CAMP-DEPENDENT PROTEIN-KINASE DIFFERENTIALLY REGULATES PRESTALK AND PRESPORE DIFFERENTIATION DURING DICTYOSTELIUM DEVELOPMENT

We and others have previously shown that cAMP-dependent protein kinase (PKA) activity is essential for aggregation, induction of prespore ge ne expression and multicellular development in Dictyostelium. In this manuscript, we further examine this regulatory role. We have overexpre ssed the Dictyostelium PKA catalytic subunit (PKAcat) in specific cell types during the multi-cellular stages, using prestalk and prespore c ell-type-specific promoters to make PKA activity constititutive in the se cells (independent of cAMP concentration). To examine the effects o n cell-type differentiation, we co-transformed the PKAcat-expressing v ectors with reporter constructs expressing lacZ from four cell-type-sp ecific promoters: ecmA (specific for prestalk A cells); ecmB (specific for prestalk B and anterior-like cells in the slug); ecmBDELTA89 (spe cific for stalk cells); and SP60 (prespore-cell-specific). By staining for beta-galactosidase expression histologically at various stages of development in individual strains, we were able to dissect the morpho logical changes in these strains, examine the spatial localization of the individual cell types, and understand the possible roles of PKA du ring multicellular development. Expression of PKAcat from either the e cmA or ecmB prestalk promoters resulted in abnormal development that a rrested shortly after the mound stage, producing a mound with a round apical protrusion at the time of tip formation. Prestalk A and prestal k B cells were localized in the central region and the apical mound in the terminal differentiated aggregate, while prespore cells showed an aberrant spatial localization. Consistent with a developmental arrest , these mounds did not form either mature spores or stalk cells and ve ry few cells expressed a stalk-cell-specific marker. Expression of PKA cat from the prespore promoter resulted in abnormal morphogenesis and accelerated spore cell differentiation. When cells were plated on agar , a fruiting body was formed with a very large basal region, containin g predominantly spores, and a small, abnormal sorocarp. Mature spore c ells were first detected by 14 hours, with maximal levels reached by 1 8-20 hours, in contrast to 24-26 hours in wild-type strains. When cell s were plated on filters, they produced an elongated tip from a large basal region, which continued to elongate as a tubular structure and p roduce a 'slug-like' structure at the end. The slug was composed predo minantly of prestalk cells with a few prespore cells restricted to the junction between the 'slug' and tube. As the slug migrated, these pre spore cells were found in the tube, while new prespore cells appeared at the slug/tube junction, suggesting a continual differentiation of n ew prespore cells at the slug's posterior. The slug eventually produce d a fruiting body-like structure that in many cases was abnormal and s howed only a low level of SP60 expression. Models are presented of the roles cAMP-dependent protein kinase plays in regulating various aspec ts of Dictyostelium differentiation.