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First published online September 1, 2004
doi: 10.1242/10.1242/dev.01330

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GSK3 is a multifunctional regulator of Dictyostelium development

¹ School of Life Sciences, Wellcome Trust Biocentre, University of Dundee, Dundee DD1 5EH, UK
² MRC Laboratory for Molecular Cell Biology and Department of Biology, University College, Gower Street, London WC1E 6BT, UK

View larger version (24K): [in a new window]   Fig. 1. (A) Western transfer analysis of transformant clones generated using the gskA disruption construct. Axenically growing cells were harvested and GskA protein was analysed as described in methods. Protein loading and transfer onto the membrane was normalised by staining with Ponceau S. (B) GSK3 kinase activity measured in cell extracts from AX2 cells and gskA-null clones. Growing cells were harvested and GSK3 levels assayed as described (Ryves and Harwood, 1998). Each sample was assayed in triplicate and means and standard deviations are shown.

View larger version (41K): [in a new window]   Fig. 2. Terminal morphology and spore characteristics of a random integrant and an Ax2/gskA– clone developing on a bacterial lawn. (A) A random integrant control is shown at same magnification as B, an Ax2/gskA– fruiting body. The beads on the stalk of the random integrant fruiting body are water droplets. Scale bar: 100 µm. (C) The spore head of an Ax2/gskA– clone was squashed under a cover slip. (D) The temperature sensitivity of spores, derived from fruiting bodies that were generated on KK2-agar, was determined as described in the Materials and methods. The percentage of surviving spores, relative to the total number of germinating untreated spores, was determined, and means and standard deviations are shown.

View larger version (96K): [in a new window]   Fig. 3. (A) Development on non-nutrient agar of Ax2/gskA– cells pregrown in either axenic medium or bacteria. Cells of a random integrant control were compared with an Ax2/gskA– strain, grown either axenically or in the presence of a bacterial food source. They were then allowed to develop on KK2-agar and photographed from above. (B) Slug migration on water agar. Cells from a random integrant clone and an Ax2/gskA– strain were grown in axenic medium, developed to the slug stage and allowed to migrate on water agar for 24 hours and towards a unidirectional light source (indicated by arrows). Approximately 80% of the gskA-null structures developed to the slug stage. The structures and their associated slime trails were transferred onto a transparent membrane and visualised by protein staining.

View larger version (38K): [in a new window]   Fig. 4. Northern transfer assay of developmentally regulated genes in a random integrant and an Ax2/gskA– mutant. Cells were developed on KK2 agar and RNA was prepared from samples taken at 2 hour intervals over a period of 24 hours. The blot was hybridised with an ecmB- and a psA-specific gene probe. Detection of ecmA mRNA was by its cross-hybridisation with the ecmB probe. Re-probing with the constitutively expressed IG7 gene acts as a control for loading and transfer.

View larger version (53K): [in a new window]   Fig. 5. (A) Analysis of the expression pattern of cell-type specific reporter constructs in a random integrant and an Ax2/gskA– strain. Stable transformants for each of the indicated lacZ reporter constructs were allowed to develop on water agar. The stained structure for each random integrant transformant clone is shown on the left and the equivalent gskA-null mutant structure is shown on the right. The developmental promoter, or promoter subfragment, that directs expression of the lacZ gene is indicated on the left. Slug stages are shown for strains transformed with all reporter constructs, except for strains transformed with psA-lacZ, where tipped aggregates are shown. All structures derive from pools of transformants. (B) Prespore vesicle staining in a random integrant and a gskA-null mutant. Slugs were stained for prespore vesicles as described in the Materials and methods section.

View larger version (13K): [in a new window]   Fig. 6. (A) Induction of the prespore marker psA-lacZ in cells of a random integrant and an Ax2/gskA– strain. Cells were developed in suspension culture and pulsed with 300 µM cAMP. Samples were taken at t=0 (–cAMP) and t=8 hours (+cAMP), and ß-galactosidase activity was determined. All samples were measured in triplicate and the means and standard deviations are presented. The accumulation of ß-galactosidase is higher in the gskA-null mutant than in the random integrant. This is likely to be due to copy number differences for the reporter construct in the two strains. (B) Induction of spore cell differentiation by 8-Bromo-cAMP in a random integrant and an Ax2/gskA– strain. The random integrant and Ax2-derived gskA-null mutant were plated at low density (104 cells/cm2), in spore induction medium and exposed to 15 mM 8-bromo-cAMP for 24 hours (Kay, 1989). Spores were counted and expressed as a percentage of total number plated. Data are expressed as mean number±s.e.m. (n=3).

View larger version (16K): [in a new window]   Fig. 7. Induction of stalk cell differentiation by DIF-1. Comparison of the DIF-1 sensitivity of AX2 cells and an Ax2/gskA– strain. Ax2 (black circles) and gskA-null mutant cells (white circles) were plated at low density (104 cells/cm2) in stalk medium in 5 mM camp). After 24 hours incubation, cAMP was washed off and replaced with DIF at varying concentrations for a further 24 hours. Stalk cells were counted and expressed as a percentage of total number plated. Data are expressed as mean±s.e.m., and represent the combination of two clones analysed in triplicate. (B) Comparison of the effect of cAMP on AX2 cells, a random integrant and an Ax2/gskA– strain. Cells were treated as in A, but incubated in 100 nM DIF±5 mM cAMP, as indicated. (C) Comparison of the effect of cAMP on AX2 cells and an Ax2/gskA– strain using different inductive concentrations of DIF-1 Cells were treated as in A, but incubated in 100, 10 or 5 nM DIF ±5 mM cAMP, as indicated.

View larger version (94K): [in a new window]   Fig. 8. Sequence comparison for seven of the ESTs that are underexpressed in the gskA-null strain. Microarray analysis was performed as described in the Materials and methods. This yielded seven ESTs where the sequence is known. This Clustal W analysis shows that the seven ESTs are all members of the 2C gene family.

View larger version (31K): [in a new window]   Fig. 9. GskA and the regulation of stalk cell differentiation. The structure at the left is a representation of an early culminant, with prestalk cells undertaking the `reverse fountain' movement. As they pass the entrance to the stalk tube, the prestalk cells activate expression of the ecmB gene and are then termed pstAB cells (pink). Before entry to the stalk tube (blue), the ecmB promoter is inactive; high extracellular cAMP signalling acts (via the cAR3 cAMP receptor, the dual specificity kinase ZAK1 and GSK-3) to prevent the ecmB activator region (A) from functioning. In addition, Dd-STATa functions as a repressor, acting via two repressor elements (RI and RII) located distal to the ecmB cap site. At culmination, repression by Dd-STATa is also relieved by a parallel pathway involving activation of PKA.