A dual role for Axin in establishing the anterior-posterior axis in the early sea urchin embryo

The activation of Wnt/β-catenin (cWnt) signaling at the future posterior end of early embryos is a highly conserved mechanism for initiating pattern formation along the anterior-posterior (AP) axis in bilaterians. Moreover, in many bilaterian taxa, in addition, to activation of cWnt signaling at the posterior end, inhibition of cWnt signaling at the anterior end is required for normal development of anterior structures. In most cases, inhibition of cWnt signaling at the anterior end occurs around gastrulation and it is typically mediated by secreted factors that block signal transduction through the cWnt cell surface receptor-ligand complex. This phenomenon has been fairly well characterized, but the emerging role for intracellular inhibition of cWnt signaling in future anterior blastomeres—in cleavage stage embryos—to regulate correct AP patterning is less well understood. To investigate this process in an invertebrate deuterostome embryo we studied the function of Axin, a critical negative regulator of cWnt signaling, during early sea urchin embryogenesis. Sea urchin Axin is ubiquitously expressed in early embryos and by the blastula stage the expression of the gene becomes restricted to the posterior end of the embryo. Strikingly, knockdown of Axin protein levels using antisense Axin morpholinos (MO) led to ectopic nuclearization of β-catenin and activation of endomesoderm gene expression in anterior blastomeres in early embryos. These embryos developed a severely posteriorized phenotype that could be fully rescued by co-injection of Axin MO with wild-type Axin mRNA. Axin is known to negatively regulate cWnt by its role in mediating β-catenin stability within the destruction complex. Consistent with this function overexpression of Axin by mRNA injection led to the downregulation of nuclear β-catenin, inhibition of endomesoderm specification and a strong anteriorization of embryos. Axin has several well-defined domains that regulate its interaction with β-catenin and the key regulators of the destruction complex, Adenomatous Polyposis Coli (APC), Glycogen Synthase Kinase 3β(GSK-3β), and Dishevelled (Dvl). Using Axin constructs with single deletions of the binding sites for these proteins we showed that only the GSK-3βbinding site on Axin is required for its inhibition of cWnt in the sea urchin embryo. Strikingly, overexpression of the GSK-3β-binding domain alone led to embryos with elevated levels of endomesoderm gene expression and a strongly posteriorized phenotype. These results indicated that Axin has a critical global role in inhibiting cWnt signaling in the early sea urchin embryo, and moreover, that the interaction of Axin with GSK-3βis critical for this inhibition. These results also add to the growing body of evidence that Axin plays a global function in suppressing cWnt signaling in early embryos and indicates that modulation of Axin function may be a critical early step during patterning of the AP axis during bilaterian development