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Fig. 4. Shisa2 controls the position of the wavefront. (A,A′) MO targeting Shisa2 mRNA. (A) Sequences of Shisa2MO1 and MO2. (A′) MO3 was designed to block mRNA splicing. Immature and mature Shisa2 mRNA was detected by RT-PCR using F/R1 and F/R2 primer set, respectively. (B) Four-cell stage embryos were injected with 50 pg of HA-tagged Shisa2 RNA (lane 2) alone or together with Shisa2MOs (lane 3, 5mis, 40 ng per embryo; lane 4, MO1, 40 ng; lane 5, MO2, 20 ng). Lysate prepared from stage 10 embryos and probed with anti-HA antibody (upper panel) or anti-α-tubulin antibody (lower panel). (B′) Four-cell stage embryos were injected with 5mis-MO3 (lane 2, 30 ng per embryo) or MO3 (lane 3, 30 ng per embryo). Total RNA was isolated at stage18 and analyzed by RT-PCR. Note that MO3 injection inhibits splicing of Shisa2 mRNA. (C-Q) Wholemount in situ hybridization (C-N′,P,Q) or immunostaining (O,O′) of the embryos unilaterally injected with MOs (5misMO1, 10 ng; MO1, 10 ng; MO2, 5 ng; MO3, 7.5 ng; 5misMO3, 7.5 ng) into two right side blastomeres at the four-cell stage. (C-E′) MOs injection had no effect on Xbra (C-C′; 5mis, n=5/5; Mo1, n=15/15; Mo2, n=5/5) or MyoD (D-D′; 5mis, n=5/5; Mo1, n=12/12; Mo2, n=5/5) expressions at midgastrulation (stage 11). MyoD expression at early neurula (stage 18) was symmetrical in the MO-injected and uninjected side (E-E′; 5mis, n=15/15; Mo1, n=35/35; Mo2, n=21/21). (F-F′) Anterior shift of Papc; 5mis, n=0/17; MO1, n=44/59; MO2, n=21/21. (G-G′) Anterior shift of Thy1; 5mis, n=8/46; MO1, n=47/58; MO2, n=38/51. (H-H′) Anterior expansion of Mes1; 5mis, n=0/19; MO1, n=56/64; MO2, n=30/34. (I-I′) Anterior expansion of Xbra; 5mis, n=0/26; MO1, n=13/21; MO2, n=21/36. (J-J′) Anterior expansion of FGF8; 5mis, n=0/19; MO1, n=17/22; MO2, n=13/22. (K-K′) Anterior expansion of Wnt3a; 5mis, n=0/12; MO1, n=8/19; MO2, n=15/24. (L,L′) Anterior shift of Thy1; 5mis-MO3, n=0/30; MO3, n=18/36. (M,M′) Anterior expansion of Mes1; 5mis-MO3, n=0/20; MO3, n=17/30. (N,N′) Anterior extention of Arp-A; 5mis MO1, n=0/20; MO1, n=30/45. (O,O′) Anterior expansion of dp-ERK staining; 5mis MO1, n=0/11; MO1, n=12/19. (P) Anterior shift of Cyp26; MO1, n=11/29. (Q) Anterior shift of Rarg; MO1, n=10/21. Arrowheads represent S-II/Papc or S-III/Thy1 stripes. Black bars indicate the anterior border of gene expressions or dp-ERK staining. CPSM/TB, caudal PSM and tailbud; RPSM, rostral PSM. |
