Characterization of differentially expressed genes in purified Drosophila follicle cells: toward a general strategy for cell type-specific developmental analysis.
Axis formation in Drosophila depends on correct patterning of the follicular epithelium and on signaling between the germ line and soma during oogenesis. We describe a method for identifying genes expressed in the follicle cells with potential roles in axis formation. Follicle cells are purified from whole ovaries by enzymatic digestion, filtration, and fluorescence-activated cell sorting (FACS). Two strategies are used to obtain complementary cell groups. In the first strategy, spatially restricted subpopulations are marked for FACS selection using a green fluorescent protein (GFP) reporter. In the second, cells are purified from animals mutant for the epidermal growth factor receptor ligand gurken (grk) and from their wild-type siblings. cDNA from these samples of spatially restricted or genetically mutant follicle cells is used in differential expression screens employing PCR-based differential display or hybridization to a cDNA microarray. Positives are confirmed by in situ hybridization to whole mounts. These methods are found to be capable of identifying both spatially restricted and grk-dependent transcripts. Results from our pilot screens include (i) the identification of a homologue of the immunophilin FKBP-12 with dorsal anterior expression in egg chambers, (ii) the discovery that the ecdysone-inducible nuclear hormone receptor gene E78 is regulated by grk during oogenesis and is required for proper dorsal appendage formation, and (iii) the identification of a Drosophila homologue of the human SET-binding factor gene SBF1 with elevated transcription in grk mutant egg chambers.
D000818:Animals; D004330:Drosophila / Q000196:embryology* / Q000235:genetics; D029721:Drosophila Proteins; D005434:Flow Cytometry; D018507:Gene Expression Regulation, Developmental / Q000235:genetics*; D017344:Genes, Insect; D049452:Green Fluorescent Proteins; D020104:Immunophilins / Q000235:genetics; D017403:In Situ Hybridization; D019476:Insect Proteins / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D012098:Reproduction / Q000235:genetics; D015398:Signal Transduction / Q000235:genetics; D022021:Tacrolimus Binding Proteins; D016211:Transforming Growth Factor alpha; D015291:Transforming Growth Factors / Q000235:genetics