Characterization of differentially expressed genes in purified Drosophila follicle cells: Toward a general strategy for cell type-specific developmental analysis

Abstract

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 f luorescence-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 f luorescent 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.Recent years have seen an explosion in tools for analyzing differential gene expression. The development of a PCR-based differential display method by Liang et al. (1, 2) has quickly been followed by array-based methods for monitoring the expression of thousands of defined genes simultaneously (3, 4). Developmental biology, a field whose progress depends heavily on understanding regulated gene expression, stands to benefit from these methods. However, there are technical hurdles to be overcome before molecular screening methods can be widely applied to problems in development. Chief among these hurdles is purification of the tissue of interest from the complex mixture of cell types typically present in a developing system. Whereas some tissues are amenable to microdissection, others will require more sophisticated techniques such as fluorescence-activated cell sorting (FACS) of dissociated tissue (5-7). The molecular screens reported herein rely on a FACS-based approach to purify specialized follicle cells from Drosophila ovaries. The system we have chosen is particularly illustrative of the need for tissue purification: the Drosophila ovary is composed of egg chambers in which a cyst of large germ-line cells is surrounded by a thin epithelium of highly differentiated follicle cells. Patterning of subregions of the follicular epithelium involves intermingled cell groups, each comprising only a tiny fraction of the volume...

Dystroglycan down-regulation links EGF receptor signaling and anterior–posterior polarity formation in the Drosophila oocyte

“…The A62-GAL4 overexpression flies had the genotype UAS-DG͞pin; A62-GAL4͞ϩ. The A62-GAL4 stock (15) crossed to a UAS-GFP line indicates expression in all PFC beginning around stage 6 and also in the border cells beginning shortly thereafter (data not shown). From the current understanding of AP axis formation, we believe that any of the phenotypes described herein for the A62-GAL4 overexpression crosses resulted from its expression in the PFC and not from its expression in the border cells, particularly because the phenotypes described here can be observed well before the border cells have migrated to the oocyte.…”

“…To examine this possibility, we induced overexpression of DG by using a UAS construct of the full-length DG protein (14) under control of either the A62-GAL4 (a PFC driver) (15) or flip-out GAL4 driver (16). Overexpression of DG by A62-GAL4 resulted in high levels of DG in all membrane domains of the PFC (Fig.…”

Cell-type-specific profiling of protein–DNA interactions without cell isolation using targeted DamID with next-generation sequencing

“…Briefly, in the first phase tissue is collected and genomic DNA is extracted (steps 1-11) and digested with the restriction enzyme DpnI, which only cuts at adenine-methylated GATC sites. The digested DNA is column purified to exclude un-cut, genomic DNA from uninduced cells (steps [12][13][14], and adaptors for PCR-amplification are ligated to the DpnI cut fragments (steps [15][16][17][18]. To prevent unmethylated regions of DNA from being aberrantly amplified, the material is digested with DpnII, which only cuts non-methylated GATC sites (steps [19][20][21]).…”

Ras signaling modulates activity of the ecdysone receptor EcR during cell migration in the drosophila ovary

“…Trizol reagent (Invitrogen) was used to extract total RNA from dissociated FC that were collected as described by Bryant et al (1999b). Reverse transcription was performed using M-MLV Reverse Transcriptase (Fisher) using random hexamer primers (Promega …”