A Dictyostelium morphogen that is essential for stalk cell formation is generated by a subpopulation of prestalk cells

The stalk cell differentiation inducing factor (DIF) has the properties required of a morphogen responsible for pattern regulation during the pseudoplasmodial stage of Dictyostelium development. It induces prestalk cell formation and inhibits prespore cell formation, but there is as yet no strong evidence for a morphogenetic gradient of DIF. We have measured DIF accumulation by monolayers of isolated prestalk and prespore cells in an attempt to provide evidence for such a gradient. DIF is accumulated in the largest quantities by a subpopulation of prestalk cells that specifically express the DIF-inducible genes pDd56 and pDd26. Since it has been shown recently that cells that express pDd56 are localized in the central core of the prestalk cell region of the pseudoplasmodia, our current results suggest a morphogenetic gradient generated by this region.     

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.     

Morphogenesis and differentiation of Dictyostelium cells interacting with immobilized glucosides: dependence on DIF production

Previous work has shown that multicellular morphogenesis of submerged Dictyostelium cells is inhibited when they bind to glucosides covalently linked to polyacrylamide gels. The amoebae aggregate normally, but then the aggregates repeatedly disperse and reaggregate, whereas control cells go on to form tight aggregates. We have investigated the role of the stalk cell differentiation inducing factors (DIFs) in this process. In the presence of cyclic AMP, amoebae submerged at high cell density accumulate DIF and differentiate into stalk cells. We find that stalk cell differentiation is inhibited by interaction of the cells with glucoside gels in these conditions, but can be restored by the addition of exogenous DIF-1. Since the responsiveness of cells to DIF-1 is not altered, it appears likely that the effect of the glucoside gel is to block DIF-1 production. Further, the addition of DIF-1 or DIF-2 stimulates the formation of tight aggregates by cells developing on glucoside gels in the absence of cyclic AMP, thus preventing the rounds of aggregation and disaggregation otherwise seen. This suggests a role for DIF in morphogenesis as well as in controlling cell differentiation. We propose a model in which immobilized glucosides activate a specific receptor ("food sensor") which drives the amoebae toward the vegetative state and inhibits DIF accumulation. DIF, on the other hand, induces tight aggregate formation and so locks the amoebae into the developmental program.     

A possible role for DIF-2 in the formation of stalk cells during Dictyostelium development.

The differentiation inducing factor (DIF) is essential for stalk cell formation in monolayers of Dictyostelium discoideum and is necessary for the expression of several prestalk cell-specific genes. DIF activity has been fractionated into a major species, designated DIF-1, and several minor species, including DIF-2. Although DIF-1 is an excellent inducer of stalk cell formation from vegetative cells, it is a poor inducer of stalk cell formation from prestalk cells. In contrast, DIF-2 is more active for the conversion of prestalk cells into stalk cells, than for the conversion of vegetative cells to stalk cells. The same results were obtained regardless of whether chemically synthesized or naturally occurring components were utilized. In addition, stalk cell formation was three- to fourfold higher when vegetative cells were incubated with DIF-1 for a suboptimal period and then subsequently incubated with DIF-2, than when cells were incubated with DIF-2 first and then subsequently with DIF-1. These results indicate a distinct role for DIF-2 during stalk cell formation and suggest the possibility that DIF-1 and DIF-2 act sequentially.     

The requirement for DIF for prestalk and stalk cell formation in Dictyostelium discoideum: a comparison of in vivo and in vitro differentiation conditions

In Dictyostelium discoideum stalk cell formation is induced by cyclic AMP and differentiation-inducing factor (DIF) when cells are plated in in vitro monolayers (Kay et al., 1979, Differentiation 13: 7-14). The in vivo developmental stages at which cells became independent of these factors were determined. Independence was defined as the stage at which dispersed cells no longer required the factors for stalk cell formation in low density monolayers. Cyclic AMP independent cells were first detected at around 12 hr of development, a time that corresponds to the transition between the tipped aggregate and the first finger stages. In contrast cells did not become independent of DIF until late culmination. The prestalk cell-specific isozyme acid phosphatase II and a stalk cell-specific 41,000 Mr antigen (ST 41) were expressed during differentiation in low density monolayers in the presence of both cyclic AMP and DIF, but neither component was expressed in the presence of cyclic AMP alone. This result implies that DIF is essential for both prestalk and stalk cell formation. The two components were expressed within 2 hr of each other during differentiation in vitro, whereas during development in vivo acid phosphatase II was first detected at the first finger stage and ST 41 was first detected during late culmination, 8-12 hr later. These contrasting results suggest that the conversion of prestalk cells to stalk cells is unrestrained in monolayers, following directly after prestalk cell induction, but restrained in vivo until the culmination stage. This interpretation is consistent with the finding that cells become independent of DIF early during in vitro differentiation (A. Sobolewski, N. Neave, and G. Weeks, 1983, Differentiation 25, 93-100), but do not become independent of DIF until the culmination stage when differentiating in vivo.     

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)     

The induction of stalk cell differentiation in submerged monolayers of Dictyostelium discoideum

Abstract. Differentiation of Dictyostelium discoideum cells in submerged monolayers was studied and compared with in vivo development. The accumulation patterns of three developmentally regulated enzymes in cells of strain V12M2 differentiating in vivo on Millipore Filters or in vitro in monolayers at high cell-densities were found to be similar. Moreover, stalk cell formation occurred at approximately the same time in high or low cell density monolayers as it did during normal differentiation. These observations suggest that the timing of differentiation in vitro and in vivo is similar.
In vitro stalk cell formation requires exogenous cyclic AMP, and in its absence, the accumulation patterns of the three developmentally regulated enzymes are alterd. At low cell densities, in vitro stalk cell induction also requires a differentiation-inducing factor (DIF). The addition or removal of cyclic AMP or DIF during development under these conditions revealed the sequence of these two requirements. Cyclic AMP is not required for stalk cell induction for the first 8 hours of incubation, but thereafter, a gradually increasing proportion of cells are induced by cyclic AMP. After a brief delay there is a period of induction by DIF, and this period corresponds approximately to the period of DIF accumulation during in vivo development. The two induction events are clearly separate, in that each inducer can act in the absence of the other, as long as cyclic AMP induction precedes DIF induction. Cyclic AMP is only required at a concentration of 40 μM when added 8 hours after the beginning of the differentiation period.     

Culmination in Dictyostelium is regulated by the cAMP-dependent protein kinase.

We placed a specific inhibitor of cyclic AMP-dependent protein kinase (PKA) under the control of a prestalk-specific promoter. Cells containing this construct form normally patterned slugs, but under environmental conditions that normally trigger immediate culmination, the slugs undergo prolonged migration. Slugs that eventually enter culmination do so normally but arrest as elongated, hairlike structures that contain neither stalk nor spore cells. Mutant cells do not migrate to the stalk entrance when codeveloped with wild-type cells and show greatly reduced inducibility by DIF, the stalk cell morphogen. These results suggest that the activity of PKA is necessary for the altered pattern of movement of prestalk cells at culmination and their differentiation into stalk cells. We propose a model whereby a protein repressor, under the control of PKA, inhibits precocious induction of stalk cell differentiation by DIF and so regulates the choice between slug migration and culmination.     

Dictyopyrones, novel alpha-pyronoids isolated from Dictyostelium spp., promote stalk cell differentiation in Dictyostelium discoideum

Dictyopyrones A and B (DpnA and B), whose function(s) is not known, were isolated from fruiting bodies of Dictyostelium discoideum. In the present study, to assess their function(s), we examined the effects of Dpns on in vitro cell differentiation in D. discoideum monolayer cultures with cAMP. Dpns at 1-20 microM promoted stalk cell formation to some extent in the wild-type strain V12M2. Although Dpns by themselves could hardly induce stalk cell formation in a differentiation-inducing factor (DIF)-deficient strain HM44, both of them dose-dependently promoted DIF-1-dependent stalk cell formation in the strain. In the sporogenous strain HM18, Dpns at 1-20 microM suppressed spore formation and promoted stalk cell formation in a dose-dependent manner. Analogs of Dpns were less effective in affecting cell differentiation in both HM44 and HM18 cells, indicating that the activity of Dpns should be chemical structure specific. It was also shown that DpnA at 2-20 microM dose-dependently suppressed spore formation induced with 8-bromo cAMP and promoted stalk cell formation in V12M2 cells. Interestingly, it was shown by the use of RT-PCR that DpnA at 10 microM slightly promoted both prespore- and prestalk-specific gene expressions in an early phase of V12M2 and HM18 in vitro differentiation. The present results suggest that Dpns may have functions (1) to promote both prespore and prestalk cell differentiation in an early stage of development and (2) to suppress spore formation and promote stalk cell formation in a later stage of development in D. discoideum.     

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.     

Plasma membrane proton pump inhibition and stalk cell differentiation in Dictyostelium discoideum

The choice of the stalk cell differentiation pathway in Dictyostelium is promoted by an endogenous substance, DIF-1, which is 1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)-1-hexanone. It is also favoured by weak acids and two inhibitors of the plasma membrane proton pumps of fungi and plants, diethylstilbestrol (DES) and zearalenone, and antagonised by ammonia and other weak bases, which promote spore differentiation. These observations led to the proposal that the choice of differentiation pathway is regulated by intracellular pH. They also prompted the conjecture that DIF-1 itself is a plasma membrane proton pump inhibitor. We report here experiments showing that DIF-1 is not a plasma membrane proton pump inhibitor. We demonstrate that diethylstilbestrol and zearalenone do inhibit the plasma membrane proton pump of Dictyostelium and we show that there is an excellent qualitative and quantitative correlation between the inhibitory activity of these agents, and of a number of other substances, and their ability to divert differentiation from the spore to the stalk pathway. We conclude that inhibition of the plasma membrane proton pump does shift the choice of differentiation pathway in Dictyostelium towards the stalk pathway, but that DIF does not act by this route, and we propose a model for the actions of DIF and plasma membrane proton pump inhibitors in which the differentiation pathway is controlled by the pH of intracellular vesicles rather than by intracellular pH itself. The model invokes a DIF- and proton-activated vesicular chloride channel whose opening permits acidification of the vesicles and lowers cytosolic Ca++ concentration.     

Studies on the accumulation of the differentiation-inducing factor (DIF) in high-cell-density monolayers of Dictyostelium discoideum

A number of factors that have been shown to influence cell type determination in Dictyostelium discoideum were assessed for their effects on the accumulation of the stalk cell differentiation-inducing factor (DIF) in high-cell-density monolayers of strain V12-M2. DIF accumulation is markedly enhanced by low pH, butyrate, and the proton pump inhibitor diethylstilbestrol (DES), conditions that induce stalk cell formation in low-cell-density monolayers in the absence of added DIF. These results are discussed in relation to a model for cell type determination recently proposed by (J.D. Gross, M.J. Peacey, and R. Pogge Von Strandmann (1988, Differentiation, 38: 91-98). DIF accumulates in high-cell-density monolayers after the cells have become independent of cyclic AMP for stalk cell formation. This accumulation is greatly enhanced by the addition of cyclic AMP. This result may explain why cyclic AMP stimulates stalk cell formation in low-density monolayers in the presence of suboptimal levels of DIF, following preincubation in the presence of saturating levels of cyclic AMP (L. Kwong, A. Sobolewski, and G. Weeks, 1988, Differentiation 37, 1-6). Adenosine has no effect on DIF accumulation in high-cell-density monolayers.     

Cyclic AMP is an inhibitor of stalk cell differentiation in Dictyostelium discoideum

Cyclic AMP and DIF-1 (1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)-1-hexanone) together induce stalk cell differentiation in vitro in Dictyostelium discoideum strain V12M2. The induction can proceed in two stages: in the first, cyclic AMP brings cells to a DIF-responsive state; in the second, DIF-1 alone can induce stalk cell formation. We report here that during the DIF-1-dependent stage, cyclic AMP is a potent inhibitor of stalk cell differentiation. Addition of cyclic AMP at this stage to V12M2 cells appreciably delays, but does not prevent, stalk cell formation. In contrast, stalk cell differentiation in the more common strain NC4 is completely suppressed by the continued presence of cyclic AMP. This fact explains earlier failures to induce stalk cells in vitro in NC4. We now consistently obtain efficient stalk cell induction in NC4 by removing cyclic AMP in the DIF-1-dependent stage. Cyclic AMP also inhibits the production of a stalk-specific protein (ST310) in both NC4 and a V12M2 derivative. Adenosine, a known antagonist of cyclic AMP action, does not relieve this inhibition by cyclic AMP and does not itself promote stalk cell formation. Finally, stalk cell differentiation of NC4 cells at low density appears to require factors in addition to cyclic AMP and DIF-1, but their nature is not yet known. The inhibition of stalk cell differentiation by cyclic AMP may be important in establishing the prestalk/prespore pattern during normal development, and in preventing the maturation of prestalk into stalk cells until culmination.     

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.     

Artificial compounds differentially control Dictyostelium chemotaxis and cell differentiation

Differentiation-inducing factor-1 and -2 (DIF-1 and DIF-2) are small lipophilic signal molecules that control both cell differentiation and chemotaxis in the cellular slime mold Dictyostelium discoideum. In this study, we examined the effects of four amide derivatives of DIF-1 on stalk cell differentiation and chemotaxis. The DIF derivatives differentially affected cell differentiation and chemotaxis, suggesting the possible existence of at least three receptors for DIFs: one receptor responsible for stalk cell induction, and two receptors responsible for chemotaxis modulation. Furthermore, our results indicate that DIF derivatives can be utilized to analyze the DIF-signaling pathways.     

Signalling pathways that direct prestalk and stalk cell differentiation in Dictyostelium

Prestalk cell differentiation in Dictyostelium is induced by DIF and two DIF-induced genes, ecmA and ecmB, have revealed the existence of multiple prestalk and stalk cell sub-types. These different sub-types are defined by the pattern of expression of subfragments derived from the ecmA and ecmB promoters. These markers have been utilised in three ways; for fate mapping in vivo, to investigate the molecular mechanisms underlying DIF signalling and to explore the relative requirement for DIF and other signalling molecules for prestalk and stalk cell differentiation in vitro. The heterogeneity of the prestalk and stalk populations seems to be reflected in differences in the cell signalling pathways that they utilise.     

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.     

盘基网柄菌中分化诱导因子信号与细胞命运决定

对盘基网柄菌发育过程中分化诱导因子(DIF)的作用及其机制进行了综述,包括DIF对盘基网柄菌前柄细胞、柄细胞分化的作用以及DIF的生物合成、DIF的诱导、降解失活、DIF对细胞命运和细胞比例的调节及其作用机制等。     

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.