Two distinct motifs for Zic-r.a drive specific gene expression in two cell lineages.

Zic-r.a, a maternal transcription factor, specifies posterior fate in ascidian embryos. However, its direct target, Tbx6-r.b, does not contain typical Zic-r.a binding sites in its regulatory region. Using an in vitro selection assay, we found that Zic-r.a binds to sites dissimilar from the canonical motif, by which it activates Tbx6-r.b in a sub-lineage of muscle cells. These sites with non-canonical motifs have weak affinity for Zic-r.a; therefore, it activates Tbx6-r.b only in cells expressing Zic-r.a abundantly. Meanwhile, we found that Zic-r.a expressed zygotically in late embryos activates neural genes through canonical sites. Because different zinc-finger domains of Zic-r.a are important for driving reporters with canonical and non-canonical sites, it is likely that the non-canonical motif is not a divergent version of the canonical motif. In other words, our data indicate that the non-canonical motif represents a motif distinct from the canonical motif. Thus, Zic-r.a recognizes two distinct motifs to activate two sets of genes at two timepoints in development.