Developmental regulation of a prestalk- and stalk-enriched protein in Dictyostelium discoideum

Abstract. Monoclonal antibodies reactive with proteins specifically present either in the prespore cells or the prestalk cells of Dictyostelium discoideum were obtained. Four of them recognized prespore-enriched proteins, as shown by both immunoblotting assays and immunofluorescent staining. The other monoclonal antibody ( mab150 ) produced more than 10 protein bands when reacted with both prespore and prestalk cell extracts in immunoblotting assays. However, a protein band with molecular weight 35 000 (st35) was specifically detected in prestalk cells as well as mature stalk cells. St35 was solubilized from the Triton X-100 insoluble fraction of mature stalks by sodium dodecyl sulfate (SDS). The purified sample gave a single spot on two-dimensional gel electrophoresis, with pI of 5.0. During development, st35 first appeared at the tipped aggregate stage and accumulated up to stalk-cell formation without modification. The protein was not lost even when slugs were disaggregated. The importance of the tipped aggregate stage for prestalk differentiation as well as prespore differentiation is discussed.     

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.     

The Dictyostelium cell cycle and its relationship to differentiation

Abstract The Dictyostelium vegetative cell cycle is characterized by a short mitotic period followed immediately by a short S-phase (less than 30 min) and a long and variable G2 phase. The cell cycle continues during differentiation despite a decrease in cell mass: DNA replication and mitosis occur early in development and also at the tipped aggregate stage. Cells that are in mitosis, S-phase or early G2, when starved differentiate into prestalk cells and cells that are in the middle of G2 differentiate into prespore cells. We postulate that there is a restriction point late in the G2 phase, about 1–2 h before mitosis, where the cells can be arrested either by starvation and the initiation of development, by growing into stationary phase, or by prolonged incubation at low temperature. During development, this block persists to the tipped aggregate stage, where it is specifically released in prespore cells, and these cells then go through one more round of cell division. Genes encoding components of the cell cycle machinery have recently been isolated and attemps to specifically block the cell cycle by reverse genetics to study the effects on differentiation have been initiated.     

A secreted factor and cyclic AMP jointly regulate cell-type-specific gene expression in Dictyostelium discoideum.

We are studying cell differentiation in Dictyostelium discoideum by examining the regulation of genes that are preferentially expressed in different cell types. A system has been established in which prestalk- and prespore-cell-specific genes are expressed in single cells in response to culture conditions. We confirm our previous results showing that cyclic AMP induces prestalk genes and now show that it is also required for prespore gene induction. The expression of both classes of genes is additionally dependent on the presence of a factor(s) secreted by developing cells which we call conditioned medium factor(s). An assay for conditioned medium factor(s) shows that it is detectable within 2.5 h after the onset of development. Conditioned medium factor(s) also promotes the expression of genes induced early in development, but has no detectable effect on the expression of actin genes and a gene expressed maximally in vegetative cells. In the presence of conditioned medium factor(s), exogenous cyclic AMP at the onset of starvation fails to induce the prespore and prestalk genes. The addition of cyclic AMP between 2 and 12 h of starvation results in rapid prestalk gene expression, whereas prespore genes are induced at an invarient time (approximately 18 h after the onset of starvation). These data suggest that cyclic AMP and conditioned medium factor(s) are sufficient for prestalk gene induction, whereas an additional parameter(s) is involved in the control of prespore gene induction. In contrast to several previous studies, we show that multicellularity is not essential for the expression of either prespore or prestalk genes. These data indicate that prespore and prestalk genes have cell-type-specific as well as shared regulatory factors.     

Spatial expression patterns of genes involved in cyclic AMP responses in Dictyostelium discoideum development

The spatial expression patterns of genes involved in cyclic adenosine monophosphate (cAMP) responses during morphogenesis in Dictyostelium discoideum were analyzed by in situ hybridization. Genes encoding adenylyl cyclase A (ACA), cAMP receptor 1, G-protein alpha2 and beta subunits, cytosolic activator of ACA (CRAC and Aimless), catalytic subunit of protein kinase A (PKA-C) and cAMP phosphodiesterases (PDE and REG-A) were preferentially expressed in the anterior prestalk (tip) region of slugs, which acts as an organizing center. MAP kinase ERK2 (extracellular signal-regulated kinase-2) mRNA, however, was enriched in the posterior prespore region. At the culmination stage, the expression of ACA, CRAC and PKA-C mRNA increased in prespore cells in contrast with the previous stage. However, no alteration in the site of expression was observed for the other mRNA analyzed. Based on these findings, two and four classes of expression patterns were catalogued for these genes during the slug and culmination stages, respectively. Promoter analyses of genes in particular classes should enhance understanding of the regulation of dynamic and coordinated gene expression during morphogenesis.     

Mutation of the Dictyostelium fbxA gene affects cell-fate decisions and spatial patterning

Cell-fate decisions and spatial patterning in Dictyostelium are regulated by a number of genes. Our studies have implicated a gene called fbxA, which codes for an F-box protein, in these pathways. The FbxA protein is one of the controls on a cAMP phosphodiesterase called RegA, mediating its degradation via ubiquitin-linked proteolysis. Using marked strains, we showed that the fbxA^– mutant has defective cell-type proportioning, with a dearth of prestalk cells compared to prespore cells. In this work, we show that this effect occurs earlier during the 24 hour developmental cycle than previously thought. The normal sorting of the prestalk and prespore cells in aggregates and mounds is not affected by the mutation. The mutant cells sort abnormally at the tipped mound stage, when prespore and prestalk cells normally distribute into their proper compartments. The fbxA^– mutant forms prestalk cells in low numbers when not in chimeras, but in the presence of wild-type amoebae the mutant preferentially forms viable spores, driving the wild type to form non-viable stalk cells. In an attempt to identify the signal transduction pathway that mediates proportionality in prestalk and prespore cells, we asked whether certain signal transduction mutants were immune to the effects of the fbxA^–cells and formed spores in chimeras.     

Two distinct classes of prestalk-enriched mRNA sequences in Dictyostelium discoideum

We have isolated cDNA clones derived from three mRNA sequences which are inducible by DIF, the putative stalk-specific morphogen of Dictyostelium. The three mRNA sequences are selectively expressed in cells on the stalk cell pathway of differentiation and we have compared them with previously characterized prestalk-enriched mRNA sequences. We find these latter sequences are expressed without a dependence on DIF, are much less highly enriched in prestalk over prespore cells and are expressed earlier during development than the DIF-inducible mRNA sequences. We propose two distinct mechanisms whereby a mRNA may become enriched in prestalk cells. An apparently small number of genes, represented by those we have isolated, is inducible by DIF and accumulates only in prestalk cells. We suggest that a second class of prestalk-enriched mRNA sequences are induced by cAMP to accumulate in all cells during aggregation and then become enriched in prestalk cells by selective loss from prespore cells.     

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.     

Use of integrational plasmid excision to identify cellular localization of gene expression during sporulation in Bacillus subtilis.

Sporulation in Bacillus subtilis is a simple developmental system involving the differentiation of two sister cells, the prespore and the mother cell. Many of the genes that regulate sporulation (spo genes) are thought to be expressed differentially. However, direct demonstration of differential gene expression, by fractionation of prespore and mother cell proteins, is possible only at a relatively late stage of development. H. De Lencastre and P. J. Piggot (J. Gen. Microbiol. 114:377-389, 1979) have described a genetic method for determining the cellular location of the requirement for spo gene expression. Here we describe a similar method based on the use of integrational plasmids that can insertionally inactivate any given spo gene. Loss of the integrated plasmid by homologous recombination leads to the restoration of spo gene function. If this occurs just before sporulation begins, the phenotypes of the progeny of heat-resistant spores should depend on whether the gene is required in the prespore or the mother cell. Thus, we show that for known prespore-specific genes, such as spoIIIG and spoVA, only phenotypically Spo+ progeny that have lost the integrated plasmid are produced. In contrast, for mother-cell-specific genes, such as spoIIIC and spoVJ, a substantial proportion of the progeny are asporogenous, having retained the integrated plasmid. On the basis of our results, the spoIID and spoIIIA genes, which are expressed soon after division, appear to be required only in the mother cell compartment.     

Developmental regulation of cell-type-enriched mRNAs in Dictyostelium discoideum

We describe sixteen new families of cDNA clones representing mRNAs that are expressed preferentially in either prespore or prestalk cells during development of Dictyostelium discoideum and two new mRNAs that are expressed in a non-cell-type-specific manner. None of the prespore-enriched mRNAs are detectable in Dictyostelium cells until 13-15 h of development but then they increase dramatically and peak at 18-22 h. Upon dissociation of developing aggregates, all these mRNAs rapidly decay to low levels. In marked contrast to data presented for prespore genes by other workers, cyclic AMP either has no effect on the mRNA levels in dissociated cells or is only weakly effective in restoring normal expression. A prestalk-enriched mRNA examined, 5G mRNA, is similarly expressed late in development but is also expressed in vegetative cells. The level of 5G mRNA is only moderately affected by cell disaggregation.     

Re-expression of 117 antigen, a cell surface glycoprotein of aggregating cells, during terminal differentiation of Dictyostelium discoideum prespore cells

117 antigen is a glycoprotein expressed on the surface of D. discoideum cells at aggregation. It then disappears and is later re-expressed on the surface of a subpopulation of cells at culmination, the terminal differentiation stage (Sadeghi et al. 1987). A cDNA clone was used to show that the appearance of cell surface 117 antigen accurately reflects the expression of the 117 gene as measured by mRNA levels. It was also shown that during multicellular development there is a reciprocal relationship between the levels of 117 mRNA and the mRNA which codes for prespore surface glycoprotein, PsA. Dual parameter flow cytometry was used to demonstrate that the 117 antigen is found on the surface of maturing prespore cells after the PsA glycoprotein disappears, but that it is not found on mature spores. Using three monoclonal antibodies which identify respectively 117 antigen, PsA, and MUD3 antigen (a spore coat glycoprotein--probably Sp96), two new stages of final spore maturation were defined. These results indicate that there is a recapitulation of at least one aggregative cell surface glycoprotein in the prespore subpopulation of cells as they rise up the stalk during final spore development. This raises the possibility that culmination, which involves complex three dimensional morphogenetic movements not unlike those observed during animal embryogenesis, involves components of the two-dimensional pattern seen during aggregation.     

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.