Functional genomic analysis of DIF signalling in Dictyostelium. (360G-Wellcome-064868_Z_01_Z)

Functional Genomic Analysis of DIF Signalling in Dictyostelium Rationale: Dictyostelium discoideum reveals a novel mechanism of patterning in which the different cell types arise intermingled with one another before sorting out into coherent tissues (patterning without positional information). Key players in the control of this developmental decision are the DIFs, small molecules released by developing cells, which can induce isolated amoebe to differentiate into stalk cells and prevent them from becoming spore cells. The cell fate choice is aided by a heterogeneity in DIF sensitivity within a cell population which is dependent on cell cycle position or growth history. However, our understanding of the DIF signal transduction pathway is rather rudimentary. Only the recent isolation of a mutant defective in DIF-1 biosynthesis and the discovery of a family of STAT transcription factors which are able to bind sequences required for DIF-1 induced gene expression, provide any clues. The isolation of other genes involved in DIF signal transduction is thus critical for our understanding of its mode of action. However, their identification by classification of mutants on the basis of their morphological phenotype (e.g. similarity to the DIFless mutant) is made impossible by the subtlety of this phenotype. To circumvent these problems requires an alternative to morphology to be used as a phenotypic readout. Aims: the proposed project will utilise the post genomic technologies of DNA microarrays and functional genomics to: a. define the transcriptional responses of cells to DIF signalling, both in cell culture and in vivo (the dmtA' DIFless mutant), by determining the target genes of each molecule using a Dictyostelium microarray. This will 1) determine if each molecule may play different roles during normal development, 2) determine which genes might be expected to be altered ini mutants affecting these pathways (transcriptional phenotype). b. Identify novel genes in DIF signaling by parallel phenotyping and transcriptional phenotype analysis. 1) Using insertional mutagenesis my sponsor laboratory has generated a library of 1000 random mutants, in which the site of insertion has been characterised. This results in a unique 'molecular barcode' for each mutant and allows the presence or absence of each mutant to be followed over many generations as they are gained or lost from the population. This will be used to define mutants with increased or decreased sensitivity to DIF in submerged cell culture where its normal effect is to drive cells from the spore cell fate (alive) to stalk cell fate (dead). Therefore DIF insensitive mutants will remain in the population through several rounds of selection whereas mutants with increased DIF sensitivity will be selectively lost. 2) As part of long term project the transcriptional phenotype of 50000 random mutants will be determined. These will be compared to the transcriptional response of cells to the DIFs in culture and to the transcriptional phenotype of the 'dmtA' DIFless mutant. Mutants showing similarities to these profiles will be tested for DIF signalling defects. C. Seek the basis of cell fate bias by comparing the expression of genes involved in DIF signalling in differently biased cells. Outcome: Understanding DIF signalling and the regulation of this signal transduction pathway will provide an important insight into a novel mechanism of pattern formation demonstrated by Dictyostelium. In the long term, a complete picture of DIF signalling and its integration with other signalling pathways is within reach using a genome based approach.