Micropattern differentiation and morphogenesis of the human ectoderm

项目总结/摘要 如何准确的细胞命运规范发生在动态组织尺度的背景下 基因重排是发育生物学中最令人兴奋的问题之一。在 我们的这个提议旨在破译命运获得之间的相互作用， 外胚层胚层的形成和形态发生 人类神经发育我们最近开发了一种强大的协议， 用于产生极其可重复的人类神经元样细胞：自组织 人类胚胎干细胞（hESC）组装， 外胚层隔室在神经形成阶段通过组织神经，神经 在同一群体内的嵴、基板和表皮种群 微图案这种自组织是非常可复制的，可以 以亚细胞分辨率和真实的时间对数百个菌落进行定量。 有了这项新技术，我们提出了三个具体目标。一是 解开细胞间信号传导驱动自我组织的机制。第二 是将信号传导与命运获得和形态发生通过肝脏整合在一起， 报告子成像和时间依赖性单细胞RNAseq。最后，第三个目标 重点是外胚层的起源和亚群的充分表征 衍生物及其体内验证，通过与 阶段匹配绒猴胚胎样品和鸡体内移植实验 胚胎大量同质的人类神经元样细胞的产生， 外胚层命运的自组织可以动态地持续一段时间 一个星期，与亚细胞分辨率，不仅解决了固有的 在脑类器官中观察到的异质性，但为我们提供了一个独特的 在人类胚胎模型中研究这些事件的机会。这将产生 在基础研究和临床应用中具有较高影响力，具有前景 已经在地平线上。 !

Project Summary/Abstract How accurate cell fate specification occurs in the context of dynamic tissue-scale rearrangements is one of the most exciting questions in developmental biology. In this proposal we aim at deciphering the interplay between fate acquisition, patterning and morphogenesis of the ectodermal germ layer in the context of human neurulation. We have recently developed a robust protocol which allows for the generation of extremely reproducible human neuruloids: self-organized human Embryonic Stem Cell (hESC) assemblies that recapitulate the organization of the ectodermal compartment at neurulation stages by organizing neural, neural crest, placodes and epidermis populations within the same colony on adhesive micropatterns. This self-organization is extremely reproducible and can be quantified with sub-cellular resolution and in real time over hundreds of colonies. Armed with this novel technology, we propose three specific aims. The first is to unravel the mechanism of cell-cell signaling driving self-organization. The second is to integrate signaling with fate acquisition and morphogenesis through live reporter imaging and time dependent single cell RNAseq. Finally, the third aim focuses on the full characterization of the origin and sub-populations of ectodermal derivatives and their in vivo validation by performing side by side comparisons with stage-matched marmoset fetal samples and grafting experiments in chick embryos. The generation of large numbers of homogenous human neuruloids, where self-organization of ectodermal fate can be followed dynamically for a period of one week, with sub-cellular resolution, not only solves the inherent heterogeneity observed in cerebral organoids, but provides us a unique opportunity to study these events in models of human embryos. This will have a high impact in both basic research as well as clinical application, a prospect already on the horizon. !