Dkk1 is a potent inducer of heart development in non-cardiogenic mesoderm, as demonstrated by the formation of ectopic hearts in normally non-cardogenic ventroposterior marginal zone (VMZ) explants of Xenopus
embryos (Foley and Mercola, 2005
; Schneider and Mercola, 2001
), in posterior lateral plate mesoderm of chick embryos (Marvin et al., 2001
), and in embryonic stem cell (ESC) cultures (Naito et al., 2006
). Other secreted Wnt antagonists, such as Wnt inhibitory factor-1 (WIF-1), Crescent, or Frz-B, are generally less potent inducers, whereas intracellular inhibitors of canonical Wnt signaling, such as GSK3β or a dominant negative version of TCF3, initiate cardiogenesis but do not stimulate the formation of beating heart muscle (Foley et al., 2006
; Marvin et al., 2001
; Schneider and Mercola, 2001
). Loss of Dkk1 function in early mouse embryos causes anencephaly (Mukhopadhyay et al., 2001
) and over-expression studies using Xenopus
and zebrafish embryos (Glinka et al., 1998
; Kazanskaya, 2000
; Shinya et al., 2000
) have confirmed its ability to induce head and anterior structures. Secondary body axes induced by combination of BMP inhibition and Dkk1 expression typically have ectopic heads with normally-positioned bilateral eyes, whereas similar expression of Frz-B, dominant negative Wnt8, or Cerberus yields cyclopic heads (Kazanskaya, 2000
), suggesting that important differences exist between these Wnt antagonists and Dkk1. Here we investigated whether the early developmental inductive and patterning properties of Dkk1 are due to a novel activity that is independent of Wnt-antagonism.
Dkk1 consists of conserved amino-terminal (N1) and carboxy-terminal (C1) cysteine-rich regions. Antagonism of canonical Wnt signaling occurs through binding of C1 to LRP5/6 proteins on the surface of the cell and subsequent disruption of the cell surface Wnt/LRP5/6/Frizzled signaling complex (reviewed in Niehrs, 2006
). Other secreted Wnt antagonists, such as WIF-1, Crescent, and Frz-B, function by binding and sequestering secreted Wnt proteins. The signaling properties of Wnts and Wnt antagonists have been extensively characterized (reviewed in Logan and Nusse, 2004
; Niehrs, 2006
). One difference that has emerged between Dkk1 (and Dkk2 and 4) and other Wnt antagonists is that, by binding LRP5/6, Dkk1/2/4 could bias signaling toward the non-canonical Wnt planar cell polarity (PCP) pathway that involves Frizzled but not LRP receptors. Another, but not mutually exclusive, explanation for Dkk1’s distinct activity is that the amino terminal cysteine-rich domain of Dkk1 (N1) might harbor a new activity that could complement the canonical Wnt antagonizing properties of C1 and trigger signaling that is required for the full patterning and morphogenetic effects of the intact protein. N1 lacks Wnt antagonizing activity (Brott and Sokol, 2002
) and no known signaling or biological function has been ascribed to this domain.
We found that the early embryological activity of Dkk1 indeed requires a novel activity that resides within the N1 domain. Deletion of the N1 domain impaired development of axial mesoderm, yet, when expressed as independent proteins, N1 synergized with C1 and other Wnt antagonists to promote development of chordal and prechordal mesoderm. N1 also synergized robustly with C1 and other Wnt antagonists to induce ectopic heart formation in non-cardiogenic mesoderm. These studies demonstrate that Dkk1 is a dual-function protein and that the C1 and N1 domains activate distinct signaling pathways. Since Dkk1 is not normally cleaved to yield independent amino- and carboxyl-terminal domains, we propose that Wnt inhibition and the novel N1 activity are needed in the intact protein for the normal induction of the heart and anterior structures.