Fig. 2.

Inhibition of vesicular acidification mimics loss of function of canonical and non-canonical mediated Wnt effects in vivo. (A–D) Bafilomycin and concanamycin A treatment results in a loss of Fgf8 transcripts in chick mb–r2 explants. These explants were isolated from HH10 chick embryos, embedded in collagen and treated with drug for 24 hours. Explants were probed for the expression of Fgf8 mRNA (A and B, blue; C and D, red) and Wnt1 mRNA (C and D, blue). Explants A and B were processed simultaneously, as were explants C and D. The differences in explant size are consistent with drug-induced inhibition of Wnt secretion and Wnt-dependent loss of fibroblast growth factor (FGF), as these are well-established mitogens in neural development (Canning et al., 2007). (E–K) Bafilomycin and concanamycin lead to a loss of axial patterning in Xenopus laevis embryos. Two-cell embryos were cultured for 24 hours alone (E) or in 1 μM bafilomycin (F,J) or 2 μM concanamycin (G,K). Drug treatment had no effects on embryos assessed at the 32-cell stage (E–G), demonstrating they are healthy and viable during the early stages of development. Drug-induced Wnt-dependent defects are seen as the embryos undergo gastrulation, as seen at the 24-hour time point (H–K). Control embryos (H,I) develop normally, whereas those cultured in the presence of bafilomycin or concanamycin A have disrupted axial development. (L–N) Treatment of whole embryos with 20 mM LiCl rescues the effect of treatment (as above) with 1 μM bafilomycin. (O) The Wnt targets Siamois and Xnr3 are induced by Xenopus Wnt8 but are reduced to 45% and 48%, respectively, in the presence of 1 μM bafilomycin. Animal caps were injected with control (not shown) or Xenopus Wnt8. Gene expression is measured relative to that of ODC, an internal control. (P) Treatment of whole embryos with vesicular acidification inhibitors does not abrogate all signaling events in the early embryo, as assayed by phosphorylation of Smad-1, a read-out of activation of BMP signaling. Actin serves as a loading control. (Q–T) Bafilomycin and concanamycin A treatment inhibits non-canonical-mediated convergent extension movements in Xenopus embryos. (Q) Animal caps dissected from control embryos do not undergo convergent extension movements compared with those treated with activin alone (R). Bafilomycin (S) and concanamycin A (T) treatment inhibits activin-mediated convergence and extension.