Mechanisms of epiblast and primitive endoderm segregation

摘要 胚胎发育是一个引人入胜的过程，在这个过程中，不同类型的细胞被制造并组织成 复杂和功能的结构，最终形成一个完整的有机体。如果我们看着胚胎发育，模式 以惊人的重现性出现。然而，如果我们放大到单个细胞的水平，场景， 特别是在哺乳动物的发育过程中，往往看起来很混乱。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，从这一明显的混乱中，稳健 然后出现了可复制的图案。如何在正确的时间和地点制造正确的细胞类型 比例？在这个提案中，我们将在植入前的小鼠胚胎中使用一个关键的细胞命运决定，当 内细胞团(ICM)谱系分为上胚层(EPI)和原始内胚层(PE)谱系， 将产生大多数胎儿和胚胎外细胞类型的细胞类型分别作为模型 系统，揭示分子和细胞机制的基本见解，确保强大和 可繁殖的谱系形成。虽然使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角色，这些在提供 关于内在动态过程的时间信息。此外，ICM细胞可分化为EPI和PE细胞 以一种看似随机的模式，从一个胚胎到下一个胚胎不同，这使得解释变得复杂 只在固定的时间点进行的分析。在这里，我们开发了一种新型的基因编码荧光报告 小鼠寻找与EPI和PE细胞命运有关的关键因素，这使我们能够可视化和探索 以前所未有的时空分辨率获取细胞命运的机制，使用 前沿的实时成像、计算图像处理和基因组学方法。使用这些强大的 工具，我们将研究ICM中最初对称性破缺的机制，以启动EPI/PE谱系 区分并确定是纯粹的随机波动还是更可预测的系统变化 存在于胚胎中的人负责启动这一命运决定。接下来，我们将确定细胞的命运如何 在空间和时间上传播，通过独特的可视化多个 信元间通信信令中的节点。最后，我们将使用最近发展起来的基因组学技术来探索 转录因子占有率和染色质可及性观察细胞类型特异性转录 计划是由计划免疫/PE血统隔离期间的关键因素建立的。这项工作将揭示根本 可变的发育过程可导致强健和可复制的发育的机制 早期哺乳动物胚胎中的花纹。

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.