Adenylyl cyclase A expression is tip-specific in Dictyostelium slugs and directs StatA nuclear translocation and CudA gene expression.

cAMP oscillations, generated by adenylyl cyclase A (ACA), coordinate cell aggregation in Dictyostelium and have also been implicated in organizer function during multicellular development. We used a gene fusion of the ACA promoter with a labile lacZ derivative to study the expression pattern of ACA. During aggregation, most cells expressed ACA, but thereafter expression was lost in all cells except those of the anterior tip. Before aggregation, ACA transcription was strongly upregulated by nanomolar cAMP pulses. Postaggregative transcription was sustained by nanomolar cAMP pulses, but downregulated by a continuous micromolar cAMP stimulus and by the stalk-cell-inducing factor DIF. Earlier work showed that the transcription factor StatA displays tip-specific nuclear translocation and directs tip-specific expression of the nuclear protein CudA, which is essential for culmination. Both StatA and CudA were present in nuclei throughout the entire slug in an aca null mutant that expresses ACA from the constitutive actin15 promoter. This suggests that the tip-specific expression of ACA directs tip-specific nuclear translocation of StatA and tip-specific expression of CudA.

Two distinct signaling pathways mediate DIF induction of prestalk gene expression in Dictyostelium.

During Dictyostelium development, the differentiation inducing factor (DIF) triggers expression of the prestalk gene ecmB and induces stalk cell differentiation, a form of programmed cell death. The effects of DIF are mediated by a sustained increase in cytosolic Ca2+ levels. The Ca2+ ATPase inhibitor BHQ causes a similar rise in Ca2+ levels and also induces prestalk gene expression. We show here that Ca2+ is a specific intermediate for prestalk gene induction, since BHQ represses transcription of the cAMP-inducible aggregative gene PDE, the postaggregative gene CP2, and the prespore gene D19. The prestalk gene ecmA is also induced by DIF, but induction appears to occur in two steps, which occur within 1 h and after 2 h, respectively. The slow step shows the same kinetics as ecmB induction and similar to ecmB induction, this step is BHQ inducible and requires an initial round of protein synthesis. The fast step does not require protein synthesis and cannot be induced by BHQ. This indicates that in addition to the slow Ca2+-mediated pathway, there is probably a second fast Ca2+-independent signal transduction pathway for DIF.

Functional promiscuity of gene regulation by serpentine receptors in Dictyostelium discoideum.

Serpentine receptors such as smoothened and frizzled play important roles in cell fate determination during animal development. In Dictyostelium discoideum, four serpentine cyclic AMP (cAMP) receptors (cARs) regulate expression of multiple classes of developmental genes. To understand their function, it is essential to know whether each cAR is coupled to a specific gene regulatory pathway or whether specificity results from the different developmental regulation of individual cARs. To distinguish between these possibilities, we measured gene induction in car1 car3 double mutant cell lines that express equal levels of either cAR1, cAR2, or cAR3 under a constitutive promoter. We found that all cARs efficiently mediate both aggregative gene induction by cAMP pulses and induction of postaggregative and prespore genes by persistent cAMP stimulation. Two exceptions to this functional promiscuity were observed. (i) Only cAR1 can mediate adenosine inhibition of cAMP-induced prespore gene expression, a phenomenon that was found earlier in wild-type cells. cAR1's mediation of adenosine inhibition suggests that cAR1 normally mediates prespore gene induction. (ii) Only cAR2 allows entry into the prestalk pathway. Prestalk gene expression is induced by differentiation-inducing factor (DIF) but only after cells have been prestimulated with cAMP. We found that DIF-induced prestalk gene expression is 10 times higher in constitutive cAR2 expressors than in constitutive cAR1 or cAR3 expressors (which still have endogenous cAR2), suggesting that cAR2 mediates induction of DIF competence. Since in wild-type slugs cAR2 is expressed only in anterior cells, this could explain the so far puzzling observations that prestalk cells differentiate at the anterior region but that DIF levels are actually higher at the posterior region. After the initial induction of DIF competence, cAMP becomes a repressor of prestalk gene expression. This function can again be mediated by cAR1, cAR2, and cAR3.

Transformation with vectors harboring the NEOR selection marker induces germination-specific adenylylcyclase activity in Dictyostelium cells.

Dictyostelium cells express an aggregative adenylylcyclase (ACA), responsible for oscillatory cAMP signaling, and a spore germination-specific adenylylcyclase (ACG). Overexpression of PKA regulatory (R) subunits blocks oscillatory cAMP signaling but increases basal cAMP levels, while neither ACA nor ACG mRNA could be detected. To test whether a novel type of adenylylcyclase (AC) was responsible for cAMP synthesis, dominant-negative PKA-R subunits (PKA-RM) and control R-subunits (PKA-RC) were overexpressed in ACA null mutants. Both transformations as well as transformation with an unrelated vector, carrying the same NEOR selection marker, induced an AC activity in growing cells with the biochemical characteristics of ACG. Similar vectors with a different URA selection marker did not increase AC activity. We conclude that the amino-glycoside phosphotransferase encoded by the very commonly used NEOR selection marker induces ectopic ACG activity in Dictyostelium cells.