Identification and evolution of molecular domains involved in differentiating the cement gland-promoting activity of Otx proteins in Xenopus laevis.

Otx genes are a class of vertebrate homeobox genes, homologous to the orthodenticle gene of Drosophila melanogaster, that play a crucial role in anterior embryo patterning and sensory organ formation. In the frog, Xenopus laevis, at least three members of this class have been isolated: otx1, otx2 and otx5 (crx); they are involved in regulating both shared and differential processes during frog development. In particular, while otx2 and otx5 are both capable to promote cement gland (CG) formation, otx1 is not. We performed a molecular dissection of Otx5 and Otx1 proteins to characterize the functional parts of the proteins that make them differently able to promote CG formation. We show that a CG promoting domain (CGPD) is localized at the Otx5 C-terminus, and is bipartite: CGPD1 (aa210-255) is the most effective domain, while CGPD2 (aa177-209) has a lower activity. A histidine stretch disrupts CGPD1 continuity in Otx1 determining its loss of CG promoting activity; this histidine-rich region acts as an actively CG repressing domain. Another Otx1 specific domain, a serine-rich stretch, may also be involved in repressing Otx1 potential to trigger CG formation, though at a much lower level. This is the first evidence that these domains, specific of the Otx1 orthology group, play a role during development in differentiating Otx1 action compared to other Otx family members. We discuss the potential implications of their appearance in light of the evolution of Otx functional activities.