions in biology with far-reaching consequences for human health and regenerative medicine. While much of our current understanding of how cell-fate decisions are made is based on either temporally-resolved and non-destructive methods (e.g., time-lapse microscopy) or high-throughput but destructive genomic assays (e.g., single-cell RNA-seq), a new method that allows continual observation of each cell throughout the developmental process will fill the major gaps existing in our understanding of cell-fate transitions during mammalian development. Here we propose to develop molecular recording methods that enable the concurrent, non-destructive, high-throughput measurements of past cellular events and the current cell-type. Our recent methods, DNA Typewriter and ENGRAM, use precision genome editing to record cell lineage information and key transcriptional signaling events to the cell’s genome, which are recovered along with the transcriptome at the single-cell level. During the mentored K99 phase, I will further improve our methods by increasing the lineage recording efficiency (Aim 1) and testing it in the synthetic mammalian embryo systems (Aim 2). After I transition to independence in the R00 phase, I will expand the molecular recorder platform to concurrently capture diverse key cellular events (Aim 3). As our preliminary data on DNA Typewriter and ENGRAM demonstrate, we are in a strong position to carry out described molecular recording in model development systems. We anticipate that molecular recording of lineage and key signaling events in the synthetic embryo systems will deepen our model of early mammalian development. Together, our proposal will serve as a strong foundation as I transition into my independence and continue developing a general molecular recording platform.

项目概要 在哺乳动物发育过程中，单个细胞产生数以千计的多样化且具有功能的细胞。 不同的细胞类型。了解每种细胞类型在发育过程中是如何决定的是核心之一 对人类健康和再生医学具有深远影响的生物学问题。虽然很多 我们目前对细胞命运决定如何做出的理解是基于暂时解决的和 非破坏性方法（例如延时显微镜）或高通量但破坏性的基因组分析 （例如，单细胞 RNA-seq），一种允许在整个过程中持续观察每个细胞的新方法 发育过程将填补我们对细胞命运转变的理解中存在的主要空白 哺乳动物的发育。 在这里，我们建议开发分子记录方法，以实现并发、非破坏性、 对过去的细胞事件和当前的细胞类型进行高通量测量。我们最近的方法，DNA 打字机和ENGRAM，利用精准基因组编辑记录细胞谱系信息和密钥 细胞基因组的转录信号事件，与转录组一起恢复 单细胞水平。在指导K99阶段，我将通过增加谱系来进一步改进我们的方法 记录效率（目标 1）并在合成哺乳动物胚胎系统中进行测试（目标 2）。之后我 在R00阶段过渡到独立，我将把分子记录器平台扩展到同时 捕获不同的关键细胞事件（目标 3）。作为我们关于 DNA 打字机和 ENGRAM 的初步数据 证明，我们有能力在模型开发中进行所描述的分子记录 系统。我们预计合成胚胎中谱系和关键信号事件的分子记录 系统将深化我们早期哺乳动物发育的模型。总之，我们的提案将成为强有力的 为我过渡到独立并继续开发通用分子记录奠定了基础 平台。