Fig. 1. Cells memorise activin concentration for at least a few hours. (A) Design of the experiment; results shown in B. Dissociated animal cap cells were treated with a concentration of 4 ng/ml of activin for 10 minutes, either at stage 8.5 or at stage 9, washed to remove the unbound activin, and cultured for various lengths of time (from 1 hour 20 minutes up to 6 hours). The double arrows indicate a 10 minute exposure to activin and the black lane represents the culture time length. (B) Cells remember their activin treatment for over 3 hours. RNAse protection analysis of Xbra expression. WE, whole embryo. Un, untreated cells. (C) Prediction of the Eomes:Apod expression ratio over time, assuming that accumulation of transcripts increases linearly with time, that degradation of transcripts is 30% per hour, that Apod and Eomes mRNAs stabilities are similar, and that Apod transcription is initiated at a quarter the activin concentration required to initiate Eomes transcription. (D) RNAse protection analysis of Apod and Eomes expression. Dissociated animal cap cells were treated with a concentration of 4 ng/ml of activin for 10 minutes, at stage 8.75, washed to remove the unbound activin and cultured for various lengths of time (from 1 hour 20 minutes up to 4 hours). (E) Summary of three separate experiments.The Eomes:Apod expression ratio does not change with time from activin treatment. Dissociated animal cap cells were treated with either of 4 ng/ml or 10 ng/ml of activin for 10 minutes at stage 8.5-9, washed to remove the unbound activin, and cultured for various lengths of time (from 40 min up to 4 hours 40 minutes). The expression of Apod and Eomes was assayed by RNase protection and quantitated. (F) Cells memorise activin concentration in the absence of protein synthesis. Dissociated animal cap cells pretreated (+) or not (-) with cycloheximide for 1 hour, were then treated with a concentration of 4 ng/ml of activin for 10 minutes at stage 9, washed to remove the unbound activin, cultured for 3 hours and gene expression was assayed by RNase protection. The Eomes:Apod expression ratio is shown on the right. (G) Chordin activation is repressed by cycloheximide. Animal caps, pretreated or not with cycloheximide for 1 hour, were then cultured with a concentration of 4 ng/ml of activin until stage 10.5 and gene expression was assayed by RNase protection.