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

项目摘要 在哺乳动物的发育过程中，一个单一的细胞产生了成千上万种不同的、具有不同功能的 不同的细胞类型。了解每种细胞类型在发育过程中是如何确定的是核心之一 生物学中对人类健康和再生医学具有深远影响的问题。虽然有很多 我们目前对细胞命运决定是如何做出的理解是基于时间分辨的和 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)，这是一种新的方法，允许在整个过程中连续观察每个细胞 发育过程将填补我们在理解细胞命运转变过程中存在的主要空白 哺乳动物的发育。 在这里，我们建议开发分子记录方法，使并发的、非破坏性的 过去蜂窝事件和当前蜂窝类型的高通量测量。我们最新的方法，DNA 打字机和Engram，使用精确的基因组编辑来记录细胞谱系信息和关键字 细胞基因组的转录信号事件，这些信号与转录组一起在 单细胞水平。在指导K99阶段，我将通过增加血统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)。在我 在R00阶段过渡到独立，我将把分子记录仪平台扩展到并发 捕捉各种关键的细胞活动(目标3)。作为关于DNA打字机和英格拉姆的初步数据 演示，我们在模型开发中进行描述的分子记录处于有利地位 系统。我们预计合成胚胎中的谱系和关键信号事件的分子记录 这些系统将深化我们早期哺乳动物发育的模式。总而言之，我们的建议将成为一个强有力的 基金会，因为我过渡到我的独立和继续开发通用分子记录 站台。

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.