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Figure 3.
In Vivo Requirement of Shisa in Head Formation
(A) shisa-MO (20 ng) specifically inhibited the translation of overexpressed shisa-Flag RNA (100 pg) in animal halves explanted at gastrula stage. Tubulin and Actin proteins were served as specificity controls.
(B–D) shisa-MO reduced ectodermal otx2 expression but not that of endomesoderm at mid-gastrula (Stage 11.5). Twenty nanograms of shisa-MO (C) or shisa-MO in combination with 10 pg of shisa RNA (D) was injected into both blastomeres at 2-cell stage. Embryos injected with COMO served as a control (B). White arrowheads indicate the prospective head ectoderm.
(E–I) shisa-MO injection reduced expression of otx2 and six3 but not that of sox2 at the end of gastrulation. Twenty nanograms of COMO (F and H) or shisa-MO (E, G, and I), together with nuclear lacZ mRNA (200 pg), were injected at the 2-cell stage into a single blastomere. β-galactosidase activity was visualized by red staining. Probes used were otx2 (E), six3 (F and G), and sox2 (H and I).
(J–L‴) shisa-MO injections suppressed head formation. Lateral (J, J′, K, K′, L, and L′) and frontal (J′, J‴, K′, K‴, L′, and L‴) views of tail bud stage. Embryos were injected with COMO (J–J‴), shisa-MO (K–K‴), or shisa-MO with shisa RNA (L–L‴) as described in (B)–(D). In situ hybridizations with otx2 and krox20 are (J′), (J‴), (K′), (K‴), (L′), and (L‴). White arrows and arrowheads in (J′) and (J‴) indicate otx2 expression in the telencephalon and midbrain, respectively. Black arrows and arrowheads indicate krox20 in the rhombomeres 3 and 5, respectively.
(M–N′) Partially restored otx2 expression in shisa-MO injected embryo at early neurula. shisa-MO was radially injected at 4-cell stage and otx2 expression was analyzed at stage 11.5 (M′) and stage 15 (N′). (M) and (N) give the expression in control embryos.
(O) Wild-type embryo at tadpole stage.
(P) Embryo injected shisa-MO as in (B).
(Q) Animal radial injection of shisa-MO at 4-cell stage.
(R) Vegetal radial injection of shisa-MO. |
