Fig. 6. Interaction of Shisa2 function and RA, Wnt and FGF signaling in the positioning of the wavefront. (A-F) Whole-mount in situ hybridization of Mes1 probe (A-C, posterior view, dorsal toward the top) or Thy1 probe (D-F, dorsal view, anterior toward the top). MO1 was injected as described in Fig. 4. At stage 14, embryos were treated with the indicated drug for 1.5 hours at 22°C and then fixed. (A,D) Embryos treated with DMSO. The unilateral depletion of Shisa2 resulted in the anterior expansion of Mes1, n=20/20, and anterior shift of Thy1 stripes, n=24/30. (B,E) Embryos treated with RA. Knockdown of Shisa2 maintained Mes1 expression (n=20/25) and suppressed the ectopic Thy1 induction (n=28/34) in the caudal PSM. The arrowheads in E and F indicate ectopic Thy1 expression. The RA-mediated enhancement of Thy1 expression in the rostral PSM (white brackets in E) remained intact in the MO1-injected side (n=30/34). (C,F) Embryos treated with SU5402. Knockdown of Shisa2 maintained Mes1 expression (n=32/40) and suppressed the ectopic Thy1 induction in the caudal PSM (n=30/34). (G) Summary of the expression pattern of Thy1 (gray) and Mes1 (green) shown in A-F. eThy1; ectopic Thy1 expression in the caudal PSM. (H) Bar graph shows the effect of RA and SU5402 treatment on the Mes1 expression. Abolished or maintained Mes1 expression is indicated by an orange and green column, respectively. Uninj: uninjected side. (I) Bar graph shows the effect of RA and SU5402 treatment on the Thy1 expression. The distance of S-III/Thy1 stripe between the MO1-injected and uninjected side was evaluated as symmetrical (red), one somite distance (blue), two somite distance (gray). (J-J'') Whole-mount in situ hybridization of Shisa2 probe. The endogenous Shisa2 expression was unaffected by RA (J') or SU5402 (J'') treatment. (K-K'') Whole-mount in situ hybridization of Cyp26 probe. Cyp26 expression was induced by RA (K') and this induction was not inhibited by MO1 injection (K'). Ubiquitous expression of Cyp26; uninjected, n=30/30; MO1 injected n=32/32. (L) RT-PCR analysis of the tailbud explants treated with SU5402. Tailbud region was dissected at stage 15 from the control or embryos radially injected with Shisa2MOs (5mis and MO1, 40 ng per embryo; MO2, 20 ng per embryo) and cultured for 3 hours. SU5402 treatment was carried out for 1.5 hours at the end of the culture period. Note that SU5402 treatment reduced Mes1 expression in the 5misMO-injected explants (lane 2) but in neither the MO1 nor MO2 explants (lanes 3, 4). (M) Western blot analysis of MAPK phosphorylation in whole embryos treated with SU5402 for 1.5 hours. Shisa2MOs were injected as described in J and treated with SU5402 for 1.5 hours from stage 14. MAPK phosphorylation was analyzed by anti-dp-ERK Ab (upper panel) and total MAPK by anti-ERK Ab (lower panel).