Project description:Transcriptional profiling comparing adult wild-type indirect flight muscle (IFM) with wild-type leg muscle and salm RNAi IFM (Mef2-GAL4, UASsalmIR). We used 2 different salm hairpin constructs for the experiments, TF3029 and TF101052, both available from the VDRC Drosophila stock centre.
Project description:Transcriptional profiling comparing adult wild-type indirect flight muscle (IFM) with wild-type leg muscle and salm RNAi IFM (Mef2-GAL4, UASsalmIR). We used 2 different salm hairpin constructs for the experiments, TF3029 and TF101052, both available from the VDRC Drosophila stock centre. Experiments were done in biological duplicates + 1 technical replicate (1 labeled sample was hybridized on a different array)
Project description:In Drosophila, fibrillar flight muscles (IFMs) enable flight, while tubular muscles mediate other body movements. Here, we use RNA-sequencing and isoform-specific reporters to show that spalt major (salm) determines fibrillar muscle physiology by regulating transcription and alternative splicing of a large set of sarcomeric proteins. We identify the RNA binding protein Arrest (Aret, Bruno) as downstream of salm. Aret shuttles between cytoplasm and nuclei, and is essential for myofibril maturation and sarcomere growth of IFMs. Molecularly, Aret regulates IFM-specific transcription and splicing of various sarcomeric targets, including Stretchin and wupA (TnI), and thus maintains muscle fiber integrity. As Aret and its sarcomeric targets are evolutionarily conserved, similar principles may regulate mammalian muscle morphogenesis. 9 samples from Drosophila melanogaster were analyzed in duplicate: control dissected wildtype flight muscle at 30h APF, 72h APF and 0 day adult, jump muscle and whole leg from 1d adult and RNAi/mutant conditions for salm (1d flight muscle) and aret (30h, 72h and 1d flight muscle)
Project description:To complement our existing data on developmental gene expression changes in flight muscle (IFM) development in Drosophila (GSE107247, GSE63707), we performed mRNA-Seq on dissected leg samples at three stages during pupal development (30, 50 and 72h APF). We further sequenced an additional timepoint at 24h APF for RNAi knockdown of aret (Bru1) in flight muscle. Comparison of splicing and expression profiles of sarcomeric genes allowed us to identify muscle-type specific differences in gene and isoform expression between fibrillar flight muscle and tubular leg muscle. We can further trace the dynamics of exon usage in sarcomere genes across the developmental timecourse, allowing us to identify events the switch during muscle differentiation and maturation.
Project description:We performed a forward genetic screening and identifid the transcription factor, salm. To identify potential downstream targets, we sorted the developing muscle cells from stage 11 embryo using GFP tagged by TEY-gal4 in wild type vs salm[340] mutants. This approach successfully identified the potential SALM-target genes.
Project description:Expression profiling of IFMs from 1-2 day old adult male flies of 3 genotypes: Canton-S, IFM-specific actin null (Act88FKM88) and IFM-specific myosin null (Mhc7). Results provide insight into how muscles respond to the absence of major scaffold proteins, and whether these transcriptional responses are filament-specific or generic to the tissue.
Project description:In Drosophila, fibrillar flight muscles (IFMs) enable flight, while tubular muscles mediate other body movements. Here, we use RNA-sequencing and isoform-specific reporters to show that spalt major (salm) determines fibrillar muscle physiology by regulating transcription and alternative splicing of a large set of sarcomeric proteins. We identify the RNA binding protein Arrest (Aret, Bruno) as downstream of salm. Aret shuttles between cytoplasm and nuclei, and is essential for myofibril maturation and sarcomere growth of IFMs. Molecularly, Aret regulates IFM-specific transcription and splicing of various sarcomeric targets, including Stretchin and wupA (TnI), and thus maintains muscle fiber integrity. As Aret and its sarcomeric targets are evolutionarily conserved, similar principles may regulate mammalian muscle morphogenesis.
Project description:Mitochondrial networks provide coordinated energy distribution throughout muscle cells. However, pathways specifying mitochondrial network-type separately from contractile fiber-type remain unclear. Here, we show that natural energetic demands placed on Drosophila melanogaster muscles yield native cell-types among which contractile and mitochondrial network-types are regulated independently. Proteomic analyses of indirect flight, jump, and leg muscles together with muscles misexpressing known fiber-type specification factor salm identified transcription factors H15 and cut as potential mitochondrial network regulators. We demonstrate H15 operates downstream of salm regulating flight muscle contractile and mitochondrial network-type. Conversely, H15 regulates mitochondrial network configuration but not contractile type in jump and leg muscles. Further, we find that cut regulates salm expression in flight muscles and mitochondrial network configuration in leg muscles. These data indicate cell type-specific regulation of muscle mitochondrial network organization separately from contractile type, mitochondrial content, and mitochondrial size through an evolutionarily conserved pathway involving cut, salm, and H15.