Maya Allalouf

During early embryogenesis, mechanical constraints and localized biochemical signals co-occur around anteroposterior axis determination and symmetry breaking. Their relative roles, however, are hard to tease apart in vivo. Using Brachyury (Bra), a primitive streak and mesendoderm marker in EBs, we study how contact, biochemical and neighboring cell cues affect the positioning of a primitive streak-like locus, determining the AP axis. We show that a Bra-competent layer must be formed in the EB before Bra expression initiates, and that Bra onset locus position is biased by contact points of the EB with its surrounding, likely through chemical cues modulation rather than by mechanical signaling. We can push or pull Bra onset away from contact points by introducing a separate localized Wnt signal source, maneuver Bra onset to a few loci, or to an isotropic peripheral pattern. Finally, we show Foxa2+ cells are predictive of the future location of Bra onset, demonstrating an earlier symmetry-breaking event. Our analysis of factors affecting symmetry breaking and spatial fate choice during this developmental process may prove valuable for in vitro differentiation and organoid formation.

Abstract During early embryogenesis, mechanical signals, localized biochemical signals and neighboring cell layers interaction coordinate around anteroposterior axis determination and symmetry breaking. Deciphering their relative roles, which are hard to tease apart in vivo, will enhance our understanding of how these processes are driven. In recent years, in vitro 3D models of early mammalian development, such as embryoid bodies (EBs) and gastruloids, were successful in mimicking various aspects of the early embryo, providing high throughput accessible systems for studying the basic rules shaping cell fate and morphology during embryogenesis. Using Brachyury (Bry), a primitive streak and mesendoderm marker in EBs, we study how contact, biochemical and neighboring cell cues affect the positioning of a primitive streak-like locus, determining the AP axis. We show that a Bry-competent layer must be formed in the EB before Bry expression initiates, and that Bry onset locus selection depends on contact points of the EB with its surrounding. We can maneuver Bry onset to occur at a specific locus, a few loci, or in an isotropic peripheral pattern. By spatially separating contact and biochemical signal sources, we show these two modalities can be integrated by the EB to generate a single Bry locus. Finally, we show Foxa2+ cells are predictive of the future location of Bry onset, demonstrating an earlier symmetry-breaking event. By delineating the temporal signaling pathway dependencies of Bry and Foxa2, we were able to selectively abolish either, or spatially decouple the two cell types during EB differentiation. These findings demonstrate multiple inputs integration during an early developmental process, and may prove valuable in directing in vitro differentiation.