Embryonic stem cell differentiation: A key to unraveling the cellular mechanisms of patterning

在受精卵发育成胚胎的过程中，受精卵的形式和功能发生了变化，这一过程的复杂性、复杂性和有效性是生物学中最吸引人和最基本的问题之一。在过去的几十年里，对早期胚胎发育至关重要的遗传网络和信号通路已经被确定并详细描述。然而，单个通路的完全复杂性，特别是它们对时间的相互作用和依赖性，迄今为止阻止了将时间信号线索明确地归因于细胞的特定行为的尝试。该提案提出了一种策略，通过建议一组专门的实验，其中一个定量模型的信号通路的活性和细胞的命运，在细胞培养的人胚胎干细胞（hESC）是适合的，以缩小这一差距。该项目建立在教授的主办实验室的两个最新发现的基础上。美国纽约洛克菲勒大学的Siggia和Brivanlou。在最近的实验中，他们可以证明，将生长因子均匀地应用于圆形限制的干细胞集落足以重建人类胚胎胚层的空间模式，突出了通过细胞间通讯和集落几何形状进行自组织的重要性。在理论方面，Siggia教授的研究小组最近开发了一个框架，用于将一个简约但定量的模型与秀丽隐杆线虫的实验数据拟合，以描述细胞命运决策作为两种信号通路的函数。这种方法避开了分子的途径的细节，并描述了他们的有效行动与最小数量的聚合变量。我们提出了一组实验，将服从上述框架内的量化。人胚胎干细胞将在不同的限制几何形状中生长，并且将使用免疫荧光染色和细胞命运和途径活性的活荧光报告物在细胞分辨率下确定途径（特别是TGF-β和Wnt）的活性以及细胞命运。这些数据将以空间和时间分辨的方式提供信号通路活性与细胞命运的映射，并且还将提供关于导致特定命运模式的细胞间通信的前所未有的信息。一个适用于胚胎干细胞情况的细胞命运的定量数学模型将与数据拟合。这项拟议中的研究不仅将为早期哺乳动物胚胎发育提供独特的见解，特别是在人类中。由于表征的途径在物种之间高度保守，并与成人中广泛的细胞过程相关，因此拟议研究的影响从胚胎发生扩展到器官发生，再生医学和癌症等多种主题。

The complexity, intricacy and efficacy of the process that orchestrates the changes in form and function of the fertilized egg as it develops to the embryo presents one of the most fascinating and fundamental problems of biology. Over the past decades, the genetic networks and signaling pathways that are key to the development of the early embryo have been identified and described in detail. However, the unmitigated complexity of the individual pathways, in particular their interaction and dependence on time have so far prevented attempts to unambiguously attribute temporal signaling cues to a specific behavior of a cell. This proposal presents a strategy for closing this gap by suggesting a dedicated set of experiments to which a quantitative model of signaling pathway activity and cell fate in cell cultures of human embryonic stem cells (hESC) is to be fitted. The project builds on two recent findings of the host laboratories of Profs. Siggia and Brivanlou at Rockefeller University, New York, USA. In recent experiments, they could show that uniform application of growth factors to circularly confined stem cell colonies is sufficient to recreate the spatial patterning into the germ layers of the human embryo, highlighting the importance of self-organization through cell-to-cell communication and the geometrical shape of the colonies. On the theoretical side, Prof. Siggia's group has recently developed a framework for fitting a parsimonious, yet quantitative model to experimental data from Caenorhabditis elegans in order to describe cell fate decision making as a function of two signaling pathways. This approach circumvents the molecular details of the pathways and describes their effective action with a minimal number of aggregate variables. We propose a set of experiments that will be amenable to quantification within the framework described above. Human embryonic stem cells will be grown in different confining geometries and the activity of pathways, in particular TGF-ß and Wnt, as well as the cell fates will be determined at cellular resolution using immunofluorescent staining and live fluorescent reporters of cell fate and pathway activity. This data will provide a mapping of the signaling pathway activity to cell fates in a space and time resolved manner and will also provide unprecedented information about the cell-to-cell communication that leads to a particular fate pattern. A quantitative mathematical model of cell fate adapted to the case of embryonic stem cells will then be fitted to the data. The proposed research not only will provide unique insight into early mammalian embryogenesis, in particular in the human. Because of the characterization of pathways highly conserved across species and relevant for a wide range of cellular processes in the adult, the impact of the proposed research extends from embryogenesis to topics as diverse as organogenesis, regenerative medicine, and cancer.