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Xenopus blastula cells show a morphogen-like response to activin by expressing different genes according to the concentration of activin to which they are exposed. To understand how cells recognize their position in a concentration gradient, it is essential to know whether each cell responds individually to activin concentration. An alternative idea, proposed by previous work, is that cells need to interact with their neighbours to generate a concentration-related response. To distinguish between these ideas, we have cultured blastula cells under conditions which provide different degrees of contact with other cells, allowing nil to maximum communication with their neighbours. The cultures include cells attached to fibronectin and cells resting unattached on an agarose base. The cultures also include cells that have no contact with any cell except their clonal progeny, cells that have lateral contact to neighbouring cells, and cells that are completely enveloped by other cells in a reaggregate. We have used RNase protection and in situ hybridization to assay the expression of the activin-responsive Xenopus genes Xbra, Xgsc, Xeomes, Xapod, Xchordin, Mix1, Xlim1 and Cerberus. We find no difference in gene expression between cells attached to fibronectin and those unattached on agarose. Most importantly, we find that cells respond to activin in a concentration-related way irrespective of their degree of contact with other cells. Therefore interaction among cells is not required for the interpretation of morphogen concentration, at least in the case of the early genes studied here. We conclude that isolated blastula cells can sense and respond individually to activin by expressing genes in a concentration-dependent way.
Fig. 1. Configurations of cell culture. Cells from stage 8.5-9 animal caps are dissociated, incubated in activin at the required concentration and washed, as described by Dyson and Gurdon (1998). The two substrates used are 1% agarose or fibronectin (see Materials and Methods). In all cases the MBS culture medium contains Ca2+, Mg2+ and 0.1% bovine serum albumin. (A) Single cells on agarose are distributed sparsely so that, initially, nearly all cells are present as single cells or as sister pairs (inset). After culture, each cell or sister-pair has formed well separated clonal groups of 2, 4 or 8 cells or, more often, has remained as a single cell. (B) To form reaggregates, several thousand cells are centrifuged into a pellet, which is then transferred to an agarose substrate. (C-E) Cells are distributed at low (C), medium (D) or high density (E) on to a fibronectin substrate to which they attach. The righthand column for Figs C-E show cells stained with rhodamine-phalloidin and TOTO-3 (see Methods). At the equivalent of stage 10.5, cells are frozen or fixed.
Fig. 2. RNase protection analysis of gene expression under low density culture conditions. Xbra is activated maximally at 0.45% activin. Xgsc and Xeomes reach maximum activation at 1.35% activin. WE, whole embryo.
Fig. 3. Graphic representation of gene activation response to activin concentration, for (A) low density and (B) reaggregated cell cultures. The values shown were obtained by phosphorimager assessment of gel analyses by RNase protection. B is taken from Dyson and Gurdon (1998, with permission), since the results shown in A and B are from the same experimental series. Comparable results were obtained in two other experimental series.
Fig. 4. Single cell analysis of gene activation in low density cell cultures. Low density preparations of cells attached to a fibronectin substrate
(as in Fig. 1C) were in situ-hybridized to the probes stated (Xbra, Xapod, Xeomes and Xchordin). The colour reaction is BM purple. Each figure shows a representative clonal colony of 1-4 cells, fixed at stage 10.5. The + or – values under each figure are based on densitometry readings (see Materials and Methods). −, background value; ±, density value 2; +, ++, +++, density values 3, 4 and 5 or more respectively (see Fig. 6). Altogether seven independent experimental series gave results similar to these (see text)
Fig. 5. Low-power views of in situ hybridization to low density cultures on fibronectin slides. There is a uniformity of gene expression among cells at each activin concentration, although these low magnification views do not reveal the more precise differences in staining intensity seen in Fig. 4, and quantitated in Fig. 6
Fig. 6. Summary of in situ hybridization results with low density cell preparations on a fibronectin substrate. For each probe and for each concentration, 40 single cells were scored for the intensity of stain (see Materials and Methods). The cells scored were present as single cells or in clonal colonies of 2-4 cells, but the density of stain was recorded over single cells. Standard deviations are shown. These results derive from three independent experimental series.
