Fig. 3. Smad BiFC constructs respond to Activin signaling.

(A) Diagram comparing wild-type VC155-Smad2 with the control protein VC155–Smad2ΔC. (B) The response of these Smad BiFC constructs to Activin. Embryos were injected at the one cell stage with RNA encoding the indicated BiFC reporter constructs, together with RNA encoding mRFP1 as a lineage marker. Animal pole regions were dissected at the midblastula stage and labelled cells were identified by mRFP1 fluorescence (not shown), disaggregated, and plated on a fibronectin coated substrate in the presence or absence of Activin. They were cultured to the equivalent of the early gastrula stage. In the absence of Activin (left-hand column of images) cytoplasmic fluorescence is detectable that derives from Smad2 homodimers (panel 1), Smad2-Smad4 heterodimers (panel 3) and Smad4 homodimers (panel 9). Some auto-fluorescent yolk platelets are detectable in cells expressing VC155-Smad2ΔC rather than VC155-Smad2 (panels 3,7). Note that levels of fluorescence in these samples is exaggerated because the cells are round; Activin treatment (right-hand column of images) causes cells to flatten (Smith et al., 1990). We note that some flattening also occurs in cells co-injected with RNA encoding Smad2 and Smad4 fusion proteins (Fig. 3B5), consistent with the ability of these constructs to activate expression of Xbra and goosecoid (Fig. 2B). In the presence of Activin there is slight accumulation of Smad2 homodimers in the nucleus of responding cells (panel 2) and dramatic accumulation of Smad2-Smad4 heterodimers (panel 6). Smad4 homodimers are excluded from the nucleus (panel 10). Little fluorescence is detectable in cells expressing VC155-Smad2ΔC rather than VC155-Smad2 (panels 4,8). (C) Loss of nuclear BiFC fluorescence by late gastrula stage 13. Animal pole blastomeres were isolated from embryos that had been injected with RNA encoding VC155-Smad2 and VNm9-Smad4 together with RNA encoding ECFP, or from embryos expressing just ECFP. Dissociated blastomeres were left untreated or treated with 16 U/ml Activin for 10 min and then cultured on a fibronectin-coated substrate and observed at the equivalent of stage 10.5 or stage 13. Strong nuclear BiFC fluorescence in response to Activin is detectable at stage 10.5, but by stage 13 this has decreased to the levels observed in untreated cells. Fluorescence levels in cells not expressing Smad-BiFC constructs are shown for comparison.