Fig. 5.

Order-of-addition experiment showing that Wnt/β-catenin activation at the 32-cell stage is inhibited by Ouabain and can be rescued by subsequent GSK3 inhibition in Xenopus embryos. (A) Untreated embryo. (B) Ouabain incubation (10 μM in 20% L-15 medium for 7 min at the 32-cell stage) resulted in ventralized embryos. (C) LiCl (300 mM for 7 min) dorsalized embryos resulting in expanded heads and reduced trunks. (D) The dorsalized phenotype due to LiCl treatment was blocked after subsequent incubation with Ouabain. (A′-D′) In situ hybridization of Sox2 expression at the gastrula stage showing reduction of CNS development by Ouabain and its expansion by LiCL in dorsal views. The radial expanded CNS phenotype by treating first with LiCl incubation was inhibited by secondary Ouabain incubation. (A″-D″) Anterior views of the same embryos shown in A′-D′. (E-H) Embryos at the 32-cell stage were treated with Ouabain for 7 min (10 µM in 20% L-15 culture medium without serum), washed twice in 0.1 MMR saline, then immersed in 300 mM LiCl in MMR for an additional 7 min, all within the 32-cell stage. Note that treatment with LiCL secondly restored head structures. (E′-H′) In situ hybridizations of the neural marker Sox2 at the gastrula stage show that the ventralized phenotype due to Ouabain was rescued by subsequent LiCl treatment. (E″-H″) Anterior views from the same embryos are shown in panels E′ to H′. (I-I′) Quantitative RT-PCR (qPCR) for the Wnt target genes Siamois and Xnr3 at blastula stage 9 (about 7 h of development), shows that the phenotypic effects are due to the early activation of the Wnt pathway. The numbers of embryos analyzed are as follows: A: 82, 100%; B: 95, 96%; C: 110, 99%; D: 87, 94%; five independent experiments; A′: 33, 100%; B′: 29, 92%; C: 34, 100%; D: 31, 92%; E: 78, 100%; F: 83, 95%; G: 92, 99%; H: 85, 94%; E′: 28, 100%; F′: 33, 96%; G′: 30, 100%; H′: 35, 95%. Scale bars: 500 μm. Error bars denote SEM (n≥3) (**P<0.01).