Essential role of Dkk3 for head formation by inhibiting Wnt/β-catenin and Nodal/Vg1 signaling pathways in the basal chordate amphioxus.

To dissect the molecular mechanism of head specification in the basal chordate amphioxus, we investigated the function of Dkk3, a secreted protein in the Dickkopf family, which is expressed anteriorly in early embryos. Amphioxus Dkk3 has three domains characteristic of Dkk3 proteins-an N-terminal serine rich domain and two C-terminal cysteine-rich domains (CRDs). In addition, amphioxus Dkk3 has a TGFβ-receptor 2 domain, which is not present in Dkk3 proteins of other species. As vertebrate Dkk3 proteins have been reported to regulate either Nodal signaling or Wnt/β-catenin signaling but not both in the same species, we tested the effects of Dkk3 on signaling by these two pathways in amphioxus embryos. Loss of function experiments with an anti-sense morpholino oligonucleotide (MO) against amphioxus Dkk3 resulted in larvae with truncated heads and concomitant loss of expression of anterior gene markers. The resemblance of the headless phenotype to that from upregulation of Wnt/β-catenin signaling with BIO, a GSK3β inhibitor, suggested that Dkk3 might inhibit Wnt/β-catenin signaling. In addition, the Dkk3 MO rescued dorsal structures in amphioxus embryos treated with SB505124, an inhibitor of Nodal signaling, indicating that amphioxus Dkk3 can also inhibit Nodal signaling. In vitro assays in Xenopus animal caps showed that Nodal inhibition is largely due to domains other than the TGFβ domain. We conclude that amphioxus Dkk3 regulates head formation by modulating both Wnt/β-catenin and Nodal signaling, and that these functions may have been partitioned among various vertebrate lineages during evolution of Dkk3 proteins.