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Activin induces the expression of different genes in a concentration-dependent manner. In this paper, we show that the initial response of cells to activin, whether assayed in dispersed cells or in a bead-implantation regime in intact animal caps, is to activate expression of both Xbra and goosecoid. However, differential expression of the two genes, with down-regulation of Xbra, occurs very rapidly and certainly within 3 h of the initial phase of expression. This rapid refinement of gene expression can occur in dispersed cells and thus does not require cell-cell interactions. Refinement of gene expression does, however, require protein synthesis but not goosecoid function. Together, our results place the burden of threshold formation not on the initial induction of different genes but on regulatory interactions between the genes once they have been activated.
FIG. 1. Time course of induction of Xbra and goosecoid after activin treatment. Quantitation by RNase protection of RNA extracted from
(A, C) aggregates of cells or (B, D) dispersed cells at 2 (A, B) or 5 (C, D) hours after activin treatment. Expression levels are normalised to
those of EF1a. These data are representative of at least three such experiments.
FIG. 2. Spatial expression patterns of Xbra and goosecoid in activin bead sandwiches at different time points. Activin beads incubated in
0.2 units/ml activin were sandwiched between two animal caps. Conjugates were cultured for 2 h (AâD) or 5 h (EâH) until control embryos
had reached stage 10.5 or 12. They were then fixed and assayed for expression of Xbra (A, B, E, F) or goosecoid (C, D, G, H) by in situ
hybridisation. Two different conjugates are shown for each condition and are representative of at least 20 samples. Each conjugate has been
bisected with a tungsten needle after in situ hybridisation; dashed white circles indicate the position of the bead. Note that Xbra and
goosecoid are expressed in similar domains in AâD but have resolved to their definitive expression patterns in EâH.
FIG. 3. (A) Down-regulation of Xbra expression at high doses of
activin requires protein synthesis. RNase protection analysis of
cells treated with different concentrations of activin in the presence
or the absence of cycloheximide and then cultured for 5 h.
Expression levels are normalised to those of EF1a. These data are
representative of four such experiments. Note that cycloheximide
prevents the down-regulation of Xbra expression that is normally
observed at high levels of activin. (B) Interference with goosecoid
function is not sufficient to prevent down-regulation of Xbra at
high activin doses. Embryo at the one-cell stage were injected with
1 ng RNA encoding Gsc-VP16 and the response of animal cap cells
derived from such embryos was compared with that of caps from
uninjected embryos. The Xbra expression level is normalised to
that of EF1a. These data are representative of three such experiments.
Note that Gsc-VP-16 does not prevent the down-regulation
of Xbra expression that is normally observed at high levels of
activin.
