Iro3 induces dorsal and represses ventral markers in a noncell autonomous manner. Iro3 mRNA (200 pg) was injected as indicated at the two- to four-cell stage together with β-gal mRNA, and embryos were fixed at the shield stage (A–H; animal view) or 90% epibody stage (I–K; dorsal view). Organizer markers, lim1 (A and B) and axial (C and D), and ventral markers, bmp2b (E and F) and bmp4 (G and H), were stained purple by in situ hybridization, and red staining identifies the injected region. Lim1 and axial were activated in iro3-injected embryos outside the β-gal-positive region. The intrinsic expression domain (shield) and ectopic domain are indicated by filled and open arrowheads, respectively (A–D). Ventral expression of bmp2b and bmp4 was suppressed in both the injected and uninjected side (F and H), but the dorsal expression domain of the bmp2b and bmp4 was retained (arrowhead). At 90% epibody, the axial-stained notochord was expanded (J) or duplicated (K) in injected embryos.