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. !