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 .     

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.     

Stalk cell differentiation without polyketides in the cellular slime mold

Polyketides induce prestalk cell differentiation in Dictyostelium. In the double-knockout mutant of the SteelyA and B polyketide synthases, most of the pstA cells—the major part of the prestalk cells—are lost, and we show by whole mount in situ hybridization that expression of prestalk genes is also reduced. Treatment of the double-knockout mutant with the PKS inhibitor cerulenin gave a further reduction, but some pstA cells still remained in the tip region, suggesting the existence of a polyketide-independent subtype of pstA cells. The double-knockout mutant and cerulenin-treated parental Ax2 cells form fruiting bodies with fragile, single-cell layered stalks after cerulenin treatment. Our results indicate that most pstA cells are induced by polyketides, but the pstA cells at the very tip of the slug are induced in some other way. In addition, a fruiting body with a single-cell layered, vacuolated stalk can form without polyketides.     

Cell differentiation and patterning in Dictyostelium.

In Dictyostelium development, prestalk cells first differentiate at scattered positions in the aggregate and then sort out, probably by chemotaxis to cAMP. They may regulate their proportions by selective depletion of the stalk cell inducer, DIF-1. Once sorted, prestalk cells form a DIF-1 sink, which can produce gradients of DIF-1 and its metabolites in the slug. Global movements of cells in the slug may be regulated by cAMP signals, as in aggregation. Terminal differentiation of stalk and spore cells requires activation of cAMP-dependent protein kinase, possibly brought about by ammonia depletion. Finally, a technique for insertional mutagenesis promises the ready isolation of developmental genes.     

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.     

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.     

Changing patterns of gene expression in dictyostelium prestalk cell subtypes recognized by in situ hybridization with genes from microarray analyses

We used microarrays carrying most of the genes that are developmentally regulated in Dictyostelium to discover those that are preferentially expressed in prestalk cells. Prestalk cells are localized at the front of slugs and play crucial roles in morphogenesis and slug migration. Using whole-mount in situ hybridization, we were able to verify 104 prestalk genes. Three of these were found to be expressed only in cells at the very front of slugs, the PstA cell type. Another 10 genes were found to be expressed in the small number of cells that form a central core at the anterior, the PstAB cell type. The rest of the prestalk-specific genes are expressed in PstO cells, which are found immediately posterior to PstA cells but anterior to 80% of the slug that consists of prespore cells. Half of these are also expressed in PstA cells. At later stages of development, the patterns of expression of a considerable number of these prestalk genes changes significantly, allowing us to further subdivide them. Some are expressed at much higher levels during culmination, while others are repressed. These results demonstrate the extremely dynamic nature of cell-type-specific expression in Dictyostelium and further define the changing physiology of the cell types. One of the signals that affect gene expression in PstO cells is the hexaphenone DIF-1. We found that expression of about half of the PstO-specific genes were affected in a mutant that is unable to synthesize DIF-1, while the rest appeared to be DIF independent. These results indicate that differentiation of some aspects of PstO cells can occur in the absence of DIF-1.     

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.     

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

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

Separation and properties of prestalk and prespore cells of Dictyostelium discoideum

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

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.     

Monoclonal antibodies specific for stalk differentiation in Dictyostelium discoideum

We have produced two monoclonal antibodies specific to the stalk cells of Dictyostelium discoideum fruiting bodies. Both monoclonal antibodies react with high molecular weight proteins previously found to be stalk-specific by two-dimensional gel analysis. One antibody (JAb 1) is specific for a single protein of apparent molecular weight 310 000 which first appears when overt stalk differentiation begins at 20 h. The other monoclonal antibody (JAb 2) is also stalk-specific, though earlier in development it binds to proteins extracted from both prestalk and prespore cells of the migrating slug. It reacts with two proteins in stalks, one of apparent molecular weight 430 000 which is first detected during tip formation at 12 h and a lower molecular weight protein (310 000) detected from 20 h. Although several markers are available for the investigation of prespore/spore differentiation there is a distinct lack of suitable prestalk/stalk markers. The monoclonal antibodies described here are highly specific stalk markers and should prove useful in the study of cell proportioning and terminal differentiation.     

GSK3 is a multifunctional regulator of Dictyostelium development

Glycogen synthase kinase 3 (GSK3) is a central regulator of metazoan development and the Dictyostelium GSK3 homologue, GskA, also controls cellular differentiation. The originally derived gskA-null mutant exhibits a severe pattern formation defect. It forms very large numbers of pre-basal disc cells at the expense of the prespore population. This defect arises early during multicellular development, making it impossible to examine later functions of GskA. We report the analysis of a gskA-null mutant, generated in a different parental strain, that proceeds through development to form mature fruiting bodies. In this strain, Ax2/gskA-, early development is accelerated and slug migration greatly curtailed. In a monolayer assay of stalk cell formation, the Ax2/gskA- strain is hypersensitive to the stalk cell-inducing action of DIF-1 but largely refractory to the repressive effect exerted by extracellular cAMP. During normal development, apically situated prestalk cells express the ecmB gene just as they commit themselves to stalk cell differentiation. In the Ax2/gskA- mutant, ecmB is expressed throughout the prestalk region of the slug, suggesting that GskA forms part of the repressive signalling pathway that prevents premature commitment to stalk cell differentiation. GskA may also play an inductive developmental role, because microarray analysis identifies a large gene family, the 2C family, that require gskA for optimal expression. These observations show that GskA functions throughout Dictyostelium development, to regulate several key aspects of cellular patterning.     

Cell Patterning During Slug Migration and Early Culmination in Dictyostelium discoideum

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

A Temperature-Dependent Choice in Cell Differentiation

Abstract. Ascension of the cell mass during culmination was observed to be temperature-sensitive in strain HH31 of the cellular slime mold Dictyostelium discoideum . A sequence of additional developmental abnormalities was also observed both preceeding and succeeding culmination. Slugs reared at the restrictive temperature (27°C), in contrast to those reared at the permissive temperature (22°C), were deficient in the expression of six prespore markers and elevated in the production of four prestalk markers. Following attempted culmination at the restrictive temperature, the majority of cells were stalk cells according to fluorescence, phase contrast, electron microscopic, biochemical, and plating efficiency criteria. These findings were correlated with a general diminution of staining of slug cell Membrane extracts on polyacrylamide gels with FITC-wheat germ agglutinin. The effect of the higher temperature was readily reversible prior to terminal differentiation. An interdependence of several of the changes was suggested by the observation that in two derivative strains the temperature-dependence of these changes was coordinately altered. In summary, a large fraction of the slug cells of HH31 appear to be temperature-dependent in their choice of pathways of cell differentiation, this change is related to a modification of the cell surface, and the choice made in the slug is remembered by cells as they terminally differentiate.     

A study of PstB cells during Dictyostelium migration and culmination reveals a unidirectional cell type conversion process

Summary The prestalk region of the Dictyostelium slug has recently been shown by Williams and his collaborators to consist of two distinct cell types, pstA and pstB cells. Here the movement of these cells in both the slug and culmination stages has been examined with the use of vital dyes. In the slug some of the pstB cells are continually lost from the prestalk region as small clusters of cells. These cells move through the prespore region and temporarily lie in the rearguard region at the posterior end of the slug. They are finally left in the slug's slime track as single cells or groups of a few cells. When culmination is initiated the pstB cells move as a whole from the prestalk region to the base where they join the rearguard cells to form the basal disc of the fruiting body. Transplantation experiments reveal that the rearguard cells form an outer ring portion of the basal disc and the pstB cells form an inner portion to which the stalk attaches. The continuous loss of one cell type during the slug stage without any change in cell type proportions suggests that cell types are redifferentiating. Grafting and transplantation experiments reveal that there is a unidirectional flow of cells through successive steps of cell type conversion. Prespore cells redifferentiate as anterior-like cells which migrate to the prestalk region and become pstA cells. The pstA cells then replace the pstB cells that are lost from the slug.