Mechanisms of epiblast and primitive endoderm segregation

摘要 胚胎发育是一个迷人的过程，在此过程中，不同类型的细胞被制造和组织成 复杂的功能性结构，最终构建一个完整的有机体。如果我们观察胚胎发育，模式 以惊人的可重复性出现。然而，如果我们放大到单个细胞的水平，场景， 尤其是在哺乳动物的发育过程中，常常显得杂乱无章。细胞四处移动，改变形状，接触 不同的邻居，并显示其基因表达模式的波动。然而，从这种明显的混乱中， 并出现可复制的模式。如何在正确的时间和地点制造正确的细胞类型 比例？在这个建议中，我们将使用一个关键的细胞命运决定在植入前小鼠胚胎，当 内细胞团（ICM）谱系分离成外胚层（EPI）和原始内胚层（PE）谱系， 将产生大部分胎儿和胚外细胞类型的细胞类型分别作为模型 该系统揭示了分子和细胞机制的基本见解， 可再生的谱系形成。虽然采用遗传学和药理学方法的研究 建立了参与EPI和PE细胞命运的必要分子参与者，这些在提供 关于固有动态过程的时间信息。此外，ICM细胞分化为EPI和PE细胞， 这些基因型看起来是随机的，从一个胚胎到下一个胚胎都是不同的，这使得解释变得复杂起来。 仅在固定时间点进行分析。在这里，我们开发新的基因编码的荧光报告 小鼠品系中的关键因素参与EPI和PE细胞的命运，这使我们能够可视化和探测 细胞命运的采集机制，具有前所未有的空间和时间分辨率，使用 尖端的实时成像、计算图像处理和基因组学方法。利用这些强大的 工具，我们将调查的机制，驱动初始的免疫破坏在ICM启动EPI/PE谱系 区分和确定是否纯粹的随机波动或更可预测的系统变化 在胚胎中存在的是负责启动这个命运的决定。接下来，我们将确定细胞命运如何 在空间和时间上传播，以实现可再现的细胞类型比例，通过独特地可视化多个 小区间通信信令中的节点。最后，我们将使用最近开发的基因组学技术来探测 转录因子占有率和染色质可及性，以观察细胞类型特异性转录因子 计划免疫/体育谱系隔离过程中的关键因素建立了程序。这项工作将揭示基本的 可变的发育过程可以导致稳健的和可重复的发育的机制 在早期哺乳动物胚胎中形成图案。

Abstract Embryonic development is a fascinating process during which different cells types are made and organized into complex and functional structures, ultimately building an entire organism. If we watch embryos develop, patterns emerge with remarkable reproducibility. However, if we zoom in to the level of individual cells, the scene, especially during mammalian development, often seems chaotic. Cells move around, change shape, contact different neighbors and show fluctuations in their gene expression patterns. Yet from this apparent chaos, robust and reproducible patterns emerge. How are the right cell types made at the right time and place in correct proportions? In this proposal we will use a crucial cell fate decision in the preimplantation mouse embryo, when the inner cell mass (ICM) lineage segregates into the epiblast (EPI) and the primitive endoderm (PE) lineages, the cell types that will give rise to most of the fetus and extraembryonic cell types, respectively, as a model system to reveal fundamental insights into the molecular and cellular mechanisms that ensure robust and reproducible lineage formation. While studies employing genetic and pharmacological approaches have established the necessary molecular players involved in EPI and PE cell fates, these are limited in providing temporal information on an inherently dynamic process. Additionally, ICM cells differentiate into EPI and PE cell types in a seemingly random pattern, which varies from one embryo to the next, complicating the interpretation of analysis performed only at fixed time points. Here we develop novel genetically encoded fluorescent reporter mouse lines for key factors involved in EPI and PE cell fates, which allow us to visualize and probe the mechanisms of cell fate acquisition with unprecedented spatial and temporal resolution, using a combination of cutting-edge live imaging, computational image processing, and genomics approaches. Using these powerful tools, we will investigate the mechanism driving initial symmetry-breaking in the ICM to initiate EPI/PE lineage differentiation and determine whether purely stochastic fluctuations or more predictable systematic variations present in the embryo are responsible for initiating this fate decision. Next, we will determine how cell fates are propagated over space and time to achieve reproducible cell-type proportions, by uniquely visualizing multiple nodes in cell-cell communication signaling. Finally, we will use recently developed genomics techniques to probe transcription factor occupancy and chromatin accessibility to observe how cell type-specific transcriptional programs are set up by key factors during EPI/PE lineage segregation. This work will uncover fundamental mechanisms by which variable developmental process can lead to robust and reproducible developmental patterning in the early mammalian embryo.