Wnt signalling in the early chick embryo

Abstract

In amphibians, maternally inherited Wnt modulators are localised to the dorsal side of the fertilised egg and, in cooperation with vegetally expressed factors, define the Nieuwkoop centre, which in turn induces the organizer from adjacent blastomeres. In amniotes such as the chick, localisation of maternal mRNA is unlikely to be involved in early development, as zygotic gene expression begins very early. The question therefore remains about whether, and if so how, canonical Wnt signalling is involved in early amniote development. This study uses the chick as a model. In situ hybridisation of the Wnt ligand Wnt8c and targets; Lef-1 and Axin2, RNA-sequencing analysis of Wnt pathway components and immunohistochemistry of β-catenin, suggests that canonical Wnt signalling is emitted by the area opaca (AO) and received by the marginal zone (MZ) in early pre-streak embryos. When the MZ is ablated, the AO can induce a new MZ in area pellucida (AP) epiblast. We therefore investigated if Wnt underlie this inductive property of the AO. Indeed, Wnt stimulation with BIO, expands, while Wnt inhibition with IWR-1 reduces, the AO inductive effect. Moreover, Wnt activation in whole embryos induces widespread expression of MZ markers in the AP, whereas Wnt inhibition represses them. Furthermore, Wnt activation in isolated AP switches the expression of AP gap-junction protein Gja1 to the MZ- specific homolog, Gjb6. These results suggest that Wnt has an active role in MZ specification. We also show how Wnt inhibition delays and/or inhibit primitive streak formation and embryonic regulation. We examined the involvement of Wnt in organizer/node formation; and we find that in node regeneration experiments BIO caused an increase in the expression of node marker Chordin. We conclude that Wnt-signalling plays several separable roles during patterning of the early embryo and formation of the primitive streak and the organizer in amniotes.