cAMP-controlled gene expression in Dictyostelium mutants exhibiting abnormal patterns of developmental cAMP metabolism

We have previously described the developmental regulation of the M4-1 gene in Dictyostelium. M4-1 is expressed in undifferentiated, vegetative cells but is repressed after the establishment of the cAMP signal and relay system. We have suggested that regulation is dependent upon the establishment of intracellular cAMP signaling. We have now extended these studies of M4-1 developmentally regulated gene expression to a series of mutants that exhibit abnormal patterns of cAMP metabolism. These include cAMP signal deficient mutants and mutants which accumulate abnormally high or low levels of intracellular and/or extracellular cAMP. Mutant cells able to establish aggregates in the absence of normal cAMP signaling are unable to repress M4-1 expression. Rather these cells continue to express M4-1 at vegetative levels. In another mutant which displays precocious cAMP signaling, repression of M4-1 occurs more rapidly than is normal. A third mutant, with decreased signaling activity, exhibits delayed repression of M4-1 expression. Taken in conjunction with our previous results, these data suggest an intimate association between M4-1 gene regulation and changes in intracellular cAMP levels and that certain genes in Dictyostelium may be regulated by a cAMP-dependent mechanism which is common to other eukaryotes.     

Dictyostelium discoideum: a model system for differentiation and patterning

In Dictyostelium, development begins with the aggregation of free living amoebae, which soon become organized into a relatively simple organism with a few different cell types. Coordinated cell type differentiation and morphogenesis lead to a final fruiting body that allows the dispersal of spores. The study of these processes is having increasing impact on our understanding of general developmental mechanisms. The availability of biochemical and molecular genetics techniques has allowed the discovery of complex signaling networks which are essential for Dictyostelium development and are also conserved in other organisms. The levels of cAMP (both intracellular and extracellular) play essential roles in every stage of Dictyostelium development, regulating many different signal transduction pathways. Two-component systems, involving histidine kinases and response regulators, have been found to regulate intracellular cAMP levels and PKA during terminal differentiation. The sequence of the Dictyostelium genome is expected to be completed in less than two years. Nevertheless, the available sequences that are already being released, together with the results of expressed sequence tags (ESTs), are providing invaluable tools to identify new and interesting genes for further functional analysis. Global expression studies, using DNA microarrays in synchronous development to study temporal changes in gene expression, are presently being developed. In the near future, the application of this type of technology to the complete set of Dictyostelium genes (approximately 10,000) will facilitate the discovery of the effects of mutation of components of the signaling networks that regulate Dictyostelium development on changes in gene expression.     

A pleiotropic defect in cAMP-regulated gene expression in the Dictyostelium agg- mutant synag 7

During differentiation of Dictyostelium discoideum, cAMP functions as a diffusible, extracellular signal to direct chemotaxis and regulate developmental gene expression. The availability of signal-transduction mutants of Dictyostelium now makes it feasible to pursue a genetic analysis of cAMP signaling. The synag 7 mutant is defective in receptor-mediated adenylate cyclase stimulation and cannot relay a cAMP signal. To further characterize this mutant, mRNA levels of several cAMP-regulated genes were measured during development. cAMP-regulated gene expression was found to be dramatically altered in synag 7:several different genes which require cAMP for expression in wild-type cells were induced in synag 7 in the absence of cAMP. In addition, the gene-encoding discoidin I, which is normally expressed in starved cells and repressed by cAMP, is expressed at very low levels in starved synag 7 cells, possibly due to precocious repression. These results suggest that a pleiotropic regulator of cAMP-regulated gene expression is uncoupled from its normal controls during development in synag 7.     

Regulation of gene expression by the intracellular second messengers IP3 and diacylglycerol

In Dictyostelium, extracellular cAMP interacts specifically with cell-surface receptors to promote the accumulation of a variety of intracellular second messengers, such as 3'-5' cyclic adenosine monophosphate (cAMP) and 1,4,5 inositol trisphosphate (IP3). We and others have shown that activation of the cell-surface cAMP receptor can also modulate the expression of the Dictyostelium genome during development. In at least one instance, synthesis of intracellular cAMP is required for appropriate gene regulation. However, the induction of most cAMP-dependent gene expression can occur in the absence of receptor-mediated activation of adenylate cyclase and a consequent accumulation of intracellular cAMP. These results suggest that other intracellular second messengers produced in response to receptor activation may potentially act as signal transducers to modulate gene expression during development. In vertebrate cells, IP3 and diacylglycerol (DAG) are intracellular activators of specific protein kinases; they are produced in equimolar amounts by cleavage of phosphoinositol bisphosphate after a receptor-mediated activation of a membrane-bound phosphodiesterase. IP3 and, thus, by inference, diacyl-glycerol are synthesized in Dictyostelium as a response to cAMP interacting with its cell-surface receptor. Using defined conditions to inhibit the accumulation of extracellular cAMP, we have examined the effects of these compounds on the expression of genes that require cAMP for their maximal expression. Our results suggest that intracellular IP3 and DAG may in part mediate the action of extracellular cAMP on the expression of the Dictyostelium genome.     

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 Regulation of Dictyostelium Development by Transmembrane Signalling

ABSTRACT. Dictyostelium discoideum has a well characterized life cycle where unicellular growth and multicellular development are separated events. Development is dependent upon signal transduction mediated by cell surface, cAMP receptor/G protein linkages. Secreted cAMP acts extracellularly as a primary signal and chemoattractant. There are 4 genes for the distinct cAMP receptor subtypes, CAR1, CAR2, CAR3 and CAR4. These subtypes are expressed with temporally and spatially specific patterns and cells carrying null mutations for each gene have distinct developmental phenotypes. These results indicate an essential role for cAMP signalling throughout Dictyostelium development to regulate such diverse pathways as cell motility, aggregation (multicellularity), cytodifferentiation, pattern formation and cell type-specific gene expression.     

Cell-cell contact mediates cAMP secretion in Dictyostelium discoideum.

Cyclic adenosine 3':5' monophosphate (cAMP) and cell-cell contact regulate developmental gene expression in Dictyostelium discoideum. Developing D. discoideum amoebae synthesize and secrete cAMP following the binding of cAMP to their surface cAMP receptor, a response called cAMP signaling. We have demonstrated two responses of developing D. discoideum amoebae to cell-cell contact. Cell-cell contact elicits cAMP secretion and alters the amount of cAMP secreted in a subsequent cAMP signaling response. Depending upon experimental conditions, bacterial-amoebal contact and amoebal-amoebal contact can enhance or diminish the amount of cAMP secreted during a subsequent cAMP signaling response. We have hypothesized that cell-cell contact regulates D. discoideum development by altering cellular and extracellular levels of cAMP. To begin testing this hypothesis, these responses were further characterized. The two responses to cell-cell contact are independent, i.e., they can each occur in the absence of the other. The responses to cell-cell contact also have unique temperature dependences when compared to each other, cAMP signaling, and phagocytosis. This suggests that these four responses have unique steps in their transduction mechanisms. The secretion of cAMP in response to cell-cell contact appears to be a non-specific response; contact between D. discoideum amoebae and Enterobacter aerogenes, latex beads, or other amoebae elicits cAMP secretion. Despite the apparent similarities of the effects of bacterial-amoebal and amoebal-amoebal contact on the cAMP signaling response, this contact-induced response appears to be specific. Latex beads addition does not alter the magnitude of a subsequent cAMP signaling response.(ABSTRACT TRUNCATED AT 250 WORDS)     

cAMP regulation of early gene expression in signal transduction mutants of Dictyostelium

We have examined the regulation of three early developmentally regulated genes in Dictyostelium. Two of these genes (D2 and M3) are induced by pulses of cAMP and the other (K5) is repressed. Expression of these genes has been examined in a number of developmental mutants that are specifically blocked in various aspects of the signal transduction/cAMP relay system involved in aggregation and control of early development. The mutant strains include Synag mutants, which are blocked in receptor-mediated activation of adenylate cyclase and do not relay cAMP pulses; FrigidA mutants, which are blocked in receptor-mediated activation of both adenylate cyclase and the putative phosphoinositol bisphosphate (PIP2) turnover pathway and appear to be mutations in the gene encoding one of the G alpha protein subunits; and a StreamerF allele, which lacks cGMP-specific cGMP phosphodiesterase. From the analysis of the developmental expression of these genes under a variety of conditions in these mutant strains, we have drawn a number of conclusions concerning the modes of regulation of these genes. Full induction of D2 and M3 genes requires cAMP interaction with the cell surface receptor and an "oscillation" of the receptor between active and adapted forms. Induction of these genes does not require activation of the signal transduction pathway that leads to adenylate cyclase activation and cAMP relay, but does require activation of other receptor-mediated intracellular signal transduction pathways, possibly that involving PIP2 turnover. Likewise, repression of the K5 gene requires pulses of cAMP. Expression of this gene is insensitive to cAMP pulses in FrigidA mutants, suggesting that a signal transduction pathway is necessary for its repression. Results using the StreamerF mutant suggest that the rise in cGMP in response to cAMP/receptor interactions may not be directly related to control of the pulse-induced genes. In addition, we have examined the effect of caffeine, which M. Brenner and S.D. Thomas (1984, Dev. Biol., 101, 136-146) showed preferentially blocks the cAMP relay system by blocking receptor-mediated activation of adenylate cyclase. We show that in many of the mutants and in an axenic wild-type strain, caffeine causes the induction of pulse-induced gene expression to almost wild-type levels or in some cases to higher than wild-type levels. Our data suggest that caffeine works by activating some step in the signal transduction pathway that must lie downstream from both the receptor and at least one of the G proteins and thus has effects other than simply blocking the receptor-mediated cAMP relay system.     

Relationships between extracellular pH, intracellular pH, and gene expression in Dictyostelium discoideum

A variety of studies have shown that differentiation of Dictyostelium discoideum amoebae in the presence of cAMP is strongly influenced by extracellular pH and various other treatments thought to act by modifying intracellular pH. Thus conditions expected to lower intracellular pH markedly enhance stalk cell formation, while treatments with the opposite effect favor spores. To directly test the idea that intracellular pH is a cell-type-specific messenger in Dictyostelium, we have measured intracellular pH in cells exposed to either low extracellular pH plus weak acid or high extracellular pH plus weak base using 31P nuclear magnetic resonance (NMR). Our results show that there is no significant difference in intracellular pH (cytosolic or mitochondrial) between pH conditions which strongly promote either stalk cell or spore formation, respectively. We have also examined the effects of external pH on the expression of various cell-type-specific markers, particularly mRNAs. Some mRNAs, such as those of the prestalk II (PL1 and 2H6) and prespore II (D19, 2H3) categories, are strongly regulated by external pH in a manner consistent with their cell-type specificity during normal development. Other markers such as mRNAs D14 (prestalk I), D18 (prespore I), 10C3 (common), or the enzyme UDP-galactose polysaccharide transferase are regulated only weakly or not at all by external pH. In sum, our results show that modulation of phenotype by extracellular pH in cell monolayers incubated with cAMP does not precisely mimic the regulation of stalk and spore pathways during normal development and that this phenotypic regulation by extracellular pH does not involve changes in intracellular pH.     

Initiation of multicellular differentiation in Dictyostelium discoideum is regulated by coronin A

Many biological systems respond to environmental changes by activating intracellular signaling cascades, resulting in an appropriate response. One such system is represented by the social amoeba Dictyostelium discoideum. When food sources become scarce, these unicellular cells can initiate a cAMP-driven multicellular aggregation program to ensure long-term survival. On starvation, the cells secrete conditioned medium factors that initiate cAMP signal transduction by inducing expression of genes such as cAMP receptors and adenylate cyclase. The mechanisms involved in the activation of the first pulses of cAMP release have been unclear. We here show a crucial role for the evolutionarily conserved protein coronin A in the initiation of the cAMP response. On starvation, coronin A–deficient cells failed to up-regulate the expression of cAMP-regulated genes, thereby failing to initiate development, despite a normal prestarvation response. Of importance, external addition of cAMP to coronin A–deficient cells resulted in normal chemotaxis and aggregate formation, thereby restoring the developmental program and suggesting a functional cAMP relay in the absence of coronin A. These results suggest that coronin A is dispensable for cAMP sensing, chemotaxis, and development per se but is part of a signal transduction cascade essential for system initiation leading to multicellular development in Dictyostelium.     

Distinct developmental regulation and properties of the responsiveness of different genes to cyclic AMP in Dictyostelium discoideum

Cyclic AMP (cAMP) is known to be an important mediator of gene expression in eukaryotic cells. At present, little is known about the developmental events which render specific genes responsive to cAMP in distinct cell types, or about the biochemical mechanisms by which cAMP exerts these regulatory effects. By examining the effects of cAMP treatment on specific mRNA levels in Dictyostelium discoideum cells with different 'developmental histories', we defined the developmental states in which specific genes display responsiveness to cAMP. We focused on two specific rapid responses: the ability of cAMP to inhibit the expression of an 'early' developmentally regulated mRNA (discoidin-I) and to stimulate the expression of a 'late', prespore-specific mRNA (PL3). Using this approach, we showed that, for both mRNAs, the ability to respond rapidly to cAMP is absent from vegetative cells grown on bacteria, and is acquired during development on filters. Furthermore, we identified several developmental states in which the discoidin-I response to cAMP is present, but in which the PL3 response is not. In experiments designed to examine the effects of cAMP analogues on the levels of these two mRNAs, we demonstrated that the analogue specificities of the discoidin-I and PL3 responses are different, and that the specificity for the PL3 response depends on the developmental state. The developmental kinetics and analogue specificity of the PL3 response suggest a two-step mode of action of cAMP in activating the expression of this gene. We discuss possible implications of these findings for the mechanisms of action of exogenous cAMP as well as for the role of cAMP in controlling the changes in gene expression that accompany normal development.     

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.     

Overproduction of the regulatory subunit of the cAMP-dependent protein kinase blocks the differentiation of Dictyostelium discoideum.

During the aggregation of Dictyostelium discoideum extracellular cAMP is known to act as a chemotractant and as an inducer of cellular differentiation. However, its intracellular role as a second messenger remains obscure. We have constructed a fusion gene consisting of the cDNA encoding the regulatory subunit (R) of the cAMP-dependent protein kinase fused to the promoter and N-terminal-proximal sequences of a Dictyostelium actin gene. Stable transformants, containing multiple copies of this gene, overproduce the R subunit which accumulates prematurely relative to the endogenous protein. These transformants fail to aggregate. Detailed analysis has shown that they are blocked at interphase, the period prior to aggregation, and that they are severely defective in most responses to cAMP including the induction of gene expression. Our observations suggest that intracellular cAMP acts, presumably by activation of the catalytic subunit of the cAMP-dependent protein kinase, to facilitate early development.     

The novel tyrosine kinase ZAK1 activates GSK3 to direct cell fate specification

Inhibition of GSK3 by 7-TM Wnt/wg receptor signaling is critical for specifying embryonic cell fate patterns. In Dictyostelium, the 7-TM cAMP receptors regulate GSK3 by parallel, antagonistic pathways to establish a developmental body plan. We describe here a novel tyrosine kinase, ZAK1, downstream of 7-TM cAMP receptor signaling that is required for GSK3 activation during development. zak1-nulls have reduced GSK3 activity and are defective in GSK3-regulated developmental pathways. Moreover, recombinant ZAK1 phosphorylates and activates GSK3 in vitro. We propose that ZAK1 is a positive regulator of GSK3 activity required for cell pattern formation in Dictyostelium and speculate that similar mechanisms exist to antagonize Wnt/wg signaling for metazoan cell fate specification.     

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.     

Signal transduction pathways involved in the expression of the uridine diphosphoglucose pyrophosphorylase gene of Dictyostelium discoideum

The uridine diphosphoglucose pyrophosphorylase (UDPGP1) gene of Dictyostelium discoideum is an excellent marker to study the pathways that control the expression of genes during development. We have previously shown that the UDPGP1 gene is regulated by exogenous cAMP acting on cell-surface cAMP receptors. Various steps in the signal transduction pathway between receptor stimulation and the induction of the gene can now be studied. Induction does not require the synthesis of intracellular cAMP, but does require new protein synthesis. By deletion and transformation with altered genes, two cis-acting sequences that are required for UDPGP1 expression have been identified. A GC-rich palindromic sequence located between -410 and -374 is essential for induction of the gene by extracellular cAMP, but not for its basal expression. A sequence element located between -374 and -337 is required for any basal expression of this gene. When the polarity of the palindromic sequence was reversed such that it resembled the H2K enhancer element, the gene could still be induced by exogenous cAMP. Two DNA binding activities were detected in gel mobility shift assays using a fragment containing both of the regulatory sequence elements of UDPGP1 gene. Transformation with a vector that resulted in the synthesis of anti-sense UDPGP1 RNA led to almost total elimination of the enzyme antigen and no detectable enzyme activity. However, these transformants developed normally, indicating that either UDPGP is not required for development or residual synthesis of UDPGP may be sufficient for normal development.     

Galpha3 and protein kinase A represent cross-talking pathways for gene expression in Dictyostelium discoideum

Heterotrimeric G proteins and protein kinase A (PKA) are regulators of development in Dictyostelium discoideum. It has been reported that disruption of the Dictyostelium Galpha3 gene (galpha3-) blocks development and expression of several early development genes, characteristics that are reminiscent of mutants lacking the catalytic subunit of PKA (pkac-). The hypothesis that Galpha3 and PKA signaling pathways may interact to control developmental gene expression was tested by comparing the regulation of seven genes expressed early in development in the wild-type and in galpha3- and pkac- mutants, and comparing PKA activity in the wild-type and in a galpha3- mutant. The expression patterns of six genes were affected similarly by the Galpha3 and PKA mutations, while the expression of only one gene, the cAMP receptor 1 (cAR1), differed between the mutants. PKA activity, measured by phosphorylation of the PKA-specific substrate Kemptide, was higher in galpha3- cells than in wild-type cells, suggesting that Galpha3 normally exerts an inhibitory effect on PKA activity. Although some early development genes appear to require both Galpha3 and PKA for expression, the differing response of cAR1 expression and the inhibitory effect of Galpha3 on PKA activity suggest that Galpha3 and PKA are members of interacting pathways controlling gene expression early in development.