Phosphorylation states change Otx2 activity for cell proliferation and patterning in the Xenopus embryo

Abstract

The homeodomain transcription factor Otx2 plays essential roles in head and eye formation by negative and positive regulations of its target genes, but it remains elusive how this dual activity of Otx2 plays roles in cellular functions. We first demonstrated that both exogenous and endogenous Otx2 are phosphorylated at multiple sites. Using Xenopus embryos, we identified three possible Cdk sites and one Akt site, and analysed biological activities of phosphomimetic (4E) and non-phosphorylatable (4A) mutants for those sites. In the neuroectoderm, the 4E but not 4A mutant downregulated the Cdk inhibitor gene p27xic1 and posterior genes, and promoted cell proliferation, possibly forming a positive feedback loop consisting of Cdk, Otx2, and p27xic1 for cell proliferation, together with anteriorization. Conversely, the 4A mutant functioned as an activator on its own and upregulated the expression of eye marker genes, and later enlarged eyes. Consistent with these results, the interaction of Otx2 with the corepressor Tle1 is suggested to be phosphorylation-dependent. These data provide the possibility that Otx2 can orchestrate cell proliferation, anteroposterior patterning, and eye formation through its phosphorylation state.