MEMOIR: Recording, and in situ readout of cell lineage and transcriptional history

摘要 单个细胞的细胞命运、谱系和分子历史如何控制细胞的发育和分化？ 组织的生理功能这个问题在整个生物医学科学中至关重要。 然而，进展长期以来受到两个看似棘手和相互关联的挑战的限制： 仍然没有方法来确定发育组织中细胞之间的谱系关系树， 本土的空间背景。其次，我们无法确定细胞外信号的序列， 在发育组织中每个细胞在分化时经历的细胞内分子事件。延时 成像已经成为对生命系统中的生物过程进行成像的主要方法。然而，一个大的 生物学和医学中的许多系统不允许活细胞成像方法，因为它们 难以接近或光学不透明的，如小鼠大脑和胚胎。因此，迫切需要发展 强大的新方法，可以实现从静态推断谱系信息和细胞事件历史， 终点测量。在这里，我们建议开发一个平台，使细胞能够记录谱系， 在它们自己的基因组中，在复杂的发育组织中，动态的基因表达历史。这个平台将 联合收割机结合了两种最近开发的工具：第一，基因组编辑工具，可以“记录”谱系信息或 第二，基于单分子显微镜的原位技术， 顺序多重荧光原位杂交（seqFISH），可读出记录的信息 在单个细胞中，而不需要将它们从组织中分离出来。我们将此方法称为MEMOIR（Memory through） 用光学原位读出增强的诱变）。类似于使用序列变异构建系统发育树 分析，这种方法将使我们能够重建细胞群体的谱系树，以及 基于掺入靶中的突变的层次，在那些细胞内发信号通知事件历史 地区我们已经在小鼠胚胎干细胞（mESC）中进行了原理验证实验， 将这项工作扩展到小鼠胚胎。我们设想MEMOIR将允许我们绘制血统和信号 直接在复杂组织中发生的事件，如脑和转移性肿瘤中的轨道细胞。

Abstract How do the cell fates, lineages, and molecular histories of individual cells control the development and physiological function of tissues? This problem is of central importance throughout biomedical science. However, progress has long been limited by two seemingly intractable, and inter-related, challenges: First, we still have no method to determine the tree of lineage relationships among cells in developing tissues within their native spatial context. Second, we have no way to determine the sequence of extracellular signals and intracellular molecular events experienced by each cell in the developing tissue as it differentiates. Time-lapse imaging has been the principal go-to method to image biological processes in living systems. However, a large number of systems in biology and medicine do not permit live cell imaging methods because they are inaccessible or optically opaque, such as mouse brains and embryos. Thus, there is a crucial need to develop powerful new methods that can achieve inference of lineage information and cellular event histories from static end-point measurements. Here we propose to develop a platform that enables cells to record lineage and dynamic gene expression histories in their own genomes, within complex developing tissues. This platform will combine two recently developed tools: first, genome editing tools that can “record” lineage information or cellular events into the genome, and second, a single molecule microscopy based in situ technology called sequential multiplexed Fluorescence In situ Hybridization (seqFISH) that can read out the recorded information in single cells without dissecting them out from tissues. We call this method MEMOIR (Memory through Enhanced Mutagenesis with Optical In situ Readout). Akin to using sequence variation for phylogenetic tree analysis, this method will allow us to reconstruct the lineage tree for a population of cells as well as the signaling event history within those cells based on the hierarchy of mutations incorporated into the target region. We have performed the proof-of-principle experiments in mouse embryonic stem cells (mESCs) and will extend the work to mouse embryos. We envision that MEMOIR will allow us to map lineages and signaling events directly in complex tissues such as the brain and track cells in metastatic tumors.