Cells from the pseudoplasmodial stage of Dictyostelium discoideum differentiation were dispersed and separated on Percoll gradients into prestalk and prespore cells. The requirements for stalk cell formation in low-density monolayers from the two cell types were determined. The isolated prespore cells required both the Differentiation Inducing Factor (DIF) and cyclic AMP for stalk cell formation. In contrast, only part of the isolated prestalk cell population required both cyclic AMP and DIF, the remainder requiring DIF alone, suggesting the possibility that there were two populations of prestalk cells, one independent of cyclic AMP and one dependent on cyclic AMP for stalk cell formation. The finding that part of the prestalk cell population required only a brief incubation in the presence of DIF to induce stalk cell formation, whilst the remainder required a considerably longer incubation in the presence of both DIF and cyclic AMP was consistent with this idea. In addition, stalk cell formation from cyclic-AMP-dependent prestalk cells was relatively more sensitive to caffeine inhibition than stalk cell formation from cyclic-AMP-independent prestalk cells. The latter cells were enriched in the most anterior portion of the migrating pseudoplasmodium, indicating that there is spatial segregation of the two prestalk cell populations. The conversion of prespore cells to stalk cells took longer and was more sensitive to caffeine when compared to stalk cell formation from cyclic-AMP-dependent prestalk cells.

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