Fig. S1. Cellular organization of an unincubated chick embryo before Brachyury gene expression and experimental setup for subgerminal cavity injection. (A) An unincubated chick embryo. (B) Apical (epiblast-side) view. Embryo fixed in ovo and removed of excessive yolk. (C) Yolk-side view. Black lines indicate regions shown in immunofluorescence stained sections in D and in Fig. 1A. (D) Sagittal section views of regions shown in C, stained for phalloidin (red) and DAPI (cyan). Low-magnification views are shown in Fig. 1A. (E) Sagittal view of an unincubated chick embryo, showing also the subgerminal cavity and the yolk cell. ap, area pellucida; mz, marginal zone; ao, area opaca. (F) Examples of embryos injected subgerminally with a solution containing growth factor and dye. (G) After injection, the egg is filled up with albumen, leaving no air space, covered with a thin plastic film and sealed with plastic rings. (H) Side view. (I) Eggs are incubated horizontally.

Fig. S2. Phenotype of Wnt3a, activin A, FGF2- and FGF8-injected embryos. (A) Wnt3a-injected embryo forms a streak with normal Brachyury expression. (B,C) Activin A-injected embryo stained for Brachyury (B) or Goosecoid (C). Yolk-side view. Goosecoid is strongly induced in a central epiblast pocket by activin A, with its rim region weakly positive for Brachyury. (D) The phenotype of activin A injection. (E) FGF2-injected embryo stained for Brachyury. (F) FGF8-injected embryo stained for Brachyury. ap, area pellucida; ao, area opaca; mz, marginal zone.

Fig. S3. True mesoderm cells are generated in FGF4-induced mesoderm ring. (A) Section of an induced embryo, stained for Brachyury, at the level indicated in Fig. 1C (middle panel), showing three centers of gastrulation. The middle one (1) represents the streak-associated gastrulation movement. The left (2) and right (3) centers represent gastrulation events associated with the induced ring. Ring-associated mesoderm cells are indistinguishable in morphology and migratory behavior from streak-associated ones. (B-E) Time-lapse imaging of gastrulation movement in induced mesoderm ring. The movie is shown in supplementary material Movie 1. (B) Embryo is injected with FGF4 subgerminally, grown in ovo for 10 hours (HH2). It is then electroporated ex ovo with a GFP-expression DNA construct (green), and cultured in New culture setup. Gastrulation movement of cells in the induced ring is imaged using time-lapse fluorescence microscopy. After imaging, the embryo is stained for GFP protein and Brachyury. (C) Whole-mount view of post-imaging embryo with GFP (brown) and Brachyury (blue) staining, showing the endogenous streak, induced ring and regions of GFP-expressing cells. Black lines indicate section levels shown in D (magnified in D′) and E (magnified in E′). Labeled cells are clearly seen in the mesoderm cell population.

Fig. S4. Roles of Wnt and TGFβ pathways in mesoderm ring induction. (A,B) Wnt8C expression in unincubated embryo (A) and incubated (to late HH1) embryo (B). (C,D) Both Vg1 (C) and Nodal (D) are induced in a ring pattern by FGF4 (left, control injection; right, FGF4 injection). (E) Co-injection of FGF4 with a strong TGFβ pathway inhibitor SB431542 still leads to formation of the Brachyury ring. White line indicates section level of F. (F) In this treatment, Brachyury+ cells are present mainly in the epiblast (arrowhead) and few true mesoderm cells migrate away from the ingression site (arrow). (G) Magnified view of F.

Fig. S5. Differentiation of induced mesoderm and the putative endogenous source of FGF. (A) Hemangioblast marker Lmo2. Left, control injection; right, FGF4 injection. (B) Smooth muscle marker dHAND. Left, control injection; right, FGF4 injection; (C) Expression of endogenous FGF8 in a late HH1 stage chicken embryo. Sagittal section shown in Fig. S5C′. (D) Expression of endogenous FGF8 in a late HH1 stage quail embryo. Sagittal section shown in Fig. S5D′. Arrowheads, hypoblast expression; arrows, posterior marginal zone expression. Hypoblast cells close to the posterior marginal zone are strongly positive for FGF8 (arrowheads). Epiblast cells in the posterior marginal zone are initially negative for FGF8 in unincubated embryos (not shown), and at late HH1 also become FGF8 positive (arrows).

Movie 1. Gastrulation-like movement of cells in the induced mesoderm ring. Movie taken as schematized in Fig. S3B. Total duration is ~6 hours (merge of three movies with no time interruption). Total number of frames is 127. Time interval between frames is 3 minutes. Movie taken with the endoderm side of the embryo facing up in New culture setting (embryo in Fig. S5B is shown with ectoderm side upwards). The streak is located in the lower-middle part (not labeled with GFP). Two patches of labeled cells in the induced mesoderm ring can be seen undergoing gastrulation-like movement, by first moving towards and then moving away from presumptive ingression sites.