Dual Effects of cAMP on the Stability of Prespore Vesicles and 8-bromo cAMP-enhanced Maturation of Spore and Stalk Cells of Dictyostelium discoideum

It is well known that interconversion between prestalk and prespore cells occurs in 3-dimensional (3–D) isolates of Dictyostelium. The present work was undertaken to examine whether or not the interconversion occurs even in monolayer sheets. The results suggested that in monolayer sheets of either prespore or prestalk cells, the interconversion does not occur. Furthermore, effects of cAMP were examined in relation to the formation or loss of prespore vesicles (PSVs). In monolayer sheets, prespore cells retain their PSVs in the presence of cAMP, though they lose them in its absence. In 3–D masses, however, cAMP induces the conversion into stalk cells, stimulating PSV loss. In the case of prestalk cells, cAMP induces the maturation of prestalk cells to stalk cells in 3–D masses, but it does not induce stalk differentiation in monolayer sheets.
8-Bromo cAMP stimulates the maturation of prespore and prestalk cells into spore and stalk cells, respectively. However, the vegetative and the aggregative cells remain amoeboid even in its presence. These observations suggest that 8-bromo cAMP stimulates the maturation rather than inducing prespore and prestalk differentiation.     

The anterior-like cells in Dictyostelium are required for the elevation of the spores during culmination

 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. Received: 26 January 1998 / Accepted: 27 May 1998     

Unexpected localisation of cells expressing a prespore marker of Dictyostelium discoideum

Abstract. We show that the anterior, prestalk region of the Dictyostelium slug contains cells which express, or have expressed, a prespore-specific marker. We term these cells "prespore-like cells" (PLC). In newly formed slugs there is a sharp prespore/prestalk boundary, with very few PLC, but after several days of migration the clear demarcation between prespore and prestalk zones breaks down because the number of PLC increases dramatically. This is consistent with previous observations showing there to be rapid interchange of cells between the prestalk and prespore regions. This is not, however, their only source, as a scattering of PLC appear when separate prestalk and prespore regions first become apparent at the time of tip formation. Also, at culmination, there is respecification of "prespore" cells at the pre-stalk/prespore boundary to form part of the mature stalk. The existence of these cells, and of PLC, may explain why we find prespore-specific mRNAs in mature stalk cells.     

Different synthetic profiles and developmental fates of prespore versus prestalk proteins of Dictyostelium

Abstract. Depending upon environmental conditions, developing cells of the cellular slime mold Dictyostelium discoideum may enter a slug stage in which the cell mass migrates in response to gradients of light and temperature. This developmental stage has often been used to study the divergent differentiation of the cells that will subsequently form spores and stalk in the mature fruiting body. However, still debated is the extent to which the differentiation evident in slug cells is a precondition for development of the mature cells in fruits. Using two-dimensional gel electrophoresis of polypeptides, we have examined the proteins made by prespore and prestalk cells of migrating slugs and by maturing spore and stalk cells. The data indicate that many of the cell-type specific polypeptides in prespore cells of slugs persist as cell-type specific polypeptides of mature spores. Prestalk slug cells, in contrast, do not contain significant amounts of stalk-specific proteins; these proteins appear only during culmination. The precursor cell types also differ in the times and rates of synthesis of cell-specific proteins: prestalk proteins appear much earlier in development than do the prespore, but never reach the levels of expression that the prespore proteins do later in culmination. These findings may explain the well established ability of prespore cells to regulate their cell type more rapidly than do prestalk cells. There are also implications for our general understanding of what is a 'prestalk' gene product.     

Developmental regulation of a prestalk- and stalk-enriched protein in Dictyostelium discoideum

Abstract. Monoclonal antibodies reactive with proteins specifically present either in the prespore cells or the prestalk cells of Dictyostelium discoideum were obtained. Four of them recognized prespore-enriched proteins, as shown by both immunoblotting assays and immunofluorescent staining. The other monoclonal antibody ( mab150 ) produced more than 10 protein bands when reacted with both prespore and prestalk cell extracts in immunoblotting assays. However, a protein band with molecular weight 35 000 (st35) was specifically detected in prestalk cells as well as mature stalk cells. St35 was solubilized from the Triton X-100 insoluble fraction of mature stalks by sodium dodecyl sulfate (SDS). The purified sample gave a single spot on two-dimensional gel electrophoresis, with pI of 5.0. During development, st35 first appeared at the tipped aggregate stage and accumulated up to stalk-cell formation without modification. The protein was not lost even when slugs were disaggregated. The importance of the tipped aggregate stage for prestalk differentiation as well as prespore differentiation is discussed.     

Cell Patterning During Slug Migration and Early Culmination in Dictyostelium discoideum

Abstract. The effects of migration and culmination on patterning of presumptive (prespore and prestalk) cells and mature (spore and stalk) cells of D. discoideum were investigated. The ratio of prespore to total cells, as determined by staining with fluorescein-conjugated antispore globulin, was constant (77%) up until 8 h of slug migration, but then decreased to a level (64%) which thereafter remained unchanged during migration. Cells which lost prespore antigen during migration were located in the posterior (prespore) part next to the agar surface.
Upon induction of culmination, however, the ratio of prespore cells quickly increased to the normal level (77%) within 1–2 h. During the transition between migration and culmination prestalk and prespore cells were considerably intermixed within the cell mass, before the normal prestalk-prespore pattern was reestablished at the preculmination (Mexican hat) stage. Spore: stalk ratios within fruiting bodies were normal irrespective of the lengths of slug migration.     

Potential morphogens involved in pattern formation during Dictyostelium differentiation.

Upon starvation, Dictyostelium amoebae aggregate together and then differentiate into either the stalk or spore cells that, respectively, form the stalk and sorus of the fruiting body. During differentiation, the prestalk and prespore cells become spatially segregated in a clearly defined developmental pattern. Several low molecular weight molecules that influence cell type determination during in vitro differentiation have been identified. The possible role of these molecules as morphogens, responsible for the formation of the developmental pattern, is discussed.     

Pattern Formation in Dictyostelium discoideum: Temporal and Spatial Distribution of Prespore Vacuoles

Cell differentiation, cell determination and pattern formation in the pseudoplasmodium of Dictyostelium discoideum , was investigated using the prespore specific vacuole (PV) as a morphological marker. Concomitantly, measurements of cell movement within the pseudoplasmodium were made by tracing radioactively labelled cells. The main results indicate that 1) prespore cells appear first during late aggregation and occur randomly throughout the pseudoplasmodium with the exception of the very tip which stays free of prespore cells throughout development; 2) after late aggregation the number of prespore cells increases over a period of several hours; 3) each prespore cell takes on a progressively more prespore-like character as judged by the increase in number of PVs it contains; 4) establishment of the distribution pattern of prespore and prestalk cells takes place within the first 2 h, mainly by a sorting out mechanism; 5) presumptive spore areas are likely to contain a small proportion of cells lacking PVs (prestalk-cells?) while presumptive stalk cell areas are homogeneous throughout; 6) maintenance of the pattern during migration may be facilitated by a circulation at low level of prestalk cells between prestalk and prespore areas; and 7) during the development of this organism the events of cell determination, cell differentiation and pattern formation overlap substantially in time.     

Stalk cell formation in monolayers from isolated prestalk and prespore cells of Dictyostelium discoideum: evidence for two populations of prestalk cells

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.     

A prespore gene, Dd31, expressed during culmination of Dictyostelium discoideum

During culmination of Dictyostelium fruiting bodies, prespore and prestalk cells undergo terminal differentiation to form spores and a cellular stalk. A genomic fragment was isolated by random cloning that hybridizes to a 1.4-kb mRNA present during culmination. Cell type separations at culmination showed that the mRNA is present in prespore cells and spores, but not in prestalk or stalk cells. After genomic mapping, an additional 3 kb of DNA surrounding the original 1-kb fragment was cloned. The gene was sequenced and named Dd31 after the size of the predicted protein product in kilodaltons. Accumulation of Dd31 mRNA occurs immediately prior to sporulation. Addition of 20 mM 8-Br-cAMP to cells dissociated from Mexican hat stage culminants induced sporulation and the accumulation of Dd31 mRNA, while 20 mM cAMP did not. Dd31 mRNA does not accumulate in the homeotic mutant stalky in which prespore cells are converted to stalk cells rather than spores. Characterization of Dd31 extends the known temporal dependent sequence of molecular differentiations to sporulation.     

Cell Sorting daring Pattern Formation in Dictyostelium

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.     

Separation and properties of prestalk and prespore cells of Dictyostelium discoideum

We describe a method of separating prestalk and prespore cells of Dictyostelium discoideum slugs using a self-generating Percoll gradient. This method gives quantitative recovery of cells and good purity. Separated prestalk and prespore cells possess different levels of the enzymes UDP galactose :polysaccharide transferase, cAMP phosphodiesterase and glycogen phosphorylase. We have used this method, as well as mechanical dissection of slugs, to examine the fate of separated prestalk and prespore cells in Dictyostelium strains that are able to give rise to mature stalk and spore cells in cell monolayers. The results from such experiments provide direct evidence that prestalk and prespore cells from the migrating slug stage are programmed to differentiate into stalk and spore cells respectively.     

Cell-type conversion in normally proportioned and prestalk-enriched populations of slug cells in Dictyostelium discoideum

Abstract. Conversion of prestalk cells to prespore cells was investigated in normally proportioned as well as prestalk-enriched cell populations under two different conditions: in slugs migrating on agar plates and in suspension cultures of dissociated slug cells in the presence of cAMP. In most experiments, prestalk cells labelled with a fluorescent dye (TRITC) and unlabelled prespore cells were combined together by grafting (for migrating slugs) or by mixing (for suspension cultures) to distinguish conversion of prestalk cells to prespore cells. In both migrating and dissociated slugs, minimal conversion of prestalk to prespore cells was observed when the proportion of prespore cells in the whole population was maintained at a normal level. When the prespore proportion in the initial population was lowered, a considerable fraction of prestalk cells underwent cell-type conversion to become prespore cells or spores. These results indicate that the presence of prespore cells somehow prevents prestalk cells from becoming prespore.     

Prestalk/prespore Differentiation of Dictyostelium Cells under the Conditions Favoring Stalk or Spore Cell Formation*

The differentiation processes of Dictyostelium discoideum cells under the conditions which favored either stalk or spore cell formation were examined by the use of prestalk- and prespore-specific antibodies. In stalk cell-forming conditions, cells reactive with prestalk-specific monoclonal antibody (C1) increased rapidly early in development and later differentiated into stalk cells. No or only a few cells became reactive with prespore-specific monoclonal (B6) and polyclonal (antispore) antibodies. Despite the fact that most cells terminally became spores under spore cell-forming conditions, cells were first stained with the C1 antibody before becoming reactive with the B6 antibody. Unlike the case of normal development where cells coincidentally become reactive with the B6 and antispore antibodies, the appearance of the cells reactive with the latter was either delayed or suppressed. In conclusion, under either spore or stalk cell-forming conditions, the appearance of the prestalk antigen preceded that of the prespore one, which is consistent with normal development.     

Glycoantigen expression is regulated both temporally and spatially during development in the cellular slime molds Dictyostelium discoideum and D. mucoroides

Six monoclonal antibodies were isolated which react with common antigens shared by multiple glycoconjugate species in the cellular slime mold Dictyostelium discoideum. Based on competition of antibody binding by glycopeptides and simple sugars, and inhibition of antibody binding by antigen pretreatment with Na periodate, it is argued that at least five of the six antibodies recognize epitopes which contain carbohydrate. These epitopes are consequently referred to as glycoantigens (GAs).Three of the GAs are expressed during growth and throughout the developmental cycle, but are eventually enriched in prestalk and stalk cells. The remaining three are expressed only during and/or after aggregation and are exclusively expressed or highly enriched in prespore cells and spores. These conclusions are derived from Western blot immunoanalysis of purified cell types, immunofluorescence, and EM immunocytochemistry.The two GAs found only in prespore cells appear to be exclusively enclosed within prespore vesicles. The third GA of this type, which is only enriched in prespore cells compared to prestalk cells, is also found in other vesicle types as well as on the cell surface.Two of the GAs enriched in prestalk cells are initially found in all cells of the slug. They are undetectable in spores and prominent in stalk cells. The third GA, though found in the interiors of both prestalk and prespore cells, is enriched on the cell surface of prestalk cells.The chief characteristics of expression of four of these GAs are conserved in the related species D. mucoroides. This species is characterized by continuous trans differentiation of prespore cells into prestalk cells. This shows that the prespore cells maintain specific mechanisms for turning over their cell type specific GAs and that prestalk cells express a specific mechanism for inducing at least one of their cell-type specific GAs.These observations identify specific carbohydrate structures (as GAs) whose synthesis, subsequent localization and turnover are developmentally regulated. The exclusive association of two GAs with prespore vesicles identifies these GAs as markers for this organelle and raises questions regarding the functional significance of this association. The restricted cell surface localization of the other four GAs, together with data from cell adhesion studies, suggest the possibility of a potential role for these GAs in intercellular recognition leading to cell sorting.This paper is dedicated to the memory of the late Daniel McMahon.     

Tissue-specific G1-phase cell-cycle arrest prior to terminal differentiation in Dictyostelium

The cell cycle status of developing Dictyostelium cells remains unresolved because previous studies have led to conflicting interpretations. We propose a new model of cell cycle events during development. We observe mitosis of about 50% of the cells between 12 and 18 hours of development. Cellular DNA content profiles obtained by flow cytometry and quantification of extra-chromosomal and chromosomal DNA suggest that the daughter cells have half the chromosomal DNA of vegetative cells. Furthermore, little chromosomal DNA synthesis occurs during development, indicating that no S phase occurs. The DNA content in cells sorted by fluorescent tissue-specific reporters indicates that prespore cells divide before prestalk cells and later encapsulate as G1-arrested spores. Consistent with this, germinating spores have one copy of their chromosomes, as judged by fluorescence in situ hybridization and they replicate their chromosomes before mitosis of the emergent amoebae. The DNA content of mature stalk cells suggests that they also attain a G1 state prior to terminal differentiation. As prestalk cells appear to be in G2 up to 22 hours of development, our data suggest that they divide just prior to stalk formation. Our results suggest tissue-specific regulation of G1 phase cell cycle arrest prior to terminal differentiation in Dictyostelium.     

Fractionation of the differentiated types of cells constituting the pseudoplasmodia of the cellular slime molds

The observation of Milleret al. (1969) that the two types of cells (the prestalk and prespore cells) constituting the slug ofDictyostelium are separated by isopicnic centrifugation was reexamined by using more reliable methods both for dissociation of the slug and for identification of the cell type. Dissociated cells of slugs which had been grown on a standard culture medium formed two distinct bands after centrifugation through a Urografin density gradient. Contrary to Miller's findings, however, the light band consisted of the prestalk cells and the heavy band of the prespore cells. When the culture medium was modified, a population of spores of different buoyant density newly appeared during the subculture. Slug cells derived from such a spore had different buoyant densities and formed extra bands in a Urografin gradient. However, the prespore fraction was always heavier than the prestalk fraction derived from the same type of spores.     

Mutation of the Dictyostelium fbxA gene affects cell-fate decisions and spatial patterning

Cell-fate decisions and spatial patterning in Dictyostelium are regulated by a number of genes. Our studies have implicated a gene called fbxA, which codes for an F-box protein, in these pathways. The FbxA protein is one of the controls on a cAMP phosphodiesterase called RegA, mediating its degradation via ubiquitin-linked proteolysis. Using marked strains, we showed that the fbxA^– mutant has defective cell-type proportioning, with a dearth of prestalk cells compared to prespore cells. In this work, we show that this effect occurs earlier during the 24 hour developmental cycle than previously thought. The normal sorting of the prestalk and prespore cells in aggregates and mounds is not affected by the mutation. The mutant cells sort abnormally at the tipped mound stage, when prespore and prestalk cells normally distribute into their proper compartments. The fbxA^– mutant forms prestalk cells in low numbers when not in chimeras, but in the presence of wild-type amoebae the mutant preferentially forms viable spores, driving the wild type to form non-viable stalk cells. In an attempt to identify the signal transduction pathway that mediates proportionality in prestalk and prespore cells, we asked whether certain signal transduction mutants were immune to the effects of the fbxA^–cells and formed spores in chimeras.