Molecular dissection of Otx1 functional domains in the zebrafish embryo

Otx proteins are involved in the induction of neurectoderm patterning and morphogenetic movements, leading to the formation of the vertebrate central nervous system. Despite lack of homology of sequence outside the homeodomain, a large body of evidence has shown that the Otx/Otd class of proteins has similar functions in many animal phyla. Thus, characterization of functional domains in proteins of this family would help in understanding how this functional equivalence operates. Our previous analysis using the zebrafish embryo (Bellipanni et al., 2000, Dev Biol 223:339-353), has suggested that induction of cell aggregation is a morphoregulatory role of Otx/Otd factors in embryonic development. We now use the induction of cell aggregation as an in vivo assay to examine the functional requirement for particular domains of the zOtx1 protein. We demonstrate that zOtx1 induces cell aggregation by acting as a transcriptional activator through its C-terminal region. Further, we show that a region of 37 amino acids in the C-terminal third of zOtx1 is necessary but not sufficient for this activation potential. The effects of selective deletion of each of the three homeodomain alpha-helices of zOtx1 on cell aggregation were also tested. Surprisingly, we find that helix 3, which is required for binding to DNA, is dispensable for stimulation of cell aggregation. Our results suggest that for transcriptional activation of at least one gene in the cell aggregation pathway, zOtx1 need not bind directly to DNA, but does require helix 1 and 2 of its homeodomain to interact with an as yet undefined DNA binding protein.