The effects of presumptive morphogens on prestalk and prespore cell gene expression in monolayers of Dictyostelium discoideum

Abstract. The expression of three prestalk cell-specific genes ( ecm A, ecm B and pDd26) was examined during in vitro differentiation in cell monolayers, in an attempt to explain the spatial heterogeneity of the prestalk region of migrating Dictyostelium pseudoplasmodia. Under these conditions ecm A, ecm B and pDd26 mRNAs were expressed sequentially in response to the addition of differentiation inducing factor-1 (DIF)-1, a temporal sequence similar to that observed during normal development. ecm A and ecm B mRNAs reached a maximum level 2–4 h after DIF-1 supplementation and then declined, whereas pDd26 mRNA levels increased more slowly but remained high 24 h after DIF addition. The increases in expression in response to increasing concentrations of either DIF-1 or DIF-2 were identical for the three genes, suggesting that neither alteration in DIF concentration nor species was an important determinant of spatial heterogeneity. Ammonia had the same inhibitory effect on the expression of all three prestalk cell-specific genes and stimulated the expression of the prespore cell-specific gene, D19. These results indicate that ammonia is also not responsible for the spatial heterogeneity of the prestalk cell region. In contrast, cyclic AMP had a differential effect on the expression of the prestalk cell specific genes: ecm A expression was variably stimulated, pDd26 expression was inhibited and ecm B expression was sometimes stimulated and sometimes inhibited. These results are difficult to explain in terms of a gradient of cyclic AMP in the prestalk region. We postulate that temporal responses are more important than spatial responses to cyclic AMP in regulating stalk cell differentiation.     

Combinatorial control of cell differentiation by cAMP and DIF-1 during development of Dictyostelium discoideum

At least three distinct types of cell arise from a population of similar amoebae during Dictyostelium development: prespore, prestalk A and prestalk B cells. We report evidence suggesting that this cellular diversification can be brought about by the combinatorial action of two diffusible signals, cAMP and DIF-1. Cells at different stages of normal development were transferred to shaken suspension, challenged with various combinations of signal molecules and the expression of cell-type-specific mRNA markers measured 1-2 h later. pDd63, pDd56 and D19 mRNAs were used for prestalk A, prestalk B and prespore cells respectively. We find the following results. (1) Cells first become responsive to DIF-1 for prestalk A differentiation and to cAMP for prespore differentiation at the end of aggregation, about 2 h before these cell types normally appear. (2) At the first finger stage of development, when the rate of accumulation of the markers is maximal, the expression of each is favoured by a unique combination of effectors: prespore differentiation is stimulated by cAMP and inhibited by DIF-1; prestalk A differentiation is stimulated by both cAMP and DIF-1 and prestalk B differentiation is stimulated by DIF-1 and inhibited by cAMP. (3) Half-maximal effects are produced by 10-70 nM DIF-1, which is in the physiological range. (4) Ammonia and adenosine, which can affect cell differentiation in other circumstances, have no significant pathway-specific effect in our conditions. These results suggest that cell differentiation could be brought about in normal development by the localized action of cAMP and DIF-1.     

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.     

Nature and distribution of the morphogen DIF in the Dictyostelium slug

The Dictyostelium slug contains a simple anterior-posterior pattern of prestalk and prespore cells. It is likely that DIF, the morphogen which induces stalk cells, is involved in establishing this pattern. Previous work has shown that a number of distinct species of DIF are released by developing cells and that cell-associated DIF activity increases rapidly during the slug stage of development. In this paper we describe a comparison of the DIF extracted from slugs with the DIF released into the medium. Analysis by high-pressure liquid chromatography (HPLC) using different solvent systems shows that the major species of DIF activity extracted from slugs coelutes with DIF-1, the major species of released DIF and is similarly sensitive to sodium borohydride reduction. Since DIF specifically induces the differentiation of prestalk cells, the anterior cells of the slug, it could be anticipated that DIF is localized in the prestalk region. We have therefore determined the distribution of DIF within the slug. Migrating slugs from strain V12M2 were manually dissected into anterior one-third and posterior two-third fragments and the DIF activity extracted. Surprisingly, we found that DIF was not restricted to the prestalk fragment. Instead there appears to be a reverse gradient of DIF in the slug with at least twice the specific activity of total DIF in the prespore region than in the prestalk region.     

Production and turnover of cAMP signals by prestalk and prespore cells in Dictyostelium discoideum cell aggregates

Dictyostelium discoideum prestalk cells and prespore cells from migrating slugs and culminating cell aggregates were isolated by Percoll density centrifugation. Several activities relevant to the generation, detection, and turnover of extracellular cyclic AMP (cAMP) signals were determined. It was found that: the two cell types have the same basal adenylate cyclase activity; prespore cells and prestalk cells are able to relay the extracellular cAMP signal equally well; intact prestalk cells show a threefold higher cAMP phosphodiesterase activity on the cell surface than prespore cells, whereas their cytosolic activity is the same; intact prestalk cells bind three to four times more cAMP than prespore cells; no large differences in cAMP metabolism and detection were observed between cells derived from migrating slugs and culminating aggregates. The results are discussed in relation to the possible morphogenetic role of extracellular cAMP in Dictyostelium cell aggregates. On the basis of the properties of the isolated cells we assume that a gradient of extracellular cAMP exists in Dictyostelium aggregates. This gradient appears to be involved in the formation and stabilization of the prestalk-prespore cell pattern.     

Stalk cell formation in monolayers of Dictyostelium discoideum V12-M2

Stalk cell formation in low-cell-density monolayers of Dictyostelium discoideum, strain V12-M2, occurs following the sequential addition of cyclic AMP and the differentiation-inducing factor (DIF). Both cyclic AMP and DIF are essential for the appearance of the prestalk-specific isozyme alkaline phosphatase-II, which suggests that both factors are necessary for prestalk cell formation. The available evidence suggests that the cyclic AMP requirement for stalk cell formation is mediated through the cell surface cyclic AMP receptor. However, stalk cell formation is inhibited by caffeine and this inhibition is reversed by the cell-permeable analogue 8-Br-cyclic AMP, which suggests in addition a possible involvement for elevated intracellular cyclic AMP concentrations in stalk cell formation. During in vivo development cells first become independent of cyclic AMP at the tipped aggregate stage, but the acquisition of cyclic AMP independence is advanced by several hours when cells are incubated in the presence of cyclic AMP for 2 hours. Cells do not become independent of DIF until the culmination stage of development, which suggests the possibility that DIF is required for the conversion of prestalk cells to stalk cells. There is an absolute requirement for DIF for stalk cell formation in low-density monolayers of prestalk cells but only part of population exhibits a requirement for cyclic AMP, which suggests that the prestalk cell population consists of two distinct cell types. Stalk cell formation from prespore cells is totally dependent on both cyclic AMP and DIF.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Developmental characterization of the wheat germ agglutinin binding proteins, wst31 and wst34, enriched in prestalk and stalk cells of Dictyostelium discoideum

Abstract. The onset of prestalk differentiation of Dictyostelium discoideum has been thought to be triggered by differentiation inducing factor (DIF), which is secreted by differentiating cells. We characterized the cell-type specific proteins, wst31 (prestalk and stalk specific) and wst34 (stalk specific), using the mutant HM44 which is defective in DIF-production, and examined the effects of DIF and cAMP on the formation of the proteins. In the mutant HM44, wst34 was formed only in the presence of exogenous DIF as reported for other prestalk/stalk markers (e.g. pDd63 and acid phosphatase-2), which indicates the DIF-requirement for this protein. By contrast, the accumulation of wst31 in this mutant occurred in the presence of cAMP regardless of the presence of exogenous DIF. Thus, we propose a new and distinct state (or stage) in prestalk differentiation, where the expression of wst31 occurs but not that of pDd63 or acid phosphatase-2.     

Control of cell type proportions by a secreted factor in Dictyostelium discoideum

It has been shown that, in Dictyostelium discoideum, conversion of prestalk cells to prespore cells in suspension cultures is inhibited by coexisting prespore cells. To examine whether the inhibition of conversion requires direct cell contact or is mediated by substances secreted by the cells, prestalk cells and prespore cells were incubated in shaken suspension, separated from each other by a dialysis membrane, and conversion of the prestalk cells to prespore cells scored after 24 h. Prestalk-to-prespore conversion was significantly inhibited if the density of the prespore cells was sufficiently high. In contrast, prestalk cells had little influence on prestalk-to-prespore conversion. Media conditioned by prespore cells, but not by prestalk cells, also inhibited the conversion of prestalk cells. Adenosine, propionate, diethylstilboestrol and differentiation inducing factor (DIF), all of which are known to influence the prestalk/prespore differentiation, were examined for their effects on prestalk-to-prespore conversion. Among these, all except adenosine significantly inhibited the conversion. Based on these results, possible mechanisms for maintenance of the constant cell-type ratio in D. discoideum slugs were discussed.     

A model for the prestalk/prespore patterning in the slug of the slime mold Dictyostelium discoideum

Abstract. We propose that the prestalk/prespore pattern in Dictyostelium is generated in two steps: In a first process, an intermingled, non-position dependent prestalk/prespore pattern is generated by a cell-restricted autocatalysis and the antagonistic action of a long-ranging substrate which becomes depleted during this autocatalysis. By computer simulations we show that the assumed interaction accounts for several experimentally observed features of the prestalk/ prespore pattern: The size-independent ratio of both cell types, the pattern regulation after removal of one cell type, the development towards one or the other pathway before the slug obtains its final shape or even before aggregation is completed. Our hypothetical substrate may be identical with an experimentally found differentiation-inducing factor (DIF). Alternative molecular realizations of the basic mechanism are discussed. A second process leads to the aggregation of the prestalk cells in a particular region of the aggregate, the future tip region. Interactions which en-able tip formation and the coupling between the prestalk/prespore and the tip-forming system are discussed. Our model shows that the formation of a single large patch of differentiated cells and its size regulation requires conflicting parameters. By a separation into a mechanism which determines the position and a second one which determines the size of a structure, each mechanism can be optimized individually without requiring compromises for the other. Such a separation also seems to occur in other developmental systems.     

Folic acid stimulation of the Gα4 G protein-mediated signal transduction pathway inhibits anterior prestalk cell development in Dictyostelium

In Dictyostelium discoideum, several G proteins are known to mediate the transduction of signals that direct chemotactic movement and regulate developmental morphogenesis. The G protein alpha subunit encoded by the Galpha4 gene has been previously shown to be required for chemotactic responses to folic acid, proper developmental morphogenesis, and spore production. In this study, cells overexpressing the wild type Galpha4 gene, due to high copy gene dosage (Galpha4HC), were found to be defective in the ability to form the anterior prestalk cell region, express prespore- and prestalk-cell specific genes, and undergo spore formation. In chimeric organisms, Galpha4HC prespore cell-specific gene expression and spore production were rescued by the presence of wild-type cells, indicating that prespore cell development in Galpha4HC cells is limited by the absence of an intercellular signal. Transplanted wild-type tips were sufficient to rescue Galpha4HC prespore cell development, suggesting that the rescuing signal originates from the anterior prestalk cells. However, the deficiencies in prestalk-specific gene expression were not rescued in the chimeric organisms. Furthermore, Galpha4HC cells were localized to the prespore region of these chimeric organisms and completely excluded from the anterior prestalk region, suggesting that the Galpha4 subunit functions cell-autonomously to prevent anterior prestalk cell development. The presence of exogenous folic acid during vegetative growth and development delayed anterior prestalk cell development in wild-type but not galpha4 null mutant aggregates, indicating that folic acid can inhibit cell-type-specific differentiation by stimulation of the Galpha4-mediated signal transduction pathway. The results of this study suggest that Galpha4-mediated signals can regulate cell-type-specific differentiation by promoting prespore cell development and inhibiting anterior prestalk-cell development.     

Two distinct classes of prestalk-enriched mRNA sequences in Dictyostelium discoideum

We have isolated cDNA clones derived from three mRNA sequences which are inducible by DIF, the putative stalk-specific morphogen of Dictyostelium. The three mRNA sequences are selectively expressed in cells on the stalk cell pathway of differentiation and we have compared them with previously characterized prestalk-enriched mRNA sequences. We find these latter sequences are expressed without a dependence on DIF, are much less highly enriched in prestalk over prespore cells and are expressed earlier during development than the DIF-inducible mRNA sequences. We propose two distinct mechanisms whereby a mRNA may become enriched in prestalk cells. An apparently small number of genes, represented by those we have isolated, is inducible by DIF and accumulates only in prestalk cells. We suggest that a second class of prestalk-enriched mRNA sequences are induced by cAMP to accumulate in all cells during aggregation and then become enriched in prestalk cells by selective loss from prespore cells.     

The role of diffusible molecules in regulating the cellular differentiation of Dictyostelium discoideum

A central problem in developmental biology is to understand how morphogenetic fields are created and how they act to direct regionalized cellular differentiation. This goal is being pursued in organisms as diverse as moulds, worms, flies, frogs and mice. Each organism has evolved its own solution to the challenge of multicellularity but there appear to be common underlying principles and, once pattern formation is fully understood in any system, some general truths seem certain to be revealed. As a non-obligate metazoan, Dictyostelium discoideum has proven a particularly tractable system in which to identify and characterize cellular morphogens. Cyclic AMP and ammonia stimulate prespore cell differentiation and ammonia plays an additional role in repressing terminal cellular differentiation. Differentiation Inducing Factor (DIF) acts to direct prestalk cell differentiation and adenosine may play a synergistic role in repressing prespore cell differentiation. This review summarizes the evidence for these interactions and describes a number of models which show how this small repertoire of diffusible molecules, acting in concert, may direct the formation of a differentiated structure.     

Dictyostelium mutants lacking DIF,a putative morphogen

DIF is an endogenous extracellular signal that may control differentiation of D. discoideum cells. It is a dialyzable, lipid-like factor that induces stalk cell formation among isolated amebae incubated in vitro with cAMP. To examine the consequences of DIF deprivation, we have isolated several mutant strains that are impaired in DIF accumulation, and whose inability to make stalk cells in vitro and during normal development on agar can be corrected by the addition of exogenous DIF. Little DIF is made by the mutants, and morphological development on agar stops after the cells have aggregated, but before a slug forms. In these DIF-deprived conditions, prespore cells can differentiate, but prestalk cells cannot.     

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.