Fig. 6. The Smad2 flow volume is used by the cells to interpret changing morphogen concentration. (A) Cells can switch from a low dose to a high dose gene response, but not from a high to a low dose gene response. RNase protection analysis of Apod and Eomes expression. Dissociated animal cap cells were treated either with 1 ng/ml of activin for 15 minutes (track 1), or with 4 ng/ml for 15 minutes (track 2), or with 1 ng/ml for 15 minutes and with 4 ng/ml for 15 minutes (track 3), or with 4 ng/ml for 15 minutes and with 1 ng/ml for 15 minutes (track 4). Cells were extensively washed after each dose of activin and cultured until control embryos reached stage 10.5. WE, whole embryo. UN, untreated cells. (B) Design of the experiment; results shown in C. Dissociated animal cap cells from GFP-Smad2-injected embryos were treated with a first dose of activin (1 or 4 ng/ml) for 15 minutes and loaded onto fibronectin substrates. At time T0, a first photo of the cells is taken and the cells receive a second dose of activin (4 or 1 ng/ml) or only buffer. At T0 + 30 minutes, a second photo of the same cells is taken. (C) The amount of nuclear Smad2 increases in response to higher concentrations of activin, but does not decrease with lower concentrations. GFP-Smad2 localisation is observed by confocal microscopy in real time. The nuclear GFP-Smad2 concentration was quantitated using the public-domain NIH Image program and the values are plotted on the bottom graph.
