Opposite effects of adenosine on two types of cAMP-induced gene expression in Dictyostelium indicate the involvement of at least two different intracellular pathways for the transduction of cAMP signals

Adenosine promotes the cAMP-induced increase of mRNAs, probed with the cDNAs D11 and D14, which are preferentially expressed in prestalk cells, while it inhibits cAMP-induced prespore gene expression. Half-maximal inhibition of prespore gene expression occurs at about 300 muM, while prestalk stimulation by adenosine occurs at about 100-fold lower concentrations and requires the presence of cAMP. These results indicate that adenosine interferes with the transduction to cAMP to gene expression and suggest the involvement of two different adenosine target sites. Our data furthermore indicate that the transduction of extracellular cAMP to prespore gene or prestalk gene expression occurs via divergent pathways.     

Induction and modulation of cell-type-specific gene expression in Dictyostelium

We have identified genes that are expressed preferentially in either prestalk or prespore cells in Dictyostelium. The prestalk mRNAs are detectable at 7.5 hr prior to the completion of cell aggregation, while the prespore mRNAs are not detectable until approximately 15 hr of development. Exogenous cAMP in the absence of sustained cell contact is sufficient to induce prestalk-specific gene expression, while multicellularity is required for the induction of prespore-specific genes. A gene expressed equally in both cell types, which has the same developmental kinetics as the prestalk genes, is induced in shaking culture in the absence of either cAMP or stable cell associations. Dissociation of aggregates results in the rapid loss of prespore- and prestalk-specific mRNAs, and these can be induced to reaccumulate with the addition of cAMP. We conclude that there are substantial differences in the timing and requirements for tissue-specific gene expression in Dictyostelium.     

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.     

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.     

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.     

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-type-specific responsiveness to cAMP in cell differentiation of Dictyostelium discoideum.

In Dictyostelium discoideum, both prespore and prestalk differentiation require extracellular cAMP. We investigated the difference in inducibility of the two cell types by cAMP. Previous studies indicate that cAMP added in the early stage of development inhibits prespore differentiation, and this was confirmed using three species of prespore specific mRNAs. By contrast, early treatment with cAMP did not inhibit, but induced the expression of prestalk-specific mRNA. These results indicate that differentiation pathways of the two cell types have different processes in the early stage of 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.     

Temperature-sensitive Gbeta mutants discriminate between G protein-dependent and -independent signaling mediated by serpentine receptors.

Deletion of the single gene for the Dictyostelium G protein beta-subunit blocks development at an early stage. We have now isolated temperature-sensitive alleles of Gbeta to investigate its role in later development. We show that Gbeta is directly required for adenylyl cyclase A activation and for morphogenetic signaling during the entire developmental program. Gbeta was also essential for induction of aggregative gene expression by cAMP pulses, a process that is mediated by serpentine cAMP receptors (cARs). However, Gbeta was not required for cAR-mediated induction of prespore genes and repression of stalk genes, and neither was Gbeta needed for induction of prestalk genes by the differentiation inducing factor (DIF). cAMP induction of prespore genes and repression of stalk genes is mediated by the protein kinase GSK-3. GSK-3 also determines cell-type specification in insects and vertebrates and is regulated by the wingless/wnt morphogens that are detected by serpentine fz receptors. The G protein-dependent and -independent modes of cAR-mediated signaling reported here may also exist for the wingless/wnt signaling pathways in higher organisms.     

Combinatorial control of cell differentiation by cAMP and DIF-1 during development of Dictyostelium discoideum

At least three distinct types of cell arise from a population of similar amoebae during Dictyostelium development: prespore, prestalk A and prestalk B cells. We report evidence suggesting that this cellular diversification can be brought about by the combinatorial action of two diffusible signals, cAMP and DIF-1. Cells at different stages of normal development were transferred to shaken suspension, challenged with various combinations of signal molecules and the expression of cell-type-specific mRNA markers measured 1-2 h later. pDd63, pDd56 and D19 mRNAs were used for prestalk A, prestalk B and prespore cells respectively. We find the following results. (1) Cells first become responsive to DIF-1 for prestalk A differentiation and to cAMP for prespore differentiation at the end of aggregation, about 2 h before these cell types normally appear. (2) At the first finger stage of development, when the rate of accumulation of the markers is maximal, the expression of each is favoured by a unique combination of effectors: prespore differentiation is stimulated by cAMP and inhibited by DIF-1; prestalk A differentiation is stimulated by both cAMP and DIF-1 and prestalk B differentiation is stimulated by DIF-1 and inhibited by cAMP. (3) Half-maximal effects are produced by 10-70 nM DIF-1, which is in the physiological range. (4) Ammonia and adenosine, which can affect cell differentiation in other circumstances, have no significant pathway-specific effect in our conditions. These results suggest that cell differentiation could be brought about in normal development by the localized action of cAMP and DIF-1.     

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.     

Dual Effects of cAMP on the Stability of Prespore Vesicles and 8-bromo cAMP-enhanced Maturation of Spore and Stalk Cells of Dictyostelium discoideum

It is well known that interconversion between prestalk and prespore cells occurs in 3-dimensional (3–D) isolates of Dictyostelium. The present work was undertaken to examine whether or not the interconversion occurs even in monolayer sheets. The results suggested that in monolayer sheets of either prespore or prestalk cells, the interconversion does not occur. Furthermore, effects of cAMP were examined in relation to the formation or loss of prespore vesicles (PSVs). In monolayer sheets, prespore cells retain their PSVs in the presence of cAMP, though they lose them in its absence. In 3–D masses, however, cAMP induces the conversion into stalk cells, stimulating PSV loss. In the case of prestalk cells, cAMP induces the maturation of prestalk cells to stalk cells in 3–D masses, but it does not induce stalk differentiation in monolayer sheets.
8-Bromo cAMP stimulates the maturation of prespore and prestalk cells into spore and stalk cells, respectively. However, the vegetative and the aggregative cells remain amoeboid even in its presence. These observations suggest that 8-bromo cAMP stimulates the maturation rather than inducing prespore and prestalk differentiation.     

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.     

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.     

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.     

Cell-type-specific genes expressed late in Dictyostelium development show markedly different responses to 3'' 5'' cyclic AMP

The maintenance of late gene expression by 3'5' cyclic AMP was re-examined using several newly isolated cell-type-specific genes. Expression of all the prespore-enriched genes ceased immediately upon disaggregation of developing cells and pre-existing mRNA was rapidly degraded. For most genes, cAMP had little or no effect either alone or in combination with conditioned medium factors. The expression of the non-cell-type-specific genes 7E and 2C also ceased upon cell disaggregation but cAMP triggered a full re-induction of expression although the timing of the response differed markedly between these two genes. In contrast to earlier interpretations, these data argue that for none of these late prespore genes is cAMP alone sufficient for the maintenance of expression. The responses of the two prestalk mRNAs examined were gene-specific. Prestalk 5D mRNA decayed slowly upon disaggregation and was partially stabilized by cAMP whereas prestalk 5G mRNA increased upon disaggregation and was inhibited by cAMP.