An analysis of culmination in Dictyostelium using prestalk and stalk-specific cell autonomous markers

The ecmA (pDd63) and ecmB (pDd56) genes encode extracellular matrix proteins of the slime sheath and stalk tube of Dictyostelium discoideum. Using fusion genes containing the promoter of one or other gene coupled to an immunologically detectable reporter, we previously identified two classes of prestalk cells in the tip of the migrating slug; a central core of pstB cells, which express the ecmB gene, surrounded by pstA cells, which express the ecmA gene. PstB cells lie at the position where stalk tube formation is initiated at culmination and we show that they act as its founders. As culmination proceeds, pstA cells transform into pstB cells by activating the ecmB gene as they enter the stalk tube. The prespore region of the slug contains a population of cells, termed anterior-like cells (ALC), which have the characteristics of prestalk cells. We show that the ecmA and ecmB genes are expressed at a low level in ALC during slug migration and that their expression in these cells is greatly elevated during culmination. Previous observations have shown that ALC sort to surround the prespore cells during culmination (Sternfeld and David, 1982 Devl Biol. 93, 111-118) and we find just such a distribution for pstB cells. We believe that the ecmB protein plays a structural role in the stalk tube and its presence, as a cradle around the spore head, suggests that it may play a further function, perhaps in ensuring integrity of the spore mass during elevation. If this interpretation is correct, then a primary role of anterior-like cells may be to form these structures at culmination. We previously identified a third class of prestalk cells, pstO cells, which lie behind pstA cells in the slug anterior and which appeared to express neither the ecmA nor the ecmB gene. Using B-galactosidase fusion constructs, which give more sensitive detection of gene expression, we now find that these cells express the ecmA gene but at a much lower level than pstA cells. We also show that expression of the ecmA gene becomes uniformly high throughout the prestalk zone when slugs are allowed to migrate in the light. Overhead light favours culmination and it may be that increased expression of the ecmA gene in the pst 'O' region is a preparatory step in the process.     

The role of DIF-1 signaling in Dictyostelium development

We have constructed a mutant blocked in the biosynthesis of DIF-1, a chlorinated signal molecule proposed to induce differentiation of both major prestalk cell types formed during Dictyostelium development. Surprisingly, the mutant still forms slugs retaining one prestalk cell type, the pstA cells, and can form mature stalk cells. However, the other major prestalk cell type, the pstO cells, is missing. Normal pstO cell differentiation and their patterning in the slug are restored by development on a uniform concentration of DIF-1. We conclude that pstO and pstA cells are in fact induced by separate signals and that DIF-1 is the pstO inducer. Positional information, in the form of DIF-1 gradients, is evidently not required for pstO cell induction.     

Spatial and temporal expression of a Polysphondylium spore-specific gene

In the cellular slime mold Polysphondylium spherical masses of cells are periodically released from the base of the culminating sorogen. These whorls undergo a morphogenetic transformation from spherical to radial symmetry, marked by the early emergence of a radially symmetric prepattern on the whorl surface. In previous experiments, morphogenesis was followed by observing prestalk cell markers. Here we describe the isolation and characterization of a spore coat gene whose expression pattern is the negative image of the prestalk pattern. To study the molecular mechanism of sp-45 gene regulation, we have cloned and analyzed the sp-45 promoter. Deletion analysis localized a single positive regulatory element (PRE) to a 106-bp fragment between positions -246 and -352 of the upstream coding sequence. This fragment can be further divided into a promoter-proximal and promoter-distal PRE and a 29-bp sequence between them. The distal PRE can regulate prespore expression when fused to a nonfunctioning basal promoter. The distal PRE contains two adjacent essential elements, a Gr box (GTGATATAGTGG) and a TA box (TAATATATT). Each element can drive prespore cell-specific reporter gene expression independently when incorporated into a nonfunctional promoter. Our results also show that prespore cell-specific gene expression is solely under positive regulation, with no evidence for spore-specific enhancers or cis-acting negative regulatory elements. By fusing GFP to the C-terminus of sp-45, we have demonstrated that the graded gene expression of SP45 in the sorogen is regulated by a sequence lying within the sp-45 coding sequence. The temporal and spatial expression pattern of this protein, taken together with the prestalk expression pattern, demonstrates unambiguously that the radial symmetries that emerge in the whorl are established by a system of positional coordinates and that cell sorting plays little if any role in this process.     

Immuno-localization and separation of multiple prestalk cell types in Dictyostelium

Abstract. The ecm A and ecm B genes of Dictyostelium encode closely related extracellular matrix proteins. The major prestalk cell population, pstA cells, expresses the ecm A gene but not the ecm B gene. PstAB cells, a minor prestalk cell population that we show to express both the ecm A and ecm B genes, form a core in the centre of the slug tip. The rear, prespore region of the slug contains amoebae, termed anterior-like cells (ALC), that display many of the properties of prestalk cells. The ecm A and B genes are weakly expressed in about 30% of the ALC and these comprise a mixture of pstA cells, pstAB cells and a third class, pstB cells. The latter cell type express the ecm B gene but show no detectable expression of the ecm A gene. The demonstration of the existence of pstB cells suggests a separate pathway of ecm B gene induction, wherein expression of the ecm A gene is absent or at a very low level. Pst A, AB and B cells most probably differ in their surface properties because they are partially separable by Counter Current Distribution (CCD), a chromatographic technique which, in the conditions used, is dependent upon differences in cell surface hydrophobicity.     

Expression of activated Ras during Dictyostelium development alters cell localization and changes cell fate

There is now a body of evidence to indicate that Ras proteins play important roles in development. Dictyostelium expresses several ras genes and each appears to perform a distinct function. Previous data had indicated that the overexpression of an activated form of the major developmentally regulated gene, rasD, caused a major aberration in morphogenesis and cell type determination. We now show that the developmental expression of an activated rasG gene under the control of the rasD promoter causes a similar defect. Our results indicate that the expression of activated rasG in prespore cells results in their transdifferentiation into prestalk cells, whereas activated rasG expression in prestalk causes gross mislocalization of the prestalk cell populations.     

Expression pattern of alkaline phosphatase in Dictyostelium

We used two different methods to study the expression pattern of alkaline phosphatase (alp) in Dictyostelium. In situ staining of the endogenous enzyme activity at different stages of development showed that the enzyme was active early in the aggregation stage and localized to the area where the tip of the first finger was initiated. The activity was localized to the anterior region of developing slugs, then became restricted to the region between the prestalk and prespore cells at the culmination stage. In the complete fruiting body, the activity was confined to the lower and upper cup. A second method to study alp expression utilized a beta-galactosidase reporter gene under the control of the alp promoter. A low level of beta-galactosidase activity was observed in vegetative cells, then increased during development. Reporter gene activity was restricted to PstO cells at the slug stage. At the culmination stage, the expression was restricted to prestalk cells at the interface between the prestalk and prespore cells. In the completed fruiting body, the expression was observed in the upper and lower cup.     

Cell type proportioning in Dictyostelium slugs: lack of regulation within a 2.5-fold tolerance range

The proportion of prestalk and prespore cells in Dictyostelium discoideum slugs is often cited as an example of "almost perfect" regulation. The pattern is similar over a very wide range of cell number; furthermore, removal of either of the cell types leads to compensatory transdifferentiation. Several studies of Dictyostelium fruiting bodies, however, have suggested that proportioning in Dictyostelium differs systematically from true constancy. We have confirmed this in the slug stage using a short-lived beta-galactosidase as a reporter of the prestalk specific ecmA gene expression: the prestalk proportion decreases from 24+/-5% in slugs of 10(3) cells to 10+/-3% when 10(5) cells are present. Regeneration experiments suggest that this difference is not due to a modulation of the proportioning set-point by size, as one might have expected; instead there appears to be a regulatory "tolerance zone" at all sizes. After amputation of the whole posterior region, transdifferentiation stops after the fraction of prestalk has been reduced from 100% to 28+/-20%, well above the initial value of 10+/-3%, while after anterior removal the transdifferentiation endpoint is about 10%. Most strikingly, we find no regulation at all after partial amputations of the prespore region. It seems that any prestalk proportion is stable between a approximately 10% lower threshold and a approximately 30% upper threshold. To explain this, we propose a regulation mechanism based on a negative feedback plus cell type bistability. In both intact and regenerating slugs we find that the slug morphology is regulated so that the length-to-width ratio of the anterior region is constant.