Project description:Insulin signalling activity is well known to mediate transcriptional changes in response to nutrients and it is changed in the spargel mutant. We performed a genome-wide microarray study to investigate the role of Spargel as a transcriptional coregulator downstream of insulin receptor signalling. Experiment Overall Design: To investigate the effect of Spargel on transcriptional changes mediated by the insulin-signalling pathway we analyzed 12 samples in total: three biological replicates of the control with or without UAS-Inr (Inr_wt and ctrl_wt) and three biological replicates of the spargel mutant with or without UAS-Inr (Inr_KGand ctrl_KG).
Project description:dFOXO targets in adult Drosophila melanogaster females, and the effect of insulin signalling and stress on binding. The experimets determined the binding locations of dFOXO in the whole adult female fly using ChIP-chip. The protocol was validated using mock conditions: pre-immune serum or IP on chromatin from foxo null flies. The response of this binding to stress induced by treatment of flies with paraquat or by their exposure to starvation, as well as the response to an insulin-signalling-reducing genetic manipulation (over-expression of dominant negative form of the insulin receptor), was determined.
Project description:Transcriptional analysis of dInR and dfoxo epistasis in Drosophila melanogaster. The experiment was performed to examine which parts of the transcriptional response to a reduction in insulin signalling in an adult female fly depend on the presence of the dfoxo transcription factor. Whole fly transcriptome was determined with flies over-expressing a dominant negative form of the insulin receptor, or the wild-type fly, in presence or absence of dfoxo.
Project description:Background: Despite the prevalence and biological relevance of both signalling pathways and alternative pre-mRNA splicing, our knowledge of how intracellular signalling impacts on alternative splicing regulation remains fragmentary. We report a genome-wide analysis of changes in alternative splicing using splicing-sensitive microarrays, induced by activation of two distinct signalling pathways, insulin and wingless, in Drosophila cells in culture. Results: Alternative splicing changes induced by insulin affect more than 150 genes and more than 50 genes are regulated by wingless activation. About 40% of the genes showing changes in alternative splicing also show regulation of mRNA levels, suggesting distinct but also significantly overlapping programs of transcriptional and posttranscriptional regulation. Distinct functional sets of genes are regulated by each pathway and, remarkably, a significant overlap is observed between functional categories of genes regulated transcriptionally and at the level of alternative splicing. Functions related with carbohydrate metabolism and cellular signalling are enriched among genes regulated by insulin and wingless, respectively. Computational searches identify pathway-specific sequence motifs enriched near regulated 5â splice sites. Conclusion: Taken together, our data indicate that signalling cascades trigger pathway-specific and biologically coherent regulatory programs of alternative splicing regulation. They also reveal that alternative splicing can provide a novel molecular mechanism for cross-talk between different signalling pathways. To monitor transcriptional and alternative splicing changes induced by activation of the insulin and wingless pathways, a custom-designed microarray platform was employed featuring probes for all Drosophila genes for which different mRNA isoforms generated by alternative splicing have been described (see Blanchette M, Green RE, Brenner SE, Rio DC: Global analysis of positive and negative pre-mRNA splicing regulators in Drosophila. Genes Dev 2005, 19(11):1306-1314.). Three biological replicates of total RNA isolated after pathway activation or controls (untreated cells for insulin, control dsRNA for wingless) were purified, reverse transcribed into cDNA and labelled with Cy5 or Cy3 fluorochromes and the cDNA was hybridized to the microarray,
Project description:Background: Despite the prevalence and biological relevance of both signalling pathways and alternative pre-mRNA splicing, our knowledge of how intracellular signalling impacts on alternative splicing regulation remains fragmentary. We report a genome-wide analysis of changes in alternative splicing using splicing-sensitive microarrays, induced by activation of two distinct signalling pathways, insulin and wingless, in Drosophila cells in culture. Results: Alternative splicing changes induced by insulin affect more than 150 genes and more than 50 genes are regulated by wingless activation. About 40% of the genes showing changes in alternative splicing also show regulation of mRNA levels, suggesting distinct but also significantly overlapping programs of transcriptional and posttranscriptional regulation. Distinct functional sets of genes are regulated by each pathway and, remarkably, a significant overlap is observed between functional categories of genes regulated transcriptionally and at the level of alternative splicing. Functions related with carbohydrate metabolism and cellular signalling are enriched among genes regulated by insulin and wingless, respectively. Computational searches identify pathway-specific sequence motifs enriched near regulated 5’ splice sites. Conclusion: Taken together, our data indicate that signalling cascades trigger pathway-specific and biologically coherent regulatory programs of alternative splicing regulation. They also reveal that alternative splicing can provide a novel molecular mechanism for cross-talk between different signalling pathways.
Project description:The patterning of Drosophila retina occurs both very fast and with high precision. This process is driven by the dynamic changes in signalling activity of the conserved Hedgehog (Hh) pathway, which coordinates cell fate determination, cell cycle and tissue morphogenesis. Here we show that during Drosophila retinogenesis, the retinal determination gene dachshund (dac) is not only a target of the Hh signaling pathway, but is also a modulator of its activity. Using developmental genetics techniques, we demonstrate that dac enhances Hh signaling by promoting the accumulation of the Gli transcription factor Cubitus interruptus (Ci) parallel to or downstream of fused. In the absence of dac, all Hh-mediated events associated to the morphogenetic furrow are delayed. One of the consequences is that, posterior to the furrow, dac- cells cannot activate a Roadkill-Cullin3 negative feedback loop that attenuates Hh signaling and which is necessary for retinal cells to continue normal differentiation. Therefore, dac is part of an essential positive feedback loop in the Hh pathway, guaranteeing the speed and the accuracy of Drosophila retinogenesis.
Project description:The patterning of Drosophila retina occurs both very fast and with high precision. This process is driven by the dynamic changes in signalling activity of the conserved Hedgehog (Hh) pathway, which coordinates cell fate determination, cell cycle and tissue morphogenesis. Here we show that during Drosophila retinogenesis, the retinal determination gene dachshund (dac) is not only a target of the Hh signaling pathway, but is also a modulator of its activity. Using developmental genetics techniques, we demonstrate that dac enhances Hh signaling by promoting the accumulation of the Gli transcription factor Cubitus interruptus (Ci) parallel to or downstream of fused. In the absence of dac, all Hh-mediated events associated to the morphogenetic furrow are delayed. One of the consequences is that, posterior to the furrow, dac- cells cannot activate a Roadkill-Cullin3 negative feedback loop that attenuates Hh signaling and which is necessary for retinal cells to continue normal differentiation. Therefore, dac is part of an essential positive feedback loop in the Hh pathway, guaranteeing the speed and the accuracy of Drosophila retinogenesis. ChIP-seq against Dachshund vs input ChIP-seq. Eye-antennal imaginal discs are dissected from Grh-GFP (Bloomington stock 42269) 3rd instar larvae and fixed with formaldehyde. Chromatin is prepared and sonicated until fragments reach an average size of 500 bp. Chromatin is immunoprecipitated with an anti-GFP Ab (ab290, Abcam) and the immunocomplexes are recovered with protein A/G magnetic beads (Millipore).