DIF-1 regulates Dictyostelium basal disc differentiation by inducing the nuclear accumulation of a bZIP transcription factor

Abstract

Exposure of monolayer Dictyostelium cells to the signalling polyketide DIF-1 causes DimB, a bZIPtranscription factor, to accumulate in the nucleus where it induces prestalk gene expression. Here we analyse DimB signalling during normal development. In slugs DimB is specifically nuclear enriched in the pstB cells; a cluster of vital dye-staining cells located on the ventral surface of the posterior, prespore region. PstB cells move at culmination, to form the lower cup and the outer basal disc of the fruiting body, and DimB retains a high nuclear concentration in both these tissues. In a dimB null (dimB-) strain there are very few pstB or lower cup cells, as detected by neutral red staining, and it is known that the outer basal disc is absent or much reduced. In the dimB- strain ecmB, a marker of pstB differentiation, is not DIF inducible. Furthermore, ChIP analysis shows that DimB binds to the ecmB promoter in DIF-induced cells. These results suggest that the differentiation of pstB cells is caused by a high perceived level of DIF-1 signalling, leading to nuclear localization of DimB and direct activation of cell type-specific gene expression.

Fig. 1

A) Whole mount DimB staining of a parental (A × 2) slug Serial confocal Z sections through a whole mount of a migratory slug, fixed, stained with anti-DimB antibody and then with Alexa 488-conjugated anti-rabbit antibody. B) Whole mount DimB staining of a dimB− slug As A) but a single confocal section of a dimB− slug from the ventral surface. C) An A × 2 slug expressing GFP-DimB A migrating dimB− slug expressing GFP-DimB visualised from its ventral surface by confocal microscopy.

Fig. 2

Whole mount of a dimB− slug expressing DimBOE and stained for DimB. DimB− slugs overexpressing dimB from its own promoter via the DimBOE construct were fixed, stained with anti-DimB antibody as in Fig. 1A. Confocal sections at different Z positions are shown. The top panel shows transmitted light.

Fig. 3

Serial confocal sections of an A × 2 mound stained for DimB. GFP-DimB cells were developed to the mound stage and visualised at different Z positions in the confocal microscope.

Fig. 4

A × 2 slugs expressing GFP-DimB and stained with neutral red. GFP-DimB cells were stained with neutral red and allowed to migrate under unidirectional light until about 20 h of development. The ventral surfaces of two slugs (A and B) are visualised in the confocal microscope for both GFP fluorescence and transmitted light. The images were merged and the neutral red stained vesicles can be seen as dark granules, surrounding or flanking at one side the fluorescent nuclei.

Fig. 5

A) A slug of the dmtA− DIF biosynthesis mutant overexpressing DimB. DmtA− slugs overexpressing DimB were fixed and stained for DimB as in Fig. 1A. This is a ventral confocal section of a slug. B) A culminant of the dmtA− DIF biosynthesis mutant overexpressing DimB. As in A) except this is a culminant.

Fig. 6

A) Whole mount DimB staining of an A × 2 culminant. B) Whole mount DimB staining of a DimB overexpressing culminant. C) An A × 2 culminant expressing GFP-DimB. Culminants of A × 2 (A) or DimB overexpressing cells (B) were fixed and stained for DimB as in Fig. 1A. (C) Culminants of GFP-dimB cells.

Fig. 7

An A × 2 culminant expressing GFP-DimB and stained with neutral red. Culminants of GFP-DimB transformed cells stained with neutral red were visualised for GFP fluorescence and transmitted light as in Fig. 4.

Fig. 8

A. Serial optical sections though an A × 2 slug double-stained for ecmB-gal and DimB. Migrating A × 2 slugs transformed with ecmB:gal were fixed at around 20 h of development and double stained with anti-DimB and anti-β-galactosidase and then with Alexa 488-conjugated anti-rabbit antibody and Alexa 594-conjugated anti-mouse antibody. Confocal sections of a Z series extending from the middle to the ventral part of the slug and are expanded at three different positions along the length of the slug. B. An A × 2 culminant double-stained for ecmB-gal and DimB. As in A except that this is a single confocal section of a culminant.

Fig. 9

ChIP analysis of DimB binding to the ecmB promoter. Cells were incubated with or without DIF for 4 h and subjected to ChIP analysis as described in the Materials and methods section. The absolute recoveries from the procedure varied from experiment to experiment, (three independent experiments with triplicate Q-PCR analysis in each) but the induced signal for ecmA was always the highest. Therefore values are normalised to this and are shown with their Standard Deviations and with the Student's paired T test; applied to the ecmB analysis with and without DIF-1 and in samples immuno-precipitated from GFP-DimB transformant cells. As indicated by the asterisk the induction by DIF is significant with a P < 0.05.

Fig. 10

A × 2 parental and dimB− mutant slugs and culminants stained with neutral red. A × 2 and dimB− cells stained with neutral red were incubated under unidirectional light till 20 h of development then migrating slugs and culminants were visualised from the side by light microscopy.

Fig. 11

Enzymatic assay of ecmB-gal expression in A × 2 parental and dimB− mutant slugs and culminants. Migrating slugs or culminants of ecmB:gal transformants of A × 2 and dimB− were stained for β-galactosidase and visualised by light microscopy.