Disruption of the gene encoding the cell adhesion molecule DdCAD-1 leads to aberrant cell sorting and cell-type proportioning during Dictyostelium development

The cadA gene in Dictyostelium encodes the Ca2+-dependent cell adhesion molecule DdCAD-1, which is expressed soon after the initiation of development. To investigate the biological role of DdCAD-1, the cadA gene was disrupted by homologous recombination. The cadA-null cells showed a 50% reduction in EDTA-sensitive cell adhesion. The remaining EDTA-sensitive adhesion sites were resistant to dissociation by anti-DdCAD-1 antibody, suggesting that they were distinct adhesion sites. Cells that lacked DdCAD-1 were able to complete development and form fruiting bodies. However, they displayed abnormal slug morphology and culmination was delayed by approximately 6 hours. The yield of spores was reduced by approximately 50%. The proportion of prestalk cells in cadA(-) slugs showed a 2.5-fold increase over the parental strain. When cadA(-) cells were transfected with pcotB::GFP to label prespore cells, aberrant cell-sorting patterns in slugs became apparent. When mutant prestalk cells were mixed with wild-type prespore cells, mutant prestalk cells were unable to return to the anterior position of chimeric slugs, suggesting defects in the sorting mechanism. The wild-type phenotype was restored when cadA(-) cells were transfected with a cadA-expression vector. These results indicate that, in addition to cell-cell adhesion, DdCAD-1 plays a role in cell type proportioning and pattern formation.     

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.     

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.     

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.     

Protein tyrosine phosphatase PTP1 negatively regulates Dictyostelium STATa and is required for proper cell-type proportioning

The protein tyrosine phosphatase PTP1, which mediates reversible phosphorylation on tyrosine, has been shown to play an important regulatory role during Dictyostelium development. Mutants lacking PTP1 develop more rapidly than normal, while strains that overexpress PTP1 display aberrant morphology. However, the signalling pathways involved have not been characterised. In reexamining these strains, we have found that there is an inverse correlation between levels of PTP1 activity, the extent of tyrosine phosphorylation on Dictyostelium STATa after treatment with cAMP, and the proportion of the slug population exhibiting STATa nuclear enrichment in vivo. This suggests that PTP1 acts to attenuate the tyrosine phosphorylation of STATa and downstream STATa-mediated pathways. Consistent with this, we show that when PTP1 is overexpressed, there is increased expression of a prestalk cell marker at the slug posterior, a phenocopy of STATa null slugs. In ptp1 null strains, STATa tyrosine phosphorylation and nuclear enrichment in the slug anterior is increased. There is also a change in the prestalk to prespore cell ratio. Synergy experiments suggest that this is due to a cell-autonomous defect in forming the subset of prespore cells that are located in the anterior prespore region.     

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.     

An F-Box/WD40 repeat-containing protein important for Dictyostelium cell-type proportioning, slug behaviour, and culmination

FbxA is a novel member of a family of proteins that contain an F-box and WD40 repeats and that target specific proteins for degradation via proteasomes. In fruiting bodies formed from cells where the fbxA gene is disrupted (fbxA(-) cells), the spore mass fails to fully ascend the stalk. In addition, fbxA(-) slugs continue to migrate under environmental conditions where the parental strain immediately forms fruiting bodies. Consistent with this latter behaviour, the development of fbxA(-) cells is hypersensitive to ammonia, the signaling molecule that regulates the transition from the slug stage to terminal differentiation. The slug comprises an anterior prestalk region and a posterior prespore region and the fbxA mRNA is highly enriched in the prestalk cells. The prestalk zone of the slug is further subdivided into an anterior pstA region and a posterior pstO region. In fbxA(-) slugs the pstO region is reduced in size and the prespore region is proportionately expanded. Our results indicate that FbxA is part of a regulatory pathway that controls cell fate decisions and spatial patterning via regulated protein degradation.     

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.     

Cell-type conversion in normally proportioned and prestalk-enriched populations of slug cells in Dictyostelium discoideum

Abstract. Conversion of prestalk cells to prespore cells was investigated in normally proportioned as well as prestalk-enriched cell populations under two different conditions: in slugs migrating on agar plates and in suspension cultures of dissociated slug cells in the presence of cAMP. In most experiments, prestalk cells labelled with a fluorescent dye (TRITC) and unlabelled prespore cells were combined together by grafting (for migrating slugs) or by mixing (for suspension cultures) to distinguish conversion of prestalk cells to prespore cells. In both migrating and dissociated slugs, minimal conversion of prestalk to prespore cells was observed when the proportion of prespore cells in the whole population was maintained at a normal level. When the prespore proportion in the initial population was lowered, a considerable fraction of prestalk cells underwent cell-type conversion to become prespore cells or spores. These results indicate that the presence of prespore cells somehow prevents prestalk cells from becoming prespore.     

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.     

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.     

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.     

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.     

Cell-type Conversion at Low Temperature in Dictyostelium discoideum

The effects of low temperature (5°C) on cell-type conversion in whole slugs of Dictyostelium discoideum and their anterior prestalk- and posterior prespore-isolates were examined immunohistochemically and electronmicroscopically. When slugs were incubated for nine days at 5°C, the proportion of cells containing spore-antigens increased from about 75% to 85%. More important, the proportion of prespore and spore cells increased from about 3% to 40% in anterior prestalk isolates incubated at 5°C for 12 days, but no cell-type conversion from prespore to prestalk cells occurred in posterior prespore isolates. Therefore, the mechanism regulating the proportions of cells that operates at 21 °C does not operate at low temperature. The cells with full competence for stalk differentiation could change into stalk cells even at 5°C, because a short stalk was always formed when early culminants were transferred to low temperature. The effects of low temperature on several sequential steps of cell differentiation are discussed on the basis of these findings. The ultrastructural characteristics during the process of cell-type conversion are also described.     

Differential Cell Cohesiveness Expressed by Prespore and Prestalk Cells of Dictyostelium discoideum

Pseudoplasmodia of Dictyostelium discoideum at the culmination stage were separated into two cell populations by sedimentation in a discontinuous renografin gradient. The two lighter fractions (I and II) had enzymatic activities characteristic of the anterior prestalk cells, while the heaviest fraction (III) showed enzyme activities characteristic of the posterior prespore cells. Cell-cell adhesion among prespore cells is much more resistant to EDTA dissociation than 10-h cells and prestalk cells. Fab fragments prepared from antibodies directed against a specific cell surface glycoprotein gp150 were more effective in dissociating prespore cells than prestalk cells. In addition, prespore cells contained an approximately 2-fold higher concentration of the endogenous carbohydrate binding protein discoidin-I than prestalk cells. These differences may account for the differential cohesiveness of these two cell populations and provide a basis for cell recognition and cell sorting at the slug stage.     

Developmental age-related cell sorting inDictyostelium discoideum

Summary The sorting behavior of mixtures ofD. discoideum cells which had been developed for different lengths of time was examined. Cells developed for 4 and 8 h were mixed together and allowed to form slugs. Within the slugs, 8 h cells sorted to the anterior prestalk region while 4 h cells sorted to the posterior prespore region. These results indicate that the more developed a cell is, the more likely it is to become part of the prestalk zone in the slug. They are also consistent with the differential adhesion and chemotaxis hypotheses as a mechanism for cell sorting since cells become more adhesive and chemotactically responsive as development proceeds.     

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.     

Production and turnover of cAMP signals by prestalk and prespore cells in Dictyostelium discoideum cell aggregates

Dictyostelium discoideum prestalk cells and prespore cells from migrating slugs and culminating cell aggregates were isolated by Percoll density centrifugation. Several activities relevant to the generation, detection, and turnover of extracellular cyclic AMP (cAMP) signals were determined. It was found that: the two cell types have the same basal adenylate cyclase activity; prespore cells and prestalk cells are able to relay the extracellular cAMP signal equally well; intact prestalk cells show a threefold higher cAMP phosphodiesterase activity on the cell surface than prespore cells, whereas their cytosolic activity is the same; intact prestalk cells bind three to four times more cAMP than prespore cells; no large differences in cAMP metabolism and detection were observed between cells derived from migrating slugs and culminating aggregates. The results are discussed in relation to the possible morphogenetic role of extracellular cAMP in Dictyostelium cell aggregates. On the basis of the properties of the isolated cells we assume that a gradient of extracellular cAMP exists in Dictyostelium aggregates. This gradient appears to be involved in the formation and stabilization of the prestalk-prespore cell pattern.     

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.