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Fig. 2 Gene expression patterns around the grafts. Embryos are oriented with the animal pole to the top, except for E and I. Unless otherwise described, a black arrowhead, a red arrow and a dotted line indicate a grafted tissue, a dorsal midline of the host and the blastoderm margin, respectively. In all lateral views, dorsal is to the right. (A-E) Gastrula embryos grafted with the chicken Hensen’s node (HN, stage 4) at the shield stage (6 h) in the ventral region. (A) Lateral view of the embryo 4 hours after transplantation (late gastrula). Lateral (B) and ventral views (C) of the same host hybridized with otx2 probe. Ectopic expression of otx2 appears around the grafted HN with a sharp posterior boundary at the same latitude as that of host endogenous expression. (D) Ventral view of the host hybridized with otx2 probe, in which the graft is located more posteriorly (at the level of the hindbrain on the dorsal side) as compared with the sample shown in B and C. Ectopic expression is induced in the region just anterior to the graft but its posterior boundary remains unchanged with respect to the host endogenous expression. (E) Longitudinal section along the secondary axis induced by HN. The histological section shows neural induction as indicated by the thickened epithelium (e) around the grafted HN (arrowhead). The hypoblast (hp) and yolk syncytial nuclei (yn) are also seen in this section. (F,G) Lateral views of the host which received the HN at midblastula stage (4 h) and examined with otx2 expression at the early gastrula stage (70% epiboly, 7 h). The ectopic expression domain, which is fused with the endogenous one at the animal pole, shares the same posterior boundary as the host domain (F). By contrast, when HN of the same stage was grafted near the blastoderm margin and located in the host trunk region, no ectopic expression of otx2 is observed (G). (H-N) Host embryos that received biotin-labelled embryonic shields at early gastrula stage (6 h) in the ventral region. (H) Lateral view of the late gastrula (10 h) hybridized with otx2 probe. Ectopic expression is induced with a sharp posterior boundary at the same level as that of host endogenous expression domain. (I) Oblique animal-pole view of the hybridized sample (shown in H) in which donor cells were visualized by biotinperoxidase staining. Donor cells (brown, arrowhead) are widely distributed along the A-P axis but ectopic otx2 expression is observed only in the anterior region. (J,K) Longitudinal section and high magnification view of the host shown in I. Red arrowheads indicate the posterior boundaries of otx2 expression along both axes, showing that they are located at the same level (the secondary axis is to the left). The labelled donor cells are distributed along the A-P axis mainly in the underlying hypoblast. (L) Lateral view of the host stained with otx2, in which the donor cells (brown, arrowhead) are located in the trunk region by posterior transplantation. No induction of otx2 expression is detected. (M,N) Lateral views of the embryos hybridized with otx2 and krox-20 probes (5-somite stage, 12 h). Arrowheads indicate the anterior limit of transplanted donor cells (brown). Both otx2 and krox-20 are induced when the donor cells are located in the anterior region (M), while only krox-20 is induced when they are posteriorly located (N). (O-R) Gastrula embryos transplanted with Noggin/Chordin COS7 cells at the midblastula stage (sphere to dome stage). Lateral (O) and ventral views (P) of the host (10 h) hybridized with otx2 probe, and lateral (Q) and ventral views (R) of the host (6.5 h) hybridized with hoxa-1 probe are shown. Note that each ectopic expression domain shares the same boundary as the host domain. Scale bars, 100 μm.