Fig. 7. Inhibition of Wnt and FGF signaling abolishes Shisa2 morphant phenotype. (A-A''') Whole-mount in situ hybridization of Thy1 probe (dorsal view, anterior toward the top). Two-cell stage embryos that had radially received Gsk3 RNA (40 pg per embryo) (A',A''') were subsequently injected with Shisa2MO1 unilaterally and were treated with DMSO or SU5402. Anterior shift of S-III/Thy1 stripe in MO1-injected side; Gsk3/DMSO, 13/17; SU5402, 10/15, Gsk3/SU5402, 9/25. (B) Schematic diagram showing procedure of dorsal explant assay at stage 14. The dashed line on the left panel and the black bars on the right panel indicate the positions of cuts. bp: blastopore lip. (C-C''') Wholemount in situ hybridization of the dorsal explants with Mes1 probe (dorsal view, anterior toward the top). The explants were generated from embryos unilaterally receiving MO1 at early neurulation (stage 14), treated with SU5402 and/or a recombinant WIF1 protein for 1.5 hours as indicated at the top, and then stained for Mes1 expression. Abolished Mes1 expression in the uninjected side; SU5402, 24/26; WIF1, 12/20; SU5402/WIF1, 27/30. Abolished Mes1 expression in the MO1-injected side; SU5402, 6/26; WIF1, 0/20; SU5402/WIF1, 20/30. (D-E'') The dorsal explants from embryos unilaterally receiving MO1 were treated with SU5402 alone or together with Wnt inhibitors for 1.5 hours, and then stained for Thy1. The treatments are indicated at the top of each panel. Symmetric S-III/Thy1 stripes (indicated by arrowheads in E',E'') were observed in the explants treated with both FGF and Wnt signaling inhibitors but by neither alone (D',D'',E). Anterior shift of S-III/Thy1 stripe in MO1-injected side; BSA/DMSO, 14/16; BSA/SU5402, 14/16; WIF1/DMSO, 20/20; FzCRD/DMSO, 18/22; WIF1/SU5402, 6/20; FzCRD/SU5402, 6/22. Some explants were further cultured for 3 hours after WIF1/SU5402 treatment and fixed. Histological analysis showed the symmetric boundary formation in these explants (n=5/5). (F) Bar graph shows the effect of WIF1 and SU5402 treatment on the Mes1 expression in the dorsal explants. Abolished and maintained Mes1 expression is indicated with an orange and green column, respectively. (G) Bar graph shows the effect of WIF1, FzCRD and SU5402 treatment on the Thy1 expression in the dorsal explants. The distance between S-III/Thy1 stripe of MO1-injected side and that of uninfected side was evaluated as symmetrical (red), one somite distance (blue), two somite distance (gray).