Molecular link in the sequential induction of the Spemann organizer: direct activation of the cerberus gene by Xlim-1, Xotx2, Mix.1, and Siamois, immediately downstream from Nodal and Wnt signaling

AbstractTo elucidate the molecular basis of organizer functions in Xenopus, we sought the target genes of the LIM homeodomain protein Xlim-1, which is one of the organizer-specific transcriptional activators. We found that an activated form of Xlim-1, Xlim-1/3m, initiates ectopic expression of the head-inducing organizer factor gene cerberus in animal caps. Thus, we analyzed the cerberus promoter using reporter assays. We show that three consecutive TAAT motifs of the homeodomain-binding sites between positions −141 and −118, collectively designated the “3×TAAT element,” are crucial for the response of the cerberus promoter to Xlim-1/3m, and for its activation in the dorsal region of the embryo. Because cooperative activation of the cerberus promoter by Xnr1 and Xwnt8 also requires the 3×TAAT element, we focused on homeodomain transcriptional activators downstream from either Nodal or Wnt signaling. We found that wild-type Xlim-1 synergistically activates the cerberus promoter with Mix.1 and Siamois through the 3×TAAT element, and this synergy requires the LIM domains of Xlim-1. In contrast, Xotx2 acts synergistically with Mix.1 and Siamois through the TAATCT sequence at −95. Electrophoretic mobility shift assays revealed that Xlim-1, Siamois, and Mix.1 are likely to bind as a complex, in a LIM domain-dependent manner, to the region containing the 3×TAAT element. These data suggest that cerberus is a direct target for Xlim-1, Mix.1, Siamois, and Xotx2. Therefore, we propose a model for the molecular link in the inductive sequence from the formation of the organizer to anterior neural induction