Changing the Waddington landscape to control mesendoderm competence -- ATAC-seq. Changing the Waddington landscape to control mesendoderm competence -- ATAC-seq

As pluripotent human embryonic stem cells progress towards one germ layer fate, they lose the ability to adopt alternative fates. It is unknown how the cells’ competence for these alternative fates changes along their developmental trajectory or if this competence can be modulated. Here, we show that a differentiating stem cell’s probability of adopting a mesendodermal fate when given the appropriate signal falls sharply at a specific point along the ectodermal trajectory, and we further demonstrate that this point can be moved using genetic perturbations. Using a low-dimensional reaction coordinate to monitor progression towards ectoderm, we can determine the probability that individual cells at different points along this path can transition to the mesendodermal fate upon BMP4 and Activin A signal exposure. Knowing this probability allows us to prospectively isolate and profile differentiating cells based on their mesendoderm competence. Analysis and validation of these RNA-seq and ATAC-seq profiles identified transcription factors that can independently control the cell’s mesendoderm competence and its progression along the ectodermal developmental trajectory. In the classical picture of a Waddington landscape, these effects correspond to altering the barrier between fates and changing the cell’s location on the landscape, respectively. The ability of the underlying gene regulatory network to modulate these two aspects of the developmental landscape could allow separate control of the dynamics of differentiation and tissue size proportions. Overall design: ATAC-seq profiling of hESCs before and after they lose mesendoderm competence during ectoderm-differentiation, alongside a mesendoderm-derived outgroup to aid in the identification of fate-specific changes

当多能人类胚胎干细胞（pluripotent human embryonic stem cells, hESCs）向某一胚层命运分化时，其获取其他胚层命运的能力会逐渐丧失。目前尚不明确细胞对于这些备选胚层命运的分化潜能沿其发育轨迹会发生何种变化，以及该潜能是否可被调控。本研究发现，在接收到适宜诱导信号时，分化干细胞获取中内胚层命运的概率会在外胚层分化轨迹上的特定节点处急剧下降；同时我们进一步证实，该节点可通过遗传扰动手段发生偏移。我们借助低维反应坐标监测细胞向外胚层的分化进程，可判定该轨迹上不同节点的单个细胞，在暴露于骨形态发生蛋白4（BMP4）与激活素A（Activin A）信号后，向中内胚层命运转化的概率。基于该概率，我们可根据细胞的中内胚层分化潜能，前瞻性地分离并分析分化中的干细胞。通过对上述RNA测序（RNA-seq）与转座酶可及性测序（ATAC-seq）数据进行分析与验证，我们鉴定出可独立调控细胞中内胚层分化潜能，以及其在外胚层发育轨迹上的分化进程的转录因子。在经典的沃丁顿表观遗传景观（Waddington landscape）模型中，上述两种效应分别对应改变命运间的表观遗传壁垒，以及调整细胞在景观中的位置。下游基因调控网络对发育景观这两个维度的调控能力，可实现对分化动力学与组织大小比例的独立调控。总体设计：对外胚层分化过程中丢失中内胚层分化潜能前后的hESCs进行ATAC-seq分析，并设置中内胚层来源的外类群，以辅助鉴定命运特异性的表观遗传变化。