Prespore-to-stalk conversion involves the production of a pathway-specific glycoprotein, wst25, in the cellular slime mould Dictyostelium discoideum

We have previously identified a stalk-specific wheat germ agglutinin (WGA)-binding protein, wst34, in the cellular slime mould Dictyostelium discoideum [Biochem. Cell Biol. 68 (1990) 699]. Here, we found another stalk-specific WGA-binding protein, wst25, which was detected with two antisera that recognize wst34. Using the two marker proteins, we then analyzed and compared the pathways of prestalk-to-stalk maturation and prespore-to-stalk conversion in vitro and in vivo. Prestalk cells isolated from normally formed slugs can be converted to stalk cells (designated StI) in vitro with 8-bromo-cAMP (Br-cAMP), whereas prespore cells isolated from slugs can be converted to fully vacuolated stalk cells (designated StII) in vitro with Br-cAMP and DIF-1. During the process of prespore-to-stalk conversion, prespore-specific mRNAs, D19 and 2H3, disappeared rapidly, while prestalk-specific mRNAs, ecmA and ecmB, appeared at 2h of incubation and increased thereafter. Most importantly, however, the StII cells thus formed were biochemically different from the StI cells originated from prestalk cells; that is, StI cells expressed wst34 but not wst25, while StII cells expressed wst25 but not wst34. When prespore cells isolated from slugs were allowed to develop on a substratum, they differentiated into spores and stalk cells and formed fruiting bodies, and the stalk cells formed from prespore cells in vivo expressed wst25 but not wst34. The present results indicate that there are two types of stalk cells, StI (prestalk-origin) and StII (prespore-origin), and that wst34 and wst25 are the specific markers for StI and StII, respectively.     

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.     

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.     

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.     

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.     

A stalk-specific wheat germ agglutinin binding protein, wst34, in Dictyostelium discoideum can be detected with antiserum raised against Dictyostelium mucoroides stalk

A new stalk-specific wheat germ agglutinin (WGA) binding protein, wst34, has been identified in Dictyostelium discoideum and purified by the use of preparative sodium dodecyl sulfate - polyacrylamide gel electrophoresis and a WGA-affinity column. In normal development, wst34 appears during culmination and is maintained in stalk cells. It has a molecular mass of 34 kilodaltons and a pI value of 5.5-6.5. A polyclonal antiserum raised against stalk cell proteins of Dictyostelium mucoroides recognizes wst34 in western blots of D. discoideum proteins.     

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.     

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.     

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.     

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.     

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.     

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.     

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。     

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.