Rapid patterning and zonal differentiation in a two-dimensional Dictyostelium cell mass: the role of pH and ammonia

Recently it was demonstrated that a rapidly forming, self-organizing pattern that emerges within two-dimensional Dictyostelium discoideum cell cultures could later give rise to stripes of distinct zones, each comprising different cell types. Here we report physiological aspects of the initial rapid patterning and its relationship to cell differentiation. We found that as the temperature is lowered the characteristic length of the pattern increases. From this we estimated the activation energy of the patterning kinetics. Fluorescence of fluorescein-conjugated dextran revealed that the cytosolic pH of cells in the inside zone becomes lower than that in the outer zone facing the air. The patterning could be inhibited by addition of the plasma-membrane proton pump inhibitors diethystilbestrol (DES) or miconazole. Preincubation of cells with weak acid delayed the timing of the patterning, whereas weak base hastened it. A pH-indicating dye revealed localized accumulation of ammonia in the extracellular space. These results suggest that gradients of secreted metabolites may be directly responsible for the rapid patterning and its consequence on cell differentiation in a confined geometrical situation. Possible diffusible candidate molecules and a reaction scheme coupled to the imposed oxygen gradient are discussed.