Fig. 5. BiFC Nuclear fluorescence increases as the concentration of Activin is increased; BiFC can reveal endogenous TGF-ß signalling during normal development of Xenopus. (A) Animal pole blastomeres derived from an embryo injected with RNA encoding VNm9-Smad4 and VC155-Smad2, together with RNA encoding ECFP, were exposed to the indicated concentrations of Activin [measured in units/ml (Cooke et al., 1987)] and examined by fluorescence microscopy 4 hours later. Note that as the concentration of Activin increases, nuclei become brighter. Cytoplasmic fluorescence in cells treated with 16 U/ml activin derives from yolk platelets. (B) Quantification of the data in A. The values represent the ratio of nuclear BiFC fluorescence to ECFP fluorescence in the nucleus averaged over 10-15 cells. The error bars represent standard deviations. (C) Endogenous TGFß signalling in the Xenopus embryo revealed by BiFC. Xenopus embryos were injected with 100 pg RNA encoding VNm9-Smad4 and VC155-Smad2 together with RNA encoding ECFP-tagged histone H2B. They were cultured to the early gastrula stage before being bisected, as shown in the diagram, into animal and vegetal halves and observed under the fluorescence microscope. Note low levels of fluorescence largely confined to nuclear membrane in the animal hemisphere of the embryo, and higher levels in the nuclei of cells in the vegetal region.