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.     

Immuno-localization and separation of multiple prestalk cell types in Dictyostelium

Abstract. The ecm A and ecm B genes of Dictyostelium encode closely related extracellular matrix proteins. The major prestalk cell population, pstA cells, expresses the ecm A gene but not the ecm B gene. PstAB cells, a minor prestalk cell population that we show to express both the ecm A and ecm B genes, form a core in the centre of the slug tip. The rear, prespore region of the slug contains amoebae, termed anterior-like cells (ALC), that display many of the properties of prestalk cells. The ecm A and B genes are weakly expressed in about 30% of the ALC and these comprise a mixture of pstA cells, pstAB cells and a third class, pstB cells. The latter cell type express the ecm B gene but show no detectable expression of the ecm A gene. The demonstration of the existence of pstB cells suggests a separate pathway of ecm B gene induction, wherein expression of the ecm A gene is absent or at a very low level. Pst A, AB and B cells most probably differ in their surface properties because they are partially separable by Counter Current Distribution (CCD), a chromatographic technique which, in the conditions used, is dependent upon differences in cell surface hydrophobicity.     

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 .     

Evidence that a combined activator-repressor protein regulates Dictyostelium stalk cell differentiation.

The ecmA gene is expressed in Dictyostelium prestalk cells and is inducible by differentiation-inducing factor (DIF), a low-molecular-weight lipophilic substance. The ecmB gene is expressed in stalk cells and is under negative control by two repressor elements. Each repressor element contains two copies of the sequence TTGA in an inverted relative orientation. There are activator elements in the ecmA promoter that also contain two TTGA sequences, but in the same relative orientation. Gel retardation assays suggest that the same protein binds to the ecmB repressor and the ecmA activator. We propose that DIF induces prestalk cell differentiation by activating this protein and that the protein also binds to the promoters of stalk-specific genes, acting as a repressor that holds cells in the prestalk state until culmination is triggered.     

Origins of the prestalk-prespore pattern in Dictyostelium development

Using cell-autonomous markers we have traced the origins of prespore cells and two types of prestalk cells (pstA and pstB cells) during slug formation. We show that cell sorting and positional information both contribute to Dictyostelium morphogenesis. The initial pattern established at the mound stage is topologically quite different from that of the slug. Confirming previous studies, we find that prespore cells occupy most of the aggregate but are absent from a thin layer at the base and from the emerging tip. PstB cells are almost entirely localized to the basal region during the early stages of tip formation. Thus prespore and pstB cell differentiation appear to occur in response to localized morphogenetic signals. In the case of pstB cells, these signals presumably emanate from the base and not, as might be expected, from the tip. When first detectable, pstA cells are scattered throughout the aggregate. They then appear to migrate to the apex, where the tip forms.     

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.     

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.     

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.     

The function of PP2A/B56 in non-metazoan multicellular development

DB56, the Dictyostelium B56 homolog, displayed high sequence homology to other eukaryotic B56 subunits of the PP2A and a specific association with the PP2A catalytic subunit. Cells lacking DB56, psrA(-), displayed higher PP2A phosphatase activity compared with the wild type, approximately 10 hr of delayed expression of ecmA and ecmB prestalk markers, and inefficient culmination. The prespore marker cotB declined as wild-type cells culminate, but no such decline was observed from psrA(-) cells. Interestingly, psrA(-) cells exhibited higher GSK3 kinase activity. Furthermore, the expression of the dominant negative GSK3 mutant (K84/85M) in psrA(-) cells improved both prestalk and prespore expression patterns similarly to wild-type cells. However, culmination was not restored in psrA(-) cells expressing dominant negative GSK3, which suggests that PP2A/DB56 has an extra target during terminal differentiation. This report shows that PP2A/DB56 controls not only metazoan development, but also non-metazoan cell fate decision processes.     

Novel patterns of the gene expression regulation in the prestalk region along the antero-posterior axis during multicellular development of Dictyostelium

Simultaneous hybridization with differentially labeled fluorescent probes for in situ hybridization analysis revealed several novel expression patterns of prestalk genes during multicellular development of Dictyostelium. Seven prestalk genes and one prespore gene (pspA) were analyzed in this study. The patterns identified here indicate that prestalk cells are more heterogeneous than previously thought. Heterogeneity was observed in peripheral prestalk tissues such as the pstAO domain of a slug and the prestalk region surrounding a stalk tube of a culminant. Heterogeneity was also observed in the core pstAB cells of the slug and immature stalk cells within the stalk tube. The upper- and lower-cups of a late culminant were also composed of several subdomains.     

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.     

Prespore cell inducing factor, ψ factor,controls both prestalk and prespore gene expression in Dictyostelium development

Prespore cell‐inducing (psi, ψ) factor (PsiA), encoded by the psiA gene of Dictyostelium, is a secreted signal glycoprotein that induces prespore cell differentiation when added to monolayer cultures. In situ hybridization during normal development showed that the psiA gene is highly expressed in scattered cells at the mound stage and in prespore cells at the onset of culmination. The conventional prespore‐cell marker genes, cotC and pspA, were expressed normally in psiA^? and psiA overexpressing strains. Expressions of rnrB and cudA are repressed in the prestalk cells of a wild type slug to render prespore specific pattern. However, a promoter‐reporter fusion gene, rnrB:lacZ, showed an ectopic expression in the prestalk cells of the psiA^? strain while cudA(psp):lacZ did so in those of the psiA overexpressing strain. Overexpression of psiA delayed expression of the prestalk specific gene, ecmB, during development, while knocking out psiA promoted its expression. In addition, overexpression inhibited DIF‐1‐induced stalk formation in monolayer cultures. Together with the known prespore inducing activity, the results indicate that PsiA regulates both prespore and prestalk/stalk cell differentiation. These results indicate that PsiA is also involved in prestalk cell differentiation.     

Cell sorting within the prestalk zone of Dictyostelium discoideum

Abstract. The prestalk zone of slugs of Dictyostelium discoideum has been shown to contain three subregions in which the extracellular matrix genes ecmA and ecmB are differentially expressed; it is generally thought that these regions are defined by extracellular signals. Using β-galactosidase as a cell marker, we have shown that cells can sort specifically to all three regions. Cells from the posterior-prestalk zone ("prestalk 0 zone") which are injected into the slug tip move within 60 min back to their position of origin. When cells from the anterior prestalk zone (presumably containing a mixture of ecmA and ecmB expressers) are transplanted to the posterior prestalk zone, they move to the tip ("prestalk A zone") within 1 h and about 30 min subsequently are often found in a cone-shaped region within the tip ("prestalk B zone"). Cells transplanted to their own positions do not move significantly within this period. Since the sub-regions of the prestalk zone can be defined by sorting, it is possible that they are normally formed in this way rather than by position-dependent signals. Cells transplanted without a change in anterior-posterior position and cells which have sorted back to their positions of origin eventually spread out throughout the prestalk zone. This suggests that sorting preferences of cells are respecified. When posterior prestalk cells are transplanted to the prespore zone, respecification of sorting preference is suspended until the cells return to the prestalk zone and anterior-prestalk cells acquire posterior-prestalk sorting preferences.     

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.     

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.