Project description:When misexpressed in late Drosophila prepupae, the transcription factor Senseless (Sens) blocks death of the larval salivary glands that normally occurs in the early pupa. The aim of the experiment was to identify genes responding to Sens that might mediate the effect of the protein on cell death and other biological processes. The yeast transcription factor GAL4, expressed from a heat-inducible transgene (P{GAL4-Hsp70.PB}89-2-1), was used to drive expression of Sens from a UAS-sens transgene. After crossing the GAL4 and UAS lines, expression of GAL4 was induced by a 30-min heat shock treatment (37 Â°C) of the progeny at 9 hours after puparium formation. Salivary glands were dissected at 14 hours after puparium formation and RNA isolated for microarray analysis with Affymetrix GeneChips. Control samples were obtained from animals treated the same way carrying one copy of the GAL4 transgene (progeny of a cross between flies of the P{GAL4-Hsp70.PB}89-2-1 and w1118 strains) and w1118 animals. The microarray data identified several genes associated with programmed cell death, including caspase genes, which respond to Sens. In addition, the data show that many Drosophila genes respond to the yeast transcription factor GAL4 in a UAS-independent manner. To identify target genes of Sens that are of biological relevance, gene expression patterns in the presence of Sens were compared to gene expression patterns in both the presence and the absence of GAL4. This comparison revealed that Sens seems to preferentially downregulate targets that are upregulated by GAL4, suggesting that these genes may not necessarily constitute true transcriptional targets of Sens. Experiment Overall Design: Experimental RNA samples were obtained in 3 biological replicates from sens-expressing salivary glands (SG_sens_14APF_rep1, SG_sens_14APF_rep2, SG_sens_14APF_rep3) and compared to control samples obtained from GAL4-expressing salivary glands (SG_1P{hsGAL4}89(S)_14APF_rep1) and non-GAL4-expressing salivary glands (SG_w1118_14APF_rep1, SG_w1118_14APF_rep2). All samples were compared using dChip and normalized to the same baseline array (median intensity 150).

Project description:The yeast transcription factor GAL4 has been reported to cause cell death and to have other biological effects when expressed in Drosophila (Kramer and Staveley, 2003: Genet. Mol. Res. 2, 43; Rezaval et al., 2007: Eur. J. Neurosci. 25, 683). Using heat-shock-induced expression of GAL4 to drive expression of a UAS-senseless responder gene in transcriptional profiling experiments, we found that the underlying cause of these effects might be a genomic response to GAL4. To further characterize this response and to account for GAL4-independent changes caused by the transgene integration, GAL4 was expressed from two copies of the transgene in two independent lines, P{GAL4-Hsp70.PB}89-2-1 (short P{hs-GAL4}89) and P{hs-GAL4}X1. In addition, GAL4 was expressed from only one copy of the transgene in P{hs-GAL4}89 prepupae to account for the dosage dependence of observed effects. Prepupae carrying the hs-GAL4 transgenes were subjected to a 30-min heat shock treatment (37 Â°C) at 9 hours after puparium formation. RNA was isolated from salivary glands dissected from these and similarly treated w1118 control animals at 14 hours after puparium formation and subjected to microarray analysis with Affymetrix GeneChips. The microarray data identified an overlapping set of 1,009 genes that showed an at least 1.5-fold change in expression in both of the GAL4-expressing lines, defining a core set of GAL4-responsive genes in the salivary glands. This set includes genes involved in the control and execution of programmed cell death and in other important regulatory pathways. Experiment Overall Design: Experimental RNA samples were obtained from GAL4-expressing salivary glands of the line P{hs-GAL4}X1 in 3 biological replicates (SG_P{hsGAL4}X1_14APF_rep1, SG_P{hsGAL4}X1_14APF_rep2, SG_P{hsGAL4}X1_14APF_rep3); of the line P{hs-GAL4}89 in one replicate (SG_P{hsGAL4}89_14APF_rep1); of animals carrying only one copy of P{hs-GAL4}89 in one replicate (SG_1P{hsGAL4}89_14APF_rep1). The samples were compared to control samples obtained in two biological replicates from the salivary glands of w1118 control animals (SG_w1118(2)_14APF_rep1, SG_w1118(2)_14APF_rep2). All samples were compared using dChip and normalized to the same baseline array (median intensity 145). Statistical analysis of differences between the P{hs-GAL4}X1 and w1118 samples was carried out using GeneSpring (Agilent Technologies) and a false discovery rate (FDR) cutoff of 0.05.

Project description:When misexpressed in late Drosophila prepupae, the transcription factor Fork head (Fkh) blocks death of the larval salivary glands that normally occurs in the early pupa. The aim of the experiment was to identify genes responding to Fkh that might mediate the effect of the protein on cell death and other biological processes. Fkh was expressed in the line P[hs-Fkh111] from a heat-inducible transgene that encodes wild-type Fkh protein. Expression of Fkh was induced by incubating prepupae for 30 min in a 37 Â°C water bath, starting at 9.5 hours after puparium formation. Salivary glands were dissected at 14 hours after puparium formation and RNA isolated for microarray analysis with Affymetrix GeneChips. Control samples were obtained from w1118 animals treated the same way. The microarray analysis identified 55 genes annotated as functioning in apoptosis whose expression was at least 1.5-fold changed by Fkh. These genes include the death genes hid and reaper, which play a central role in the control of salivary gland death. Other groups of significantly enriched genes include genes functioning in autophagy, steroid-signaling pathways, salivary gland secretion, and phospholipid metabolism. In addition, the microarray data identify genes as responsive to Fkh that are known to be controlled by the FOXA counterparts of Fkh in vertebrates, indicating that target genes and biological processes controlled by Fkh are evolutionarily conserved. SUBMITTER_CITATION: Liu and Lehmann, 2008: Genes and biological processes controlled by the Drosophila FOXA orthologue Fork head. Insect Molecular Biology, 17, 91-101; link: http://www.blackwell-synergy.com/toc/imb/17/2) Experiment Overall Design: Experimental RNA samples were obtained in 3 biological replicates from fkh-expressing salivary glands (SG_Fkh_14APF_rep1, SG_Fkh_14APF_rep2, SG_Fkh_14APF_rep3) and compared to control samples obtained in two biological replicates from the salivary glands of w1118 control animals (SG_w1118(3)_14APF_rep1, SG_w1118(3)_14APF_rep2). All samples were compared using dChip and normalized to the same baseline array (median intensity 134).

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