Cell behavior during formation of prestalk/prespore pattern in submerged agglomerates of Dictyostelium discoideum

When cells dissociated from Dictyostelium discoideum slugs were cultured in roller tubes, they formed agglomerates in which prestalk cells were initially dispersed but soon sorted out to the center and then moved to the edge to reconstitute the prestalk/prespore pattern. To examine the mechanism of sorting out, individual prestalk cells were traced by a videotape recorder. The radial component of the rate of movement toward the center of the presumptive prestalk region was calculated. Prestalk cells did not move randomly, but rather directionally toward the center. Their movement was pulsatile, with a period of ca. 15 min, and accompanied by occasional formation of cell streams, thus resembling the movement observable during cell aggregation. These results favor the idea that prestalk cells sort out to the prestalk region due to differential chemotaxis rather than differential adhesiveness. After formation of the prestalk/prespore pattern, the prestalk region rotated along the circumference of the agglomerates. This appears comparable to migration of slugs on the substratum, the rate of rotation being similar to that of slug migration. To examine the processes of pattern formation during development, washed vegetative cells were cultured in roller tubes. Prespore cells identified by antispore immunoglobulin initially appeared randomly within the agglomerates, but then nonprespore cells accumulated in the center and finally moved to the edge to establish the prestalk/prespore pattern, the processes being similar to those of pattern reconstruction with differentiated prestalk and prespore cells.     

Evidence that positional information is used to establish the prestalk-prespore pattern in Dictyostelium discoideum aggregates

Two contrasting mechanisms have been proposed for the establishment of the prestalk-prespore pattern in the multicellular aggregate of the simple eukaryote Dictyostelium discoideum. One involves intermingled, non-position-dependent cell differentiation followed by sorting out which produces the pattern of prestalk cells in the anterior region and prespore cells posteriorly. The second mechanism involves patterning according to the position of cells within the aggregate, in which case intermingled cell types are not expected. Here we use a monoclonal antibody (MUD1), recognising a prespore cell surface antigen, to study the initial appearance of prespore cells in aggregates. Quantitative studies were made with a flow cytometer and frozen sections were used to localise the cells expressing the prespore antigen. This antigen first appeared at the onset of tip formation in the centre of aggregates in a position-dependent fashion. The prespore antigen was not detected in the tip region or in streams of cells entering the aggregate. We re-examined the evidence on which the non-position-dependent differentiation model is based. Our results support the positional model for pattern formation.     

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.     

Pattern Formation in Dictyostelium discoideum: Temporal and Spatial Distribution of Prespore Vacuoles

Cell differentiation, cell determination and pattern formation in the pseudoplasmodium of Dictyostelium discoideum , was investigated using the prespore specific vacuole (PV) as a morphological marker. Concomitantly, measurements of cell movement within the pseudoplasmodium were made by tracing radioactively labelled cells. The main results indicate that 1) prespore cells appear first during late aggregation and occur randomly throughout the pseudoplasmodium with the exception of the very tip which stays free of prespore cells throughout development; 2) after late aggregation the number of prespore cells increases over a period of several hours; 3) each prespore cell takes on a progressively more prespore-like character as judged by the increase in number of PVs it contains; 4) establishment of the distribution pattern of prespore and prestalk cells takes place within the first 2 h, mainly by a sorting out mechanism; 5) presumptive spore areas are likely to contain a small proportion of cells lacking PVs (prestalk-cells?) while presumptive stalk cell areas are homogeneous throughout; 6) maintenance of the pattern during migration may be facilitated by a circulation at low level of prestalk cells between prestalk and prespore areas; and 7) during the development of this organism the events of cell determination, cell differentiation and pattern formation overlap substantially in time.     

A SET/MYND chromatin re-modelling protein regulates Dictyostelium prespore patterning

SmdA is a Dictyostelium orthologue of the SET/MYND chromatin re-modelling proteins. In developing structures derived from a null mutant for smdA (a smdA- strain), prestalk patterning is normal, but using a prespore lacZ reporter fusion, there is ectopic accumulation of beta-galactosidase in the prestalk region. As wild type slugs migrate, there is continual forward movement and re-differentiation of prespore cells into prestalk cells. Thus, a potential explanation for the ectopic reporter localization in smdA null prestalk cells is an increased rate of re-differentiation and anterior movement of prespore cells. In support of this notion, analysis of an unstable lacZ reporter, driven by the prespore promoter, reveals a normal staining pattern in the smdA- strain. We suggest that one or more genes regulated by SmdA acts to repress prespore re-specification.     

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.     

Analysis of 5' nucleotidase and alkaline phosphatase by gene disruption in Dictyostelium

In Dictyostelium discoideum a phosphatase with a high pH optimum is known to increase in activity during cell differentiation and become localized to a narrow band of cells at the interface of prespore and prestalk cells. However, it was not clear if this activity is due to a classical "alkaline phosphatase" with broad range substrate specificity or to a "5'nucleotidase" with high substrate preference for 5'AMP. We attempted to disrupt the genes encoding these two phosphatase activities in order to determine if the activity that is localized to the interface region resides in either of these two proteins. During aggregation of 5nt null mutants, multiple tips formed rather than the normal single tip for each aggregate. In situ phosphatase activity assays showed that the wt and the 5nt gene disruption clones had normal phosphatase activity in the area between prestalk and prespore cell types, while the alp null mutants did not have activity in this cellular region. Thus, the phosphatase activity that becomes localized to the interface of the prestalk and prespore cells is alkaline phosphatase.     

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.     

Prestalk/prespore differentiation tendency of Dictyostelium discoideum cells as detected by a stalk-specific monoclonal antibody

By the use of a prestalk- and stalk-specific monoclonal antibody, production of prestalk antigen was examined with non-glucose grown [G(-)] and glucose grown [G(+)] cells of Dictyostelium discoideum AX2. Unlike wild type (NC4), some growth phase cells of AX2 were reactive with the antibody. However, G(-) cells contained much more antigen than G(+) cells and the difference between the two remained during the preaggregation period. Besides glucose, the addition of metabolizable, but not nonmetabolizable sugars to both growth phase and preaggregation cells suppressed the production of the prestalk antigen on the one hand and stimulated the accumulation of glycogen on the other hand. When mixed, G(-) cells which produced more prestalk antigen during the preaggregation period remained prestalk cells after aggregation, while G(+) cells which produced less antigen were converted to prespore cells. G(+) cells collected at the stationary phase [G(+)st] were stronger in prestalk sorting tendency than G(+) cells but weaker than G(-) cells. The prestalk antigen content of G(+)st cells prior to aggregation was an intermediate between those of G(-) and G(+) cells. These lead to the conclusion that the prestalk antigen content of preaggregation cells reflect the tendency of the cells toward either prestalk or prespore differentiation after aggregation.     

Cell type proportioning in Dictyostelium slugs: lack of regulation within a 2.5-fold tolerance range

The proportion of prestalk and prespore cells in Dictyostelium discoideum slugs is often cited as an example of "almost perfect" regulation. The pattern is similar over a very wide range of cell number; furthermore, removal of either of the cell types leads to compensatory transdifferentiation. Several studies of Dictyostelium fruiting bodies, however, have suggested that proportioning in Dictyostelium differs systematically from true constancy. We have confirmed this in the slug stage using a short-lived beta-galactosidase as a reporter of the prestalk specific ecmA gene expression: the prestalk proportion decreases from 24+/-5% in slugs of 10(3) cells to 10+/-3% when 10(5) cells are present. Regeneration experiments suggest that this difference is not due to a modulation of the proportioning set-point by size, as one might have expected; instead there appears to be a regulatory "tolerance zone" at all sizes. After amputation of the whole posterior region, transdifferentiation stops after the fraction of prestalk has been reduced from 100% to 28+/-20%, well above the initial value of 10+/-3%, while after anterior removal the transdifferentiation endpoint is about 10%. Most strikingly, we find no regulation at all after partial amputations of the prespore region. It seems that any prestalk proportion is stable between a approximately 10% lower threshold and a approximately 30% upper threshold. To explain this, we propose a regulation mechanism based on a negative feedback plus cell type bistability. In both intact and regenerating slugs we find that the slug morphology is regulated so that the length-to-width ratio of the anterior region is constant.     

Changes during differentiation in requirements for cAMP for expression of cell-type-specific mRNAs in the cellular slime mold, Dictyostelium discoideum

A number of genes encoding developmentally regulated mRNAs in the cellular slime mold, Dictyostelium discoideum, have been described. Many of these are regulated by cAMP. Analysis of the earliest time at which elevated levels of cAMP can induce the expression of these mRNAs reveals a more complex pattern of regulation in which genes change in their ability to be induced in response to cAMP with developmental stage. A prestalk mRNA (C1/D11) previously thought not be regulated by elevated levels of cAMP is inducible by cAMP between aggregation and loose mound stage; later in development its expression becomes independent of elevated cAMP. The early prespore genes (prespore class I) also show two modes of regulation; early in development they are induced independently of continuous elevated levels of cAMP, while later in development their expression is dependent upon elevated cAMP. The period during development when the prestalk genes are cAMP inducible precedes by 2 hr the first time at which either the early prespore class I or late prespore class II mRNAs are inducible by continuous elevated levels of cAMP. Previous analysis of these mRNAs has been carried out using Dictyostelium cells grown axenically. In this report we have studied the developmental expression of these mRNAs in cells grown on bacteria. A substantial shutoff of the class I prestalk and early prespore (class I) mRNAs not seen in axenically grown cells is observed when bacterially grown cells are plated for development. Less than 10% of the maximal level of these mRNAs remains in the cells at the time of mature spore and stalk differentiation. Additionally, in the bacterially grown cells two distinct patterns of developmental regulation are observed for mRNAs which in axenically growing cells appear to be constitutively expressed throughout growth and development.     

Interactions between adenosine and oscillatory cAMP signaling regulate size and pattern in Dictyostelium

We present evidence for the hypothesis that in multicellular structures of Dictyostelium, production of adenosine by hydrolysis of cAMP near the tip region prevents both generation of competing tips and differentiation of prespore cells near the tip, and thus establishes a "prestalk" region. We demonstrate that adenosine affects the immunological prespore specific staining pattern in slugs in a manner opposite to cAMP:cAMP induces an increase of prespore antigen; adenosine induces a decrease. When endogenous adenosine is removed from slugs, prespore vacuoles are synthesized throughout the prestalk region. Adenosine was found to inhibit the induction of prespore differentiation by cAMP in an apparently competitive manner. It was also found that adenosine specifically increased the amount of tissue controlled by one tip, probably by inhibiting generation of competing oscillators. Removing endogenous adenosine from slugs resulted in a decrease of tip dominance.     

Spatial and temporal expression of a Polysphondylium spore-specific gene

In the cellular slime mold Polysphondylium spherical masses of cells are periodically released from the base of the culminating sorogen. These whorls undergo a morphogenetic transformation from spherical to radial symmetry, marked by the early emergence of a radially symmetric prepattern on the whorl surface. In previous experiments, morphogenesis was followed by observing prestalk cell markers. Here we describe the isolation and characterization of a spore coat gene whose expression pattern is the negative image of the prestalk pattern. To study the molecular mechanism of sp-45 gene regulation, we have cloned and analyzed the sp-45 promoter. Deletion analysis localized a single positive regulatory element (PRE) to a 106-bp fragment between positions -246 and -352 of the upstream coding sequence. This fragment can be further divided into a promoter-proximal and promoter-distal PRE and a 29-bp sequence between them. The distal PRE can regulate prespore expression when fused to a nonfunctioning basal promoter. The distal PRE contains two adjacent essential elements, a Gr box (GTGATATAGTGG) and a TA box (TAATATATT). Each element can drive prespore cell-specific reporter gene expression independently when incorporated into a nonfunctional promoter. Our results also show that prespore cell-specific gene expression is solely under positive regulation, with no evidence for spore-specific enhancers or cis-acting negative regulatory elements. By fusing GFP to the C-terminus of sp-45, we have demonstrated that the graded gene expression of SP45 in the sorogen is regulated by a sequence lying within the sp-45 coding sequence. The temporal and spatial expression pattern of this protein, taken together with the prestalk expression pattern, demonstrates unambiguously that the radial symmetries that emerge in the whorl are established by a system of positional coordinates and that cell sorting plays little if any role in this process.     

Cell differentiation and fine structures in the development of the cellular slime molds

Changes in fine structures during the development of the cellular slime molds D. discoideum and D. mucoroides were studied, with emphasis on the regional differentiation between the prestalk and prespore cells of the slug. Cells in the prestalk region were in closer contact than those in the prespore region. Some differences were also noticed in the structure of plasma membrane between the two types of cells. An endoplasmic reticulum, vesicle, autophagic vacuole, and cytoplasmic fibril were found more abundantly in the prestalk cell than in the prespore cell. In the prespore cells there were observed a number of prespore specific vacuoles of ca. 0.6 μ diameter which consist of membraneous and fibrous structures. The vacuole was never found in the prestalk cells, and was a sole structure that existed only in one of the two types of cells. A possible function of such a vacuole was discussed in relation to spore differentiation. No differences in structure and distribution of mitochondria and crystal bodies were noticed between the prestalk and prespore cells, although these structures underwent considerable changes during the development. The nucleolus underwent considerable structual differentiation between the prestalk and prespore cells as well as during the course of development.     

A demonstration of pattern formation without positional information in Dictyostelium

Although positional information, conveyed by morphogen gradients, is a widely accepted way of forming patterns during development, an alternative method is conceivable, based on the intermingled differentiation of cells with different fates, followed by their sorting into discrete pattern elements. It has been proposed that Dictyostelium prestalk and prespore cells behave in this way at the mound stage of development. However, it has been difficult to conclusively demonstrate that they initially differentiate intermingled, because rapid cell movement within the mound makes it impossible to be sure where prestalk and prespore cells originate. We have taken a novel approach to address this problem by blocking cell movement at different stages in development, using the actin-depolymerizing drug, latrunculin-A. Prestalk and prespore cells differentiate with essentially normal efficiency and timing in such paralyzed structures. When movement is blocked sufficiently early, the major cell types all subsequently differentiate at scattered positions throughout the aggregate, and even in the streams leading into it. Our work strongly supports the idea that the prestalk/prespore pattern in Dictyostelium forms without positional information and demonstrate that latrunculin-A may provide a useful tool for the investigation of patterning in other organisms.     

cAMP production by adenylyl cyclase G induces prespore differentiation in Dictyostelium slugs

Encystation and sporulation are crucial developmental transitions for solitary and social amoebae, respectively. Whereas little is known of encystation, sporulation requires both extra- and intracellular cAMP. After aggregation of social amoebae, extracellular cAMP binding to surface receptors and intracellular cAMP binding to cAMP-dependent protein kinase (PKA) act together to induce prespore differentiation. Later, a second episode of PKA activation triggers spore maturation. Adenylyl cyclase B (ACB) produces cAMP for maturation, but the cAMP source for prespore induction is unknown. We show that adenylyl cyclase G (ACG) protein is upregulated in prespore tissue after aggregation. acg null mutants show reduced prespore differentiation, which becomes very severe when ACB is also deleted. ACB is normally expressed in prestalk cells, but is upregulated in the prespore region of acg null structures. These data show that ACG induces prespore differentiation in wild-type cells, with ACB capable of partially taking over this function in its absence.     

A Ca2+-dependent signal transduction system participates in coupling expression of some cAMP-dependent prespore genes to the cell surface receptor

Elevated levels of cAMP are essential for the expression of many postaggregation prespore and prestalk mRNA species and for the suppression of some growth phase mRNAs. Here we review evidence that this regulation is mediated by cAMP interacting at the cell surface receptor. These effects of cAMP on gene expression can occur under conditions where the receptor-associated adenylate cyclase is inactivated and in concentrations that are consistent with receptor-binding. A number of differences are noted in the mechanism by which cAMP regulates prespore and prestalk genes. Finally, evidence is reviewed for the role of a Ca2+-dependent signal transduction system in coupling the expression of some of the prespore mRNAs to the cAMP receptor. This signal transduction system does not appear to be involved in the expression of the cAMP-dependent prestalk gene.     

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.