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First published online 26 March 2008
doi: 10.1242/dev.020883

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Cell type specificity of a diffusible inducer is determined by a GATA family transcription factor

Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester, M13 9PT.

View larger version (27K): [in this window] [in a new window]   Fig. 1. Identification of a novel DIF resistant mutant. (A) Schematic of the selection strategy used to identify the dimC- mutant. REMI mutagenized cells were subjected to the cAMP-removal stalk-cell induction assay before HL5 growth medium was added. Surviving cells were grown up and subjected to a further two rounds of selection before cloning out. (B) dimC- cells fail to make stalk cells in the cAMP removal assay. At least three independent assays were performed with comparable results. Data are shown from a representative experiment in which plates were scored in triplicate. Representative pictures are shown on the right. Black arrows highlight vacuolized stalk cells. (C) dimC- cells make equivalent numbers of spore cells as wild type in the 8-Br-cAMP assay in the absence of DIF. In the presence of DIF, spore cell formation is repressed in both wild-type and mutant cells. At least three independent assays were performed with comparable results. Data are shown from a representative experiment in which plates were scored in triplicate. White arrows highlight representative spore cells. The pictures on the right show that the mutant cells, in contrast to wild type, still do not make stalk cells when treated with DIF. Black arrows highlight vacuolized stalk cells.

View larger version (21K): [in this window] [in a new window]   Fig. 2. The disrupted gene in the dimC- mutant is a GATA transcription factor. (A) Schematic of the site of insertion of the pBSRBam disruption vector in the dimC- mutant. The numbers refer to Dictyostelium database identifiers (www.dictybase.org). DDB0217990 is predicted to encode a ubiquitin ligase and DDB0220470 a GATA family transcription factor. (B,C) Quantitative PCR analysis of DDB0217990 (B) and DDB0220470 (C) gene expression in developing wild-type (grey bars) and dimC- mutant cells (black bars). PCR reactions were performed in triplicate and data averaged. Identical results were obtained with mRNA extracted from two independent developmental time courses.

View larger version (35K): [in this window] [in a new window]   Fig. 3. GtaC activity is regulated by DIF. (A) gtaC expression is DIF inducible and dependent on DimA and DimB. Expression of gtaC was measured by qPCR in wild-type, dimA- and dimB- cells with and without a 1 hour DIF treatment. Results shown are an average of three independent experiments. (B) dimA and dimB expression is also DIF inducible in wild-type cells. Transcript levels were measured by qPCR with and without a 1 hour DIF treatment. Results shown are an average of three independent experiments. (C) GtaC shows DIF-dependent nuclear localization. GtaC-GFP-expressing cells were starved for 4 hours before induction with DIF. Maximal nuclear localization can be seen after 5 minutes. After 15 minutes, nuclear accumulation is no longer visible. (D) Quantification of the kinetics of GtaC-GFP nuclear import in response to DIF. Results shown are an average of three independent experiments.

View larger version (20K): [in this window] [in a new window]   Fig. 4. gtaC- null cells exhibit DIF responses that are absent from dimA- mutant cells. (A) Alignment of the GATA DNA-binding domain of GtaC with the DNA-binding domain of mouse GATA1, Drosophila Pannier and Neurospera WC-2. (B) Schematic of the gtaC gene and gene disruption. The region encoding the GATA DNA-binding domain is shaded black, poly asparagine repeats are blue and poly glutamines are green. Approximately three-quarters of the gtaC-coding sequence was replaced with a blasticidin cassette, including the predicted GATA DNA-binding domain. (C) Measurement of DIF responsive gene expression in monolayer assays. Expression of prestalk markers (ecmA and ecmB) and prespore markers (psA and cotB) was measured by qPCR. Cells were treated with cAMP for 9 hours before addition of DIF for 1 (ecmA and ecmB) or 3 hours (psA and cotB). gtaC- cells exhibit responses comparable with those of wild-type cells, whereas no responses were seen in dimA- cells. Results shown are from one experiment. Comparable results were seen in at least three independent experiments. (D) DIF responsive gene expression in dissociated cell assays. Mound stage cells were dissociated before shaking in cAMP with or without DIF for 2 hours. Expression of prestalk markers (ecmA and ecmB) and prespore markers (psA and cotB) was measured by qPCR. gtaC- cells exhibit comparable responses with those of wild-type cells, whereas no responses were seen in dimA- cells. Results shown are from one representative experiment. Comparable results were seen in at least three independent experiments.

View larger version (59K): [in this window] [in a new window]   Fig. 5. Morphological defects of developing gtaC- mutant cells. Cells were developed on non-nutrient agar for the times shown. Mutant structures are smaller than those of wild type. In addition, mutant slugs were often found to break apart (arrows). At the fruiting body stage, plates appeared messy with stalks often laying on the agar surface. Scale bar: 1 mm.

View larger version (71K): [in this window] [in a new window]   Fig. 6. gtaC- null mutant cells exhibit defects in pstB cell and basal disc cell differentiation. Wild-type and gtaC- mutant cells were transformed with representative prestalk and prespore reporter genes, and developed and stained for β-galactosidase activity at the slug (A) and early culminant (B) stages. ecmB-lacZ expression was aberrant as many staining cells were found at the rear of slugs and was absent from the basal disc. Brackets highlight the pstB cell population. Arrows indicate basal disc. Expression of all other markers was normal at the slug stage. However, ecmAO and ecmA-lacZ expression was absent at the expected position of the basal disc in mutant structures. Scale bars: 0.25 mm.

View larger version (86K): [in this window] [in a new window]   Fig. 7. pstB cells do not sort to the prestalk-prespore border at any developmental stage. Wild type and gtaC- mutant ecmB-lacZ transformants were stained at the tip mound (A,D), early finger (B,E) and standing (C,F) slug stages. A collection of staining cells is visible towards the prestalk-prespore boundary in wild-type specimens as soon as morphological pattern appears. In mutant specimens, staining cells always appeared to be scattered throughout the prespore zone and became concentrated towards the rear as development progressed. Scale bars: 0.25 mm.

View larger version (76K): [in this window] [in a new window]   Fig. 8. gtaC expression at the slug and fruiting body stages in wild type. A gtaC promoter-lacZ reporter gene construct is expressed in scattered cells throughout the rear of slugs, but with highest levels of staining towards the prestalk-prespore boundary (A). At the early culminant (B) and mature fruiting body (C) stages, expression is restricted to the upper and lower cup, as well as to the stalk and basal disc. Scale bar: 0.25 mm.

View larger version (84K): [in this window] [in a new window]   Fig. 9. gtaC- mutant defects are phenocopied by DIF signalling mutants. (A) Expression of ecmB-lacZ at the slug stage. In wild-type structures clusters of high expressing cells are found at the prestalk/prespore boundary (pstB cells). In DIF signalling mutants (gtaC-, dimA-, dimB- and dmtA), these cells are mostly found clustered towards the slug rear. (B) Basal disc differentiation is aberrant in DIF signalling mutants. st-lacZ expression was used to label the stalk and basal disc. Although staining can be observed in the stalk proper in mutant fruiting bodies, no staining is present in the basal disc. (C) The basal disc is absent from DIF signalling mutants. The arrowhead shows the basal disc of a typical wild-type fruiting body. Although a morphological fruiting body with stalk and spore is formed in each DIF signalling mutant, the basal disc structure is much reduced or absent. Scale bars: 0.25 mm.

View larger version (20K): [in this window] [in a new window]   Fig. 10. Model for the role of GtaC in the regulation of DIF responses. (A) At the mound stage, stochastic differences due to growth history result in two populations of DIF-hypersensitive cells. Those with highest GtaC activity (blue) will behave as pstB cells while the rest will differentiate as pstO cells. (B) At the slug stage, pstB cells (blue) with highest GtaC activity have sorted to the prestalk/prespore boundary, with pstO cells (red) lying just anterior, where they continue to respond to DIF produced by prespore cells. (C) In the monolayer assay, DIF induces outer basal disc cell differentiation. DIF activity is dependent on the transcription factors GtaC, DimA and DimB, and is inhibited by the GskA protein kinase.

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