Canonical Wnt signalling from the Area Opaca induces and maintains the Marginal Zone in pre-primitive-streak stage chick embryos

In chick embryos prior to primitive streak formation, the outermost extraembryonic region, known as the area opaca (AO), was generally thought to act only by providing nutrients and mechanical support to the embryo. Just internal to the AO is a ring of epiblast called the marginal zone (MZ), separating the former from the inner, area pellucida epiblast. The MZ does not contribute cells to any part of the embryo but is involved in determining the position of primitive streak formation from the inner epiblast. Recently it was discovered that the AO can induce a MZ from area pellucida epiblast. Here we explore the nature of this inductive signal. We find that Wnt8c is highly expressed in the AO, whereas canonical Wnt pathway targets are enriched in the MZ, along with strong nuclear β-catenin localization. Using isolation and recombination experiments combined with gain- and loss-of-function by exogenous chemical modulators of the pathway, we reveal that Wnt signalling is essential for induction and maintenance of the MZ, as well as sufficient to induce MZ properties in area pellucida epiblast. We propose that canonical Wnt signalling is responsible for induction of the marginal zone by the area opaca.