Spatiotemporal dissection of cell specification processes during gastrulation and early organogenesis

生物学中最基本和最显著的过程之一是在短时间内将单个受精卵转化为多细胞胚胎。在小鼠胚胎中，基本的身体计划在48小时内就完成了。单层多能细胞转变为三层结构（原肠胚形成），产生主体轴和第一器官（早期器官发生）。这一过程背后的分子机制在很大程度上仍不清楚，因为细胞的基因表达谱随时间迅速变化，并且在空间上变化很大。此外，细胞命运决定来自各种细胞外和细胞内信号的相互作用，例如小信号分子、转录因子、受体和表观遗传修饰。单细胞基因组学的最新进展使得能够以前所未有的分子分辨率测量数百万个单细胞的转录组。在第一步，我将联合收割机这些技术与新的实验技术和计算方法，将允许创建准确的时空地图的基因表达的原肠胚。每个单细胞转录状态都补充了胚胎内的时间坐标和位置，相邻时间点的细胞通过轨迹相互连接。这一时空基因表达图将随后作为基础，系统地剖析外在和内在的信号参与细胞分化过程中的胚胎。为此，我将另外使用小鼠胚状体系统，该系统在体外经历原肠胚样分化过程。重点将是开发一个综合模型的原始条纹程序和理解早期中胚层图案。将使用遗传扰动实验来验证特定基因在体内和体外的作用。将通过在其发育期间的特定时间点将胚状体暴露于不同浓度水平来研究细胞外信号传导分子的作用。DNA甲基化水平分析结合DNA甲基化机制的遗传扰动将有助于阐明细胞定型的可能表观遗传驱动因素。我的长期目标是开发发育过程的定量模型，在机械和可解释的框架内容纳大量的单细胞实验数据。

One of the most fundamental and remarkable processes in biology is the transformation of a single fertilized egg into a multi-cellular embryo within a short period of time. In the mouse embryo, the basic body plan is laid out within just 48 hours. A single layer of pluripotent cells transforms into a three-layer structure (gastrulation), that gives rise to the main body axes and first organs (early organogenesis). The molecular mechanisms underlying this process remain largely unclear because gene expression profiles of cells change rapidly in time and additionally vary strongly in space. Moreover, cell fate decisions arise from an interplay of various extra- and intracellular signals such as small signaling molecules, transcription factors, receptors and epigenetic modifications. Recent advances in single-cell genomics enable to measure the transcriptomes of millions of single cells with unprecedented molecular resolution. In a first step, I will combine these technologies with new experimental techniques and computational methods that will permit to create accurate spatiotemporal maps of gene expression of the gastrulating embryo. Each single-cell transcriptional state is supplemented by a time coordinate and position within the embryo and cells of neighboring time points are connected with each other through trajectories. This spatiotemporal gene expression map will subsequently serve as the basis to systematically dissect extrinsic and intrinsic signals involved in cellular differentiation processes in the embryo. To this end, I will additionally use a mouse embryoid body system, that undergoes a gastrulation-like differentiation process in vitro. The key focus will be on developing a comprehensive model of the primitive streak program and on understanding early mesoderm patterning. Genetic perturbation experiments will be used to validate the effect of specific genes in vivo and in vitro. The effect of extracellular signaling molecules will be studied by exposing the embryoid bodies to varying concentration levels at specific time points during their development. DNA methylation level profiling in combination with genetic perturbations of the DNA methylation machinery will help to elucidate possible epigenetic drivers of cellular commitment. My longer-term goal is to develop quantitative models of developmental processes that accommodate massive single-cell experimental data within a mechanistic and interpretable framework.