Figure 2. Order-of-addition experiment showing that lysosomal acidification at 32-cell stage is required for early Wnt/β-catenin activation.

(A) Untreated embryo. (B) Baf treatment (5 μM for 7 min) inhibited endogenous axis formation resulting in ventralized embryos lacking heads at early tailbud. (C) Embryos treated with Baf, washed, and then immersed in 300 mM Lithium Chloride (LiCl) an additional 7 min formed heads, resulting in dorsalized embryos with enlarged heads and short trunks. Note that all treatments were done at 32-cell stage, which is critical for the early Wnt signal. (D) LiCl treatment alone dorsalized embryos. (E) Quantitative RT-PCR (qPCR) for Wnt target genes Siamois and Xnr3 at blastula, confirming that the phenotypic effects are due to early activation of the Wnt pathway. (F) Untreated embryo. (G) Embryos incubated with LiCl at 32-cell were dorsalized. (H) Embryos first treated with LiCl and then subsequently with Baf resulted in ventralized embryos lacking heads. (I) Baf treatment caused ventralization and small heads. (J) Quantitative RT-PCR for Wnt target genes Siamois and Xnr3 showing that inhibiting V-ATPAse at the 32-cell stage blocks the effect of earlier LiCl treatment. The numbers of embryos analyzed were as follows: A = 11 5, 100%; B = 124, 98% with phenotype; C = 132, 97%; D = 99%; F = 132, 100%; G = 135, 99%; H = 126, 97%; I = 129, 98% (scale bars, 500 μm.). Error bars denote standard error of the mean (SEM) (n ≥ 3) (**p < 0.01).