The endoderm is the cell lineage which gives rise in the 1 embryo to the organs of the respiratory and gastrointestinal 2 system. In the mouse this may be the germ layer with the 3 strongest association with its extraembryonic counterpart. 4 Uniquely indeed, endodermal tissue does not just derive from 5 descendants of the embryo proper (the epiblast) but instead 6 arises from their gradual incorporation into an extraembryonic 7 substrate (the visceral endoderm). Given the configuration of 8 the early embryo, such a paradigm requires epiblast endoder-9 mal progenitors to negotiate embryonic compartments with 10 very diverse epithelial character, a developmental contingency 11 reflected by the fact that key early endodermal markers such 12 as Foxa2 and Sox17 have been consistently found to be em-13 bedded within gene programmes involved in epithelialisation. 14 15 To explore the underlying cell biology of embryonic endo-16 derm precursors, and to explore the relationship between 17 endoderm development, epithelial identity, and extraembry-18 onic mixing, we leveraged Gastruloids, in vitro models of 19 early development. These self-organising three-dimensional 20 aggregates of mouse embryonic stem cells do not possess an 21 extraembryonic component, nor do they appear to display 22 typical tissue architecture. Yet, they generate cells expressing 23 endodermal markers. By tracking these cells throughout in 24 vitro development, we highlight a persistent and uninterrupted 25 pairing between epithelial and endodermal identity, with 26 FoxA2+/Sox17+ endoderm progenitors never transitioning 27 through mesenchymal intermediates and never leaving the 28 epithelial compartment in which they arise. We also docu-29 ment the dramatic morphogenesis of these progenitors into a 30 macroscopic epithelial primordium extending along the entire 31 anterior-posterior axis of the Gastruloid, patterned into broad 32 domains of gene expression. Corollarily we thus also reveal 33 an underappreciated epithelial component in Gastruloids, 34 and thus the spontaneous emergence in vitro of stratified 35 architectures and germ layer compartmentalisation. 36 gastruloid | endoderm development | epithelium | self-organisation 37 Correspondence: stefano.vianello@epfl.ch and matthias.lutolf@epfl.ch 38 76 fiers somehow oxymoronic, "extraembryonic" cells (i.e. cells 77 that segregated away from the epiblast early on even before 78 implantation) also contribute to the embryo, and specifically 79 to its endoderm-derived tissues. Indeed, the epiblast is not 80 isolated from other tissues within the conceptus, and it is 81 actually enveloped by a thin epithelium of so-called Visceral 82
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