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ABSTRACTWe have determined the proportions of the prespore and prestalk regions in Dictyostelium discoideumslugs 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.

ABSTRACTWe used microarrays carrying most of the genes that are developmentally regulated in Dictyosteliumto discover those that are preferentially expressed in prestalk cells. Prestalk cells are localized at the front of slugs and play crucial roles in morphogenesis and slug migration. Using whole-mount in situ hybridization, we were able to verify 104 prestalk genes. Three of these were found to be expressed only in cells at the very front of slugs, the PstA cell type. Another 10 genes were found to be expressed in the small number of cells that form a central core at the anterior, the PstAB cell type. The rest of the prestalk-specific genes are expressed in PstO cells, which are found immediately posterior to PstA cells but anterior to 80% of the slug that consists of prespore cells. Half of these are also expressed in PstA cells. At later stages of development, the patterns of expression of a considerable number of these prestalk genes changes significantly, allowing us to further subdivide them. Some are expressed at much higher levels during culmination, while others are repressed. These results demonstrate the extremely dynamic nature of cell-type-specific expression in Dictyosteliumand further define the changing physiology of the cell types. One of the signals that affect gene expression in PstO cells is the hexaphenone DIF-1. We found that expression of about half of the PstO-specific genes were affected in a mutant that is unable to synthesize DIF-1, while the rest appeared to be DIF independent. These results indicate that differentiation of some aspects of PstO cells can occur in the absence of DIF-1.

The chemoattractant cAMP, acting through serpentine cAMP receptors, results in a rapid and transient stimulation of the Dictyosteliummitogen-activated protein kinase ERK2 activity (1). In this study we show that other pathways required for aggregation, including Ras and cAMP-dependent protein kinase (PKA), are important regulators of ERK2 activation and adaptation. By examining both the level and kinetics of activation and adaptation of ERK2, we show that Ras is a negative regulator of ERK2. Activated Ras or disruption of a Ras GAP gene results in reduced ERK2 activation whereas disruption of putative Ras GEF or expression of dominant negative Ras proteins have a more rapid, higher, and extended activation. CRAC, a PH domain-containing protein required for adenylyl cyclase activation, is also required for proper ERK2 adaptation. PKA overexpression results in a more rapid, higher level of activation, whereas pkanull cells show a lower level but more extended ERK2 activation. Furthermore, we show that constitutive expression of PKA catalytic subunit bypasses the requirement of ERK2 for aggregation and later development, indicating that PKA lies downstream from ERK2 and that ERK2 may regulate one or more components of the signaling pathway required for mediating PKA function, possibly by directly regulating PKA R or a protein controlling the intracellular level of cAMP.

The effects of low temperature on morphogenesis and cellular differentiation of Dictyostelium discoideum were examined. During incubation at 5 °C, the vegetative and preaggregation cells never developed, but cell masses at the aggregation or slug stage developed to form hemispherical, or dumbbell-shaped multicellular structures. By staining with FITC-antispore IgG, the structures formed after 10 days of incubation of tipped aggregates at 5 °C were found to be composed of 90% spores, 5% prespore cells and 5% non-stained cells. Since only 20% of the total cells constituting the tipped aggregate had been prespore cells at the beginning of incubation, this showed that spore differentiation proceeded even at low temperature, while stalk differentiation was completely inhibited. Similar results were obtained when the cells were incubated at 3 °C. However, at 0°C, morphogenesis and cellular differentiation did not occur, although most of the prespore cells at the late culmination stage differentiated incompletely into spores. Possible reasons for the high proportion of spores being induced by low temperature are discussed.