No abstract

Formation of the prestalk‐prespore pattern in Dictyostelium was investigated in slugs and submerged clumps of cells. Prestalk and prespore cells were identified by staining with vital dyes, which are shown to be stable cell markers. Dissociated slug cells reaggregate and form slugs that contain a prestalk‐prespore pattern indistinguishable from the original pattern. The pattern forms by sorting out of stained prestalk cells from unstained prespore cells. Sorting also occurs in clumps of dissociated slug cells submerged in liquid or agar. A pattern arises in 2 h in which a central core of stained cells is surrounded by a periphery of unstained cells. Sorting appears to be due to differential chemotaxis of stained and unstained cells to cAMP since exogenous cAMP (>10−7 M) reverses the normal direction of sorting‐out such that stained cells sort to the periphery of the clumps. Isolated portions of slugs regenerate a new prestalk‐prespore pattern. Posterior isolates regenerate a pattern within 2 h due to sorting of a population of vitally stained ‘anterior‐like’ cells present in posteriors. Anterior‐like cells do not sort in intact slugs due to the influence of a diffusible inhibitor secreted by the anterior region. During posterior regeneration this signal is absent and anterior‐like cells rapidly acquire the ability to sort. Anterior isolates regenerate a staining pattern more slowly than posterior isolates by a process that requires conversion of stained prestalk cells to unstained prespore cells. The results suggest that pattern formation in Dictyostelium consists of two processes: establishment of appropriate proportions of two cell types and establishment of the pattern itself by a mechanism of sorting‐out.

The prestalk region of the Dictyostelium slug has recently been shown by Williams and his collaborators to consist of two distinct cell types, pstA and pstB cells. Here the movement of these cells in both the slug and culmination stages has been examined with the use of vital dyes. In the slug some of the pstB cells are continually lost from the prestalk region as small clusters of cells. These cells move through the prespore region and temporarily lie in the rearguard region at the posterior end of the slug. They are finally left in the slug's slime track as single cells or groups of a few cells. When culmination is initiated the pstB cells move as a whole from the prestalk region to the base where they join the rearguard cells to form the basal disc of the fruiting body. Transplantation experiments reveal that the rearguard cells form an outer ring portion of the basal disc and the pstB cells form an inner portion to which the stalk attaches. The continuous loss of one cell type during the slug stage without any change in cell type proportions suggests that cell types are redifferentiating. Grafting and transplantation experiments reveal that there is a unidirectional flow of cells through successive steps of cell type conversion. Prespore cells redifferentiate as anterior-like cells which migrate to the prestalk region and become pstA cells. The pstA cells then replace the pstB cells that are lost from the slug.

Shortly after initiation of Dictyostelium fruiting body formation, prespore cells begin to differentiate into non-motile spores. Although these cells lose their ability to move, they are, nevertheless, elevated to the tip of the stalk. Removal of the amoeboid anterior-like cells, located above the differentiating spores in the developing fruiting body, prevents further spore elevation although the stalk continues to elongate. Furthermore, replacement of the anterior-like cells with anterior-like cells from another fruiting body largely restores the ability to lift the spores to the top of the stalk. However, if amoeboid prestalk cells are used to replace the anterior-like cells, there is no restoration of spore elevation. Finally, when a droplet of mineral oil replaces differentiating spores, it is treated as are the spores: the mineral oil is elevated in the presence of anterior-like cells and becomes arrested on the stalk in the absence of anterior-like cells. Because a similar droplet of mineral oil is totally ignored by slug tissue, it appears that there is a dramatic transformation in the treatment of non-motile matter at this point in Dictyostelium development.

Hitherto it has not been possible to obtain spore and stalk cell differentiation of the cellular slime molds in submerged cultures. It is shown here that cells when elaced in roller tubes under an atmosphere of oxygen, will form clumps and differentiate in 48-72 hr into mature spores and stalk cells. Although differentiation occurs without the normal morphogenetic movements, there is the appearance of an anteroposterior polarity of the cells in the clump. In addition to oxygen we examined a number of other factors that affect differentiation. It has always been assumed that differentiation in the cellular slime molds could not occur under water, for development would invariably stop at aggregation (1), and that an air-water or an oil-water interface was required. The success of an oilwater interface, which was first demonstrated by Potts (2), has been ascribed to-the fact that mineral oil can be saturated with 10 times more oxygen than can water. As will be shown, if oxygen is added to roller tubes containing Dictyostelium discoideum cells suspended in a physiological salt solution, then clumps of cells will form that show both mature spore and stalk cell differentiation. This work was first presented at a meeting in May, 1976 at Cold Spring Harbor; at the same meeting I. Takeuchi reported similar results in roller tubes using different methods. Ultimately, the findings of both these studies will increase our understanding of the mechanism of cell differentiation in the cellular slime molds. MATERIALS AND METHODSAll the experiments reported here were done with Dictyosteltum discoideum NC-4 grown on Escherichia colt B/r on 1% peptone-dextrose buffered agar. The amebae were washed three times by centrifugation at 75 X g in 10-2 M salt solution (1). RESULTSTime course description of differentiation One of the most interesting aspects of this study has been the morphological events that accompany the differentiation of the clumps in the roller tubes. The following description, which is also shown in diagrammatic sequence in Fig. 1