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	Figure 1. Induction of pancreas marker expression from pluripotent ACC. (A) ACC were induced either by injecting one-cell stage embryos with mRNAs coding for VegT, ß-catenin or noggin (top panel), or by incubating dissected ACC in retinoic acid (RA) (middle panel), or a combination of both (bottom panel). ACC were dissected at blastula stage and RA treatment was performed at late gastrula stage. Treated ACC were cultured until the equivalent of tadpole stages, when pancreatic marker expression was analyzed by RT-PCR and whole mount in situ hybridization; (B) RA induces pancreatic marker expression in ACC injected with VegT and ß-catenin mRNA. Additional injection of noggin mRNA leads to an increase in pancreatic marker expression. RT-PCR analysis of pancreas, liver and intestine marker gene expression of ACC treated as described in (A) (figure modified from [15]); CE, control embryo; CC, control ACC; (C) Increasing concentrations of RA (8, 16 or 32 µM) expand pancreatic marker expression in ACC injected with VegT, ß-catenin and noggin mRNA. XPDIp (red, marks exocrine pancreas) and insulin (blue, marks endocrine pancreas) expression was analyzed by whole mount in situ hybridization.
	Figure 2. Generation of ectopic hearts by transplantation of in vitro induced ACC. (A) ACC were dissected at the blastula stage and dissociated by culturing in calcium-free medium. Dissociated cells were reaggregated by incubation in a saline solution containing calcium and activin. A fragment of the reassociated ACC was transplanted into the posterior abdomen of a neurula stage host embryo and developed to an ectopic beating heart at tadpole stage; (B) Tadpole embryo with a beating ectopic heart (white arrow); (C) Young frog displaying an ectopic in vitro induced heart. The ectopic heart is situated in the left abdomen and is filled with red blood cells (white arrow); (D) Internal anatomy of a one-year old frog with an ectopic heart (h) adjacent to the host's intestine. The blood flow from the host’s mesenteric artery (black arrow) to the anterior abdominal vein (white arrow) via the ectopic heart is indicated. (B–D) were reproduced from [37] with permission from The International Journal of Developmental Biology (Int. J. Dev. Biol. 2003, 47, 405–410). Scale bars: (B) 1 mm, (D) 5 mm.
	Figure 3. In vitro induction of functional eyes from ACC. (A) ACC were dissected from blastula stage transgenic embryos constitutively expressing yellow fluorescent protein (YFP). When transplanted into the flanks of neurula stage embryos, these ACC developed into sheets of epidermis; (B) In contrast, ACC injected with mRNA coding for a set of seven different transcription factors, collectively called the eye field transcription factors (EFTF), generated ectopic eyes after transplantation; (C) Tadpole embryo with ectopic EFTF/YFP-expressing eye in close proximity to the gut (indicated by dashed lines). Inlay shows a magnification of the ectopic eye; (D) In situ hybridization showing the expression of the retinal ganglion cell (RGC) marker, hermes, in a section of the ectopic eye. Outer nuclear (ONL), inner nuclear (INL) and ganglion cell layers (GCL) are indicated; RPE, retinal pigment epithelium; (E) Half of an animal cap from an uninjected transgenic embryo is not sufficient to generate an eye, if it is used to replace portions of the eye field; (F) In contrast, half of an animal cap expressing EFTF is able to replace the eye field and generate a functional eye. Control ACC expressing YFP only form epidermis (G), while EFTF-expressing ACC generate an eye (H); Insert in (H) shows a higher magnification of the eye. Sections of the control (I) and induced eye (J) of the same tadpole embryo. YFP fluorescence indicates that the eye originates from donor tissue. Figures (C,D and G–J) were reproduced from a PLoS Biology article by Viczian et al. [42]. Scale bar: (D) 12 µm.

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