Cellular and subcellular distribution of a cAMP-regulated prestalk protein and prespore protein in Dictyostelium discoideum: a study on the ontogeny of prestalk and prespore cells

We have analyzed a developmentally and spatially regulated prestalk-specific gene and a prespore-specific gene from Dictyostelium. The prestalk gene, pst-cathepsin, encodes a protein highly homologous to the lysosomal cysteine proteinases cathepsin H and cathepsin B. The prespore gene encodes a protein with some homology to the anti-bacterial toxin crambin and has been designated beejin. Using the lambda gtll system, we have made polyclonal antibodies directed against a portion of the protein encoded by pst-cathepsin and other antibodies directed against the beejin protein. Both antibodies stain single bands on Western blots. By immunofluorescence and Western blots, pst-cathepsin is not present in vegetative cells or developing cells during the first approximately 10 h of development. It then appears with a punctate distribution in a subset of developing cells. Beejin is detected only after approximately 15 h of development, also in a subset of cells. Pst-cathepsin is distributed in the anterior approximately 1/10 of migrating slugs and on the peripheral posterior surfaces of slugs. Beejin is distributed in the posterior region of slugs. Expression of both pst-cathepsin and beejin can be induced in subsets of isolated cultured cells by a combination of conditioned medium and extracellular cAMP in agreement with the regulation of the mRNAs encoding these proteins. We have used the antibodies as markers for cell type to examine the ontogeny and the spatial distribution of prestalk and prespore cells throughout multicellular development. Our findings suggest that prestalk cell differentiation is independent of position within the aggregate and that the spatial localization of prestalk cells within the multicellular aggregate arises from sorting of the prestalk cells after their induction. We have also found a class of cell in developing aggregates that contains neither the prestalk nor the prespore markers.     

Spatiotemporal Patterning of discoidin I and II during Development of Dictyostelium discoideum

We have examined the distribution of Dictyostelium lectins (discoidin I and II) during development by means of a sample preparation method of a whole mount. Monoclonal antibodies which were bound to discoidins revealed unique patterns of discoidin distribution. Discoidin I was localized mainly at the periphery of the aggregates, while the base of the aggregates was devoid of discoidin I staining. Discoidin I was not prominent in the body of the aggregates but when a migrating slug culminated, discoidin I staining appeared in the prestalk region, this suggested that prestalk cells begin to express discoidin I at the onset of culmination. During fruit formation we observed discoidin I staining at the foremost anterior prestalk region of the culminant, which implies a heterogeneity of discoidin I expression among prestalk cells; such a heterogenous pattern has also been found in other prestalk-specific proteins. In addition, anterior-like cells (ALC), which were sorted at the apex and basal parts of a spore mass during culmination, were also strongly stained with anti-discoidin I mAb; interestingly, we observed the staining of ALC from the slug stage through fruit formation. No discoidin II was observed in a migrating slug that had already accumulated prespore antigen ligands for discoidin II; it appeared in prespore cells after the onset of culmination. The present results indicate that, in addition to the early expression of discoidin I, both discoidin I and II are expressed during culmination, and these lectins also seem to be involved in the late development of Dictyostelium .     

Control of cell type proportioning in Dictyostelium discoideum by differentiation-inducing factor as determined by in situ hybridization

We have determined the proportions of the prespore and prestalk regions in Dictyostelium discoideum slugs by in situ hybridization with a large number of prespore- and prestalk-specific genes. Microarrays were used to discover genes expressed in a cell type-specific manner. Fifty-four prespore-specific genes were verified by in situ hybridization, including 18 that had been previously shown to be cell type specific. The 36 new genes more than doubles the number of available prespore markers. At the slug stage, the prespore genes hybridized to cells uniformly in the posterior 80% of wild-type slugs but hybridized to the posterior 90% of slugs lacking the secreted alkylphenone differentiation-inducing factor 1 (DIF-1). There was a compensatory twofold decrease in prestalk cells in DIF-less slugs. Removal of prespore cells resulted in cell type conversion in both wild-type and DIF-less anterior fragments. Thus, DIF-1 appears to act in concert with other processes to establish cell type proportions.     

Induction and modulation of cell-type-specific gene expression in Dictyostelium

We have identified genes that are expressed preferentially in either prestalk or prespore cells in Dictyostelium. The prestalk mRNAs are detectable at 7.5 hr prior to the completion of cell aggregation, while the prespore mRNAs are not detectable until approximately 15 hr of development. Exogenous cAMP in the absence of sustained cell contact is sufficient to induce prestalk-specific gene expression, while multicellularity is required for the induction of prespore-specific genes. A gene expressed equally in both cell types, which has the same developmental kinetics as the prestalk genes, is induced in shaking culture in the absence of either cAMP or stable cell associations. Dissociation of aggregates results in the rapid loss of prespore- and prestalk-specific mRNAs, and these can be induced to reaccumulate with the addition of cAMP. We conclude that there are substantial differences in the timing and requirements for tissue-specific gene expression in Dictyostelium.     

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.     

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.     

Regulation of the anterior-like cell state by ammonia in Dictyostelium discoideum

Ammonia appears to be an important regulatory signal for several aspects of the Dictyostelium life cycle. The postulated role of ammonia in the determination of the prespore pathway in cells of the slug stage has led us to examine the effect of ammonia on the prestalk/prespore ratio of migrating slugs. In the presence of 10(-3) M ammonium chloride, the volume of the prestalk region decreases by 40.8%. The kinetics of the process make it unlikely that this is due to a shift in the differentiation pathway. A test of the hypothesis that the decrease in volume of the prestalk region is due to the conversion of prestalk cells to anterior-like cells shows that the percent of anterior-like cells in the posterior region increases by the amount predicted by the hypothesis. This suggests that ammonia may be the molecular signal, produced by the tip, that prevents anterior-like cells from chemotactically migrating to the tip and thereby becoming anterior cells. The effect of enzymatic removal of ammonia from vitally stained migrating slugs is the appearance of a series of dark stripes beginning at the posterior end and progressing forward. We interpret this as a result of progressive removal of anterior-like cells from tip dominance and essentially as the formation of new potential tips. Indeed, in a few cases one or even two of the stripes separate from the posterior of the cell mass and form small fruiting bodies. We consider the phenomenon of stripe formation further evidence that the tip acts on anterior-like cells through ammonia.     

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.     

Altered cell-type proportioning in Dictyostelium lacking adenosine monophosphate deaminase.

The proportions of prespore and prestalk cells in Dictyostelium discoideum are regulated so that they are size invariant and can adjust when the ratio is perturbed. We have found that disruption of the gene amdA that encodes AMP deaminase results in a significantly increased proportion of prestalk cells. Strains lacking AMP deaminase form short, thick stalks and glassy sori with less than 5% the normal number of spores. The levels of prestalk-specific mRNAs in amdA(-) cells are more than twice as high as those in wild-type strains and prespore-specific mRNAs are reduced. Using an ecmA::lacZ construct to mark prestalk cells, we found that amdA(-) null slugs have twice the normal number of prestalk cells. The number of cells expressing an ecmO::lacZ construct was not affected by loss of AmdA, indicating that the mutation results in an increase in PST-A prestalk cells rather than PST-O cells. This alteration in cell-type proportioning is a cell-autonomous consequence of the loss of AMP deaminase since mutant cells developed together with wild-type cells still produced excess prestalk cells and wild-type cells carrying the ecmA::lacZ construct formed normal numbers of prestalk cells when developed together with an equal number of amdA(-) mutant cells.     

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.     

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.     

Cyclic AMP and NH3/NH4+ both regulate cell-type-specific mRNA accumulation in the cellular slime mold, Dictyostelium discoideum

Dictyostelium discoideum cells plated for development until aggregation stage, and then dissociated into media containing glucose, albumin, and cAMP will form into clumps and undergo prespore and prestalk differentiation. Differentiation in this in vitro system is dependent on three components: cAMP, multicellularity, and the acquisition of "differentiation competence" which the cells acquire in a period between interphase and aggregation stage when plated on Millipore filters. We have used this system to explore aspects of the multicellular environment which are involved in regulation the accumulation of the different prespore- and prestalk-specific messenger RNAs. Two classes of prespore messenger RNA, as well as a prestalk-specific messenger RNA, all require the acquisition of differentiation competence in order to be expressed in response to cAMP. Additionally, all of these messenger RNAs require agglomerate formation for maximal expression. The addition of 33 mM ammonium sulfate (NH4)2SO4, however, can entirely replace the requirement for agglomerate formation for expression of the prestalk-specific messenger RNA, and can partially substitute for agglomerate formation in inducing the expression of both classes of prespore-specific messenger RNAs. In this system, cAMP is essential for the initial induction of expression of all three classes of messenger RNAs. In this system, cAMP is essential for the initial induction of expression of all three classes of messenger RNAs while agglomerate formation or elevated NH3/NH+4 is essential only for the maintenance of the elevated levels of the messenger RNAs.     

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.     

Lithium respecifies cyclic AMP-induced cell-type specific gene expression in Dictyostelium

We investigated the effect of LiCl on pattern formation and cAMP-regulated gene expression in Dictyostelium discoideum. In intact slugs, 5 mM LiCl induces an almost complete redifferentiation of prespore into prestalk cells. We found that LiCl acts by interfering with the transduction of extracellular cAMP to cell-type-specific gene expression; LiCl inhibits the induction of prespore-specific gene expression by cAMP, while it promotes the induction of prestalk-associated gene expression by cAMP. Our results indicate that two divergent pathways transduce the extracellular cAMP signal to, respectively, prestalk and prespore gene expression.     

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.     

The regulative capacity of prespore amoebae as demonstrated by fluorescence-activated cell sorting and green fluorescent protein

The ability of prespore Dictyostelium discoideum amoebae to undergo redifferentiation so as to reestablish normal spore/stalk proportioning has been demonstrated in various ways over the years, beginning with the classic microdissection work of K. Raper. The discovery of anterior-like cells in the slug posterior, however, cast doubt on that ability, and more recent experiments using a cell-specific toxin suggested that prespore redifferentiation may not in fact occur. To reexamine this question, we performed fluorescence-activated cell sorting (FACS) upon amoebae expressing a mutated green fluorescent protein gene (S65T-GFP) under the control of a prespore-specific (PsA) promoter. FACS produced prespore cell populations with purities, measured by GFP expression, as high as 99. 5%. Sorted GFP(+) cells were developmentally competent and produced normally proportioned fruits, indistinguishable from those of "sham-sorted" (permissively gated, mixed GFP(+) and GFP(-)) amoebae. This result confirms the developmental totipotency of prespore amoebae.     

DIF-Resistant Expression of a Prespore-Specific Gene in Dictyostelium in Vitro Development

In submerged culture, the prespore-specific gene, D19, of Dictyostelium discoideum was found to be expressed in the early stages of development, even in the presence of 1 nM of DIF-1 (stalk differentiation inducing factor). This concentration of DIF-1 later caused the degradation of the previously accumulated D19 mRNA concomitant with the induction of the prestalk-specific genes ecmA/ecmB and eventually 85% of cells differentiated into stalk cells. These results suggest that prespore differentiation occurs at least transiently even in the presence of DIF-1.