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Early stages of vertebrate heart development have been linked to Wnt signaling. Here we show in both gain- and loss-of-function experiments that XDbf4, a known regulator of Cdc7 kinase, is an inhibitor of the canonical Wnt signaling pathway. Depletion of endogenous XDbf4 protein did not disturb gastrulation movements or early organizer genes but resulted in embryos with morphologically defective heart and eyes and suppressed cardiac markers. These markers were restored by overexpressed XDbf4, or an XDbf4 mutant that inhibits Wnt signaling but lacks the ability to regulate Cdc7. This indicates that the function of XDbf4 in heart development is independent of its role in the cell cycle. Moreover, our data suggest that XDbf4 acts through the physical and functional interaction with Frodo, a context-dependent regulator of Wnt signaling. These findings establish an unexpected function for a vertebrate Dbf4 homolog and demonstrate the requirement for Wnt inhibition in early cardiac specification.
Figure 1. XDbf4 Associates with Frodo and Is Expressed throughout Xenopus Development(A) Diagram of XDbf4 and XDbf4-δMC with amino acid numbers indicated. Domains N, M, and C conserved between XDbf4 and yeast Dbf4 are shown.(B) XDbf4 and XDbf4-δMC coimmunoprecipitate with Frodo. Lysates of embryos injected with Myc-tagged XDbf4 or XDbf4-δMC (δMC) and HA-tagged FrdB mRNA were precipitated with anti-HA antibody as indicated. Top panel, Myc-XDbf4 and Myc-δMC coprecipitated with FrdB was detected with anti-Myc antibodies; middle panel, precipitated HA-FrdB detected with anti-HA antibodies; bottom panel, Myc-XDbf4 and Myc-δMC detected with anti-Myc antibodies in whole lysates. C, uninjected control embryos.(C–N) In situ hybridization analysis of XDbf4 expression. (C–G, I, and J) XDbf4 antisense probe. (H and K) Chordin antisense probe as a specificity control. (C) Stage 9, animal pole view. (D) Stage 9, lateral view. (E) Stage 10.5, gastrula, dorsal view. (F) Stage 20, late neurula, dorsal view. (G) Stage 22, lateral view. (H) Stage 10, vegetal view. (E and H) Arrowheads point to dorsal lip. (I and K) Stage 26. (J) Stage 35/36. (F, G, and I–K) Anterior is to the left. (L, M, and N) Sections from the regions indicated in (I) and (K); dorsal is up. ha, heart anlage; e, eye vesicle; en, endodermal yolk mass; sa, stomodeal anlage; ms, mesencephalon; fg, foregut; nc, notochord. Bar in L, 200 μm, also corresponds to M and N.(O) Developmental expression of XDbf4 protein. Western analysis of embryo lysates with anti-XDbf4 antibodies was carried out at indicated stages. A vertical bar indicates multiple phosphorylation states of XDbf4; asterisk, a nonspecific protein band reflects loading; M.W., molecular weight markers.
