Evidence that positional information is used to establish the prestalk-prespore pattern in Dictyostelium discoideum aggregates

Two contrasting mechanisms have been proposed for the establishment of the prestalk-prespore pattern in the multicellular aggregate of the simple eukaryote Dictyostelium discoideum. One involves intermingled, non-position-dependent cell differentiation followed by sorting out which produces the pattern of prestalk cells in the anterior region and prespore cells posteriorly. The second mechanism involves patterning according to the position of cells within the aggregate, in which case intermingled cell types are not expected. Here we use a monoclonal antibody (MUD1), recognising a prespore cell surface antigen, to study the initial appearance of prespore cells in aggregates. Quantitative studies were made with a flow cytometer and frozen sections were used to localise the cells expressing the prespore antigen. This antigen first appeared at the onset of tip formation in the centre of aggregates in a position-dependent fashion. The prespore antigen was not detected in the tip region or in streams of cells entering the aggregate. We re-examined the evidence on which the non-position-dependent differentiation model is based. Our results support the positional model for pattern formation.     

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.     

Diffusion-induced morphogenesis in the development of Dictyostelium

A reaction-diffusion model for the regulation of cAMP in Dictyostelium discoideum is analyzed. As a parameter declines with starvation, the model sequentially yields pulse relaying, spiral waves, target patterns, streaming and sorting, directed locomotion, and tissue buckling, closely matching the observed morphogenetic sequence. These morphologies appear through successive bifurcations of a single reaction-diffusion system and do not require the expression of new genetic information.     

Expression pattern of 5''-nucleotidase in Dictyostelium.

In order to analyze the expression pattern of the 5′-nucleotidase (5nt) gene in Dictyostelium, we made a fusion construct in which the 5nt promoter directed the expression of β-galactosidase gene. The reporter gene was not active in vegetative amoebae but was expressed during the aggregation stage. At the slug stage, 5nt was highly expressed in pstAB cells. As the slug moved along the substratum, high activity of β-galactosidase was detected in cells that were left behind in the slime trail. In the completed fruiting body, 5nt was expressed in the lower cup, the anterior like cells (ALC) and the basal disc.     

Cell-cell interactions in Dictyostelium development

The development of an organism requires extensive cell-cell communication; however, little is known about the signals transmitted among differentiating cells. Observations of Dictyostelium amoebae reveal that transmembrane signaling systems have been highly conserved in evolution. The signals that cause these cells to differentiate are processed by mechanisms similar to those that process sensory and hormonal stimuli in higher animals.     

Plasticity in the development and dedifferentiation of Dictyostelium discoideum

Dictyostelium discoideum has served as a model for development and differentiation for over 70 years. Also regulated in Dictyostelium is the process of dedifferentiation, which consists of multiple cellular events that are separately regulated, providing an excellent model system for studying the return of partially differentiated cells to a more pluripotent state. An interesting aspect of Dictyostelium development is the plasticity between growth and development. Reversibility of the processes of differentiation and dedifferentiation exist, allowing Dictyostelium to adjust to changing conditions by reverting to the growth phase during differentiation or reinitiating development during dedifferentiation. This ability of cells to respond to environmental cues is mediated by the checkpoint-like events "commitment" and "erasure," which occur during differentiation and dedifferentiation, respectively. Our review will discuss the current state of knowledge regarding dedifferentiation and the plasticity of the developmental process in both the forward and reverse directions.     

Signals controlling cell differentiation and pattern formation in Dictyostelium

The major inducers of cell differentiation in Dictyostelium appear to be cyclic AMP and DIF-1. Recently we have chemically identified DIF-1, together with the closely related DIF-2 and -3. They represent a new chemical class of potent effector molecules, based on a phenyl alkanone with chloro, hydroxy, and methoxy substitution of the benzene ring. Previous work has shown that DIF-1 can induce prestalk-specific gene expression within 15 min, whereas it suppresses prespore differentiation. Hence, DIF-1 can control the choice of pathway of cell differentiation in Dictyostelium and is therefore likely to be involved in establishing the prestalk/prespore pattern in the aggregate. In support of this, we show that DIF treatment of slugs results in an enlarged prestalk zone. Cyclic AMP seems less likely to have such a pathway-specific role, but later in development it becomes inhibitory to stalk cell differentiation. This inhibition may be important in suppressing terminal stalk cell differentiation until culmination. Spore differentiation can be induced efficiently by high levels of Br-cyclic AMP, a permeant analogue of cyclic AMP. In this, it phenocopies certain spore-maturation mutants, and we propose that during normal development spore differentiation is triggered by an elevation in intracellular cyclic AMP levels. How this elevation in cyclic AMP levels is brought about is not known. The experiments with Br-cyclic AMP also provide the first direct evidence that elevated levels of intracellular cyclic AMP induce differentiation in Dictyostelium.     

A demonstration of pattern formation without positional information in Dictyostelium

Although positional information, conveyed by morphogen gradients, is a widely accepted way of forming patterns during development, an alternative method is conceivable, based on the intermingled differentiation of cells with different fates, followed by their sorting into discrete pattern elements. It has been proposed that Dictyostelium prestalk and prespore cells behave in this way at the mound stage of development. However, it has been difficult to conclusively demonstrate that they initially differentiate intermingled, because rapid cell movement within the mound makes it impossible to be sure where prestalk and prespore cells originate. We have taken a novel approach to address this problem by blocking cell movement at different stages in development, using the actin-depolymerizing drug, latrunculin-A. Prestalk and prespore cells differentiate with essentially normal efficiency and timing in such paralyzed structures. When movement is blocked sufficiently early, the major cell types all subsequently differentiate at scattered positions throughout the aggregate, and even in the streams leading into it. Our work strongly supports the idea that the prestalk/prespore pattern in Dictyostelium forms without positional information and demonstrate that latrunculin-A may provide a useful tool for the investigation of patterning in other organisms.     

Requirements for the adenylyl cyclases in the development of Dictyostelium

It has been suggested that all intracellular signaling by cAMP during development of Dictyostelium is mediated by the cAMP-dependent protein kinase, PKA, since cells carrying null mutations in the acaA gene that encodes adenylyl cyclase can develop so as to form fruiting bodies under some conditions if PKA is made constitutive by overexpressing the catalytic subunit. However, a second adenylyl cyclase encoded by acrA has recently been found that functions in a cell autonomous fashion during late development. We have found that expression of a modified acaA gene rescues acrA- mutant cells indicating that the only role played by ACR is to produce cAMP. To determine whether cells lacking both adenylyl cyclase genes can develop when PKA is constitutive we disrupted acrA in a acaA- PKA-C(over) strain. When developed at high cell densities, acrA- acaA- PKA-C(over) cells form mounds, express cell type-specific genes at reduced levels and secrete cellulose coats but do not form fruiting bodies or significant numbers of viable spores. Thus, it appears that synthesis of cAMP is required for spore differentiation in Dictyostelium even if PKA activity is high.     

Cell polarity and Dictyostelium development

Cell polarity is essential for unicellular and multicellular stages of Dictyostelium development. Chemotaxis during early development requires each cell to rapidly reorganize its cytoskeleton to point towards a source of cAMP. This involves a balance between local induction of F-actin polymerization and suppression of pseudopods that point in other directions. Both the lipid phosphatidylinositol (3,4,5) trisphosphate and the soluble signal cGMP have been implicated in these processes, in addition to conserved and novel proteins. During later development cells adopt newly discovered, alternative modes of movement and interact through adhesion molecules. Finally, cells polarize secretion to particular regions of their surface.     

Changes in phospholipid composition during the development of Dictyostelium discoideum

The phospholipid composition of Dictyostelium discoideum cells was determined at various stages of development by two-dimensional, thin-layer chromatography and reaction thin-layer chromatography. Major phospholipids of D. discoideum which were detectable throughout all stages of development were ethanolamine phosphoglyceride and choline phosphoglyceride. Ethanolamine phosphoglyceride and choline phosphoglyceride were found as their plasmalogen forms at 45–58 and 10–24%, respectively. There were no qualitative changes in phospholipid composition during the development, but quantitative changes did occur. The relative content of ethanolamine phosphoglyceride in the total phospholipids gradually decreased from 60% at the vegetative stage to 44% at the 1-day-sorocarp stage. In contrast, choline phosphoglyceride gradually increased from 27% at the vegetative stage to 48% at the preculmination stage, and then gradually decreased to 43% during the culmination. The decrease in ethanolamine phosphoglyceride content during the middle and late development was due mainly to the decreased amount of its plasmalogen form but the increase of choline phosphoglyceride was independent of quantitative changes of its plasmalogen form. Other minor components of phospholipid did not show significant changes in their levels. The causes of these changes in contents of ethanolamine phosphoglyceride and choline phosphoglyceride were examined by label and chase experiments with [³H]ethanolamine and [¹⁴C]choline. It seems that one-third to one-half of the increased amount of choline phosphoglyceride was due to stepwise methylation of ethanolamine phosphoglyceride, and the remaining two-thirds to one-half was caused by de novo synthesis of choline phosphoglyceride from CDP-choline and diglyceride.     

The development of the relaying competence in Dictyostelium discoideum.

The fraction, X2, of a population of D. discoideum cells competent to relay an aggregative signal has been measured as a function of time, t, spent in interphase. X2(t) is less than 0.I up to 5 h, increases slowly to 0.4 by 8 h 20 min, and saturates at I by 10 h. The hypothesis that cellular interactions might enhance X2(t) was tested by mixing populations of cells set into interphase at different times. No interactions were found. External stimulation did not affect X2(t).