Mitochondrial complex I-perturbed muscle remotely affects gene expression in the fat body in Drosophila
ABSTRACT: Purpose: Muscle injury caused by mitocondrial complex I disruption remotedly impairs mitochondrial activity and lipid homeostasis in the fat body . To gain insight into the corss talk between fat body and muscle, we performed a transcriptomic analysis in fat bodies of 3rd instar larvae with complex I-perturbed muscles. Methods: To extract total RNAs for RNA-Seq experiment, we used 10 fat bodies dissected out from both dMef2-Gal4/UAS-white-RNAi (Con) and dMef2-Gal4/UAS-ND-75-RNAi (ND-75-i) 3rd instar larvae. After assessing RNA quality with Agilent Bioanalyzer, mRNAs were enriched by poly-A pull-down. Then, sequencing libraries constructed with Illumina TruSeq RNA prep kit were sequenced using Illumina HiSeq2000 at the Columbia Genome Center (http://systemsbiology.columbia.edu/genome-center). We multiplexed samples in each lane, which yields targeted number of single-end 100 bp reads for each sample, as a fraction of 180 million reads for the whole lane. Sequence reads were Reads were mapped back to fly genome using flybase annotation r5.51 using Tophat with 4 mismatches (--read-mismatches = 4) and 10 maximum multiple hits (--max-multihits = 10). With the uniquely mapped reads, we quantified gene expression levels using Cufflinks (FPKM values) (version 2.0.2) with default settings. Next, we performed data normalization on the read counts and applied a negative binomial statistical framework using the Bioconductor package DESeq to quantify differential expression between experimental and control data. Results: Gene list enrichment analysis of the differentially expressed genes in ND-75-i larval fat body revealed a striking enrichment of multiple metabolic processes impinging on carbohydrate metabolism, amino acid metabolism, and lipid metabolism. However, half of genes that encode mitochondrial proteins are up-regulated. Interestingly, target genes of TGF-beta signaling pathways, including activin signaling and BMP signaling, are enriched in changed transcriptome in ND-75-i larval fat body . In particular, p-Mad and p-dSmad2, are validated with westenr blot or immunostaining. Conclusions: Our study represents that complex I-perturbed muscle remotely decreases mitochondrial activity and subsequent lipid mobilization in the fat body via modulation of TGF-beta signaling. Our results show that RNA-seq offers a comprehensive evaluation of signaling network and biological process in organ communication. Overall design: mRNA profiles of both dMef2-Gal4/UAS-white-RNAi (Con) and dMef2-Gal4/UAS-ND-75-RNAi (ND-75-i) 3rd instar arval fat bodies were generated by deep sequencing, in replicate, using Illumina HiSeq2000.
Project description:We characterized insulin receptor (InR)-dependent gene expression in the Drosophila fat body using transgenic RNAi. Chronic knockdown of InR in the fat body was elicited via (r4-GAL4, UAS-InRi) and RNA-seq was used to identify potential target genes. Overall design: Drosophila were reared on control (0.15 M sucrose) or high sugar (0.7 M sucrose) diets until the wandering third instar stage. Control (r4-GAL4 x w1118) offspring were compared with InRi (r4-GAL4 x UAS-InRi) using the VDRC's w1118 (#60000) or UAS-RNAi targeting InR (#992). Fat bodies were isolated, and RNA was extracted to determine the effects of reduced insulin signaling on gene expression using Illumina RNA-seq.
Project description:Mutations of the gene MEGF8 cause Carpenter syndrome in humans, and the mouse orthologue has been functionally associated with Nodal and Bmp4 signalling. Here, we have investigated the phenotype associated with loss-of-function of CG7466, a gene that encodes the Drosophila homologue of MEGF8. We generated three different frame-shift null mutations in CG7466 using CRISPR/Cas9 gene editing. Heterozygous flies appeared normal, but homozygous animals had disorganised denticle belts and died as 2nd or 3rd instar larvae. Larvae were delayed in transition to 3rd instars and showed arrested growth, which was associated with abnormal feeding behaviour and prolonged survival when yeast food was supplemented with sucrose. RNAi-mediated knockdown using the Gal4-UAS system resulted in lethality with ubiquitous and tissue-specific Gal4 drivers, and growth defects including abnormal bristle number and orientation in a subset of escapers. We conclude that CG7466 is essential for larval development and that diminished function perturbs denticle and bristle formation.
Project description:Analysis of the larval fat bodies overexpressing AKH, which activates AKH signaling in the fat body in an autocrine manner. As a homolog of human glucagon, AKH causes dysregulation of carbohydrate metabolism and hyperglycemia. Results provide insight into the AKH effects on metabolic gene expression. Overall design: The fat bodies were dissected from late 3rd instar Drosophila larvae of control (R4-Gal4/+) and AKH-overexpressing (UAS-AKH/+; R4-Gal4/+) strains.RNAs were extracted for deep sequencing, in triplicate, using Illumina Hiseq 2000.
Project description:Ehrlichia chaffeensis is a Gram-negative, obligate intracellular bacterium which causes the tick-borne disease human monocytic ehrlichiosis. In vertebrates, E. chaffeensis replicates in monocytes and macrophages. However, no clear cell or tissue tropism has been defined in arthropods. Our group identified two host genes that control E. chaffeensis replication and infection in vivo in Drosophila, Uridine cytidine kinase and separation anxiety. Using the UAS-GAL4 RNAi system, we generated F1 flies (UAS-gene of interestRNAi x tissue-GAL4 flies) that have Uck2 or san silenced in ubiquitous or tissue-specific fashion. When Uck2 or san were suppressed in the hemocytes or in the fat body, E. chaffeensis replicated poorly and caused significantly less severe infections. Silencing of these genes in the eyes, wings, or the salivary glands did not impact fly susceptibility or bacterial replication. Our data suggest that in Drosophila, E. chaffeensis replicates within the hemocytes, the insect homolog of mammalian macrophages, and in the fat body, the liver homolog of mammals.
Project description:In this study, we report the establishment of the binary Gal4/UAS system for the yellow fever mosquito Aedes aegypti. We utilized the 1.8-kb 5' upstream region of the vitellogenin gene (Vg) to genetically engineer mosquito lines with the Vg-Gal4 activator and established UAS-EGFP responder transgenic mosquito lines to evaluate the binary Gal4/UAS system. The results show that the Vg-Gal4 driver leads to a high level of tissue-, stage- and sex-specific expression of the EGFP reporter in the fat body of Vg-Gal4/UAS-EGFP hybrids after blood-meal activation. In addition, the applicability of this system to study hormonal regulation of gene expression was demonstrated in in vitro organ culture experiments in which the EGFP reporter was highly activated in isolated fat bodies of previtellogenic Vg-Gal4/UAS-EGFP females incubated in the presence of 20-hydroxyecdysone (20E). Hence, this study has opened the door for further refinement of genetic tools in mosquitoes.
Project description:We characterized constitutively active insulin receptor (InR)-dependent gene expression in the Drosophila fat body. Transient activation of InR was elicited via heat shock and RNA-seq was used to identify potential target genes. Overall design: Drosophila were reared on control diets until the third instar stage. Control (hs-GAL4 x y1w1118) offspring were compared with InRCA (hs-GAL4 x UAS-InR.A1325D). Fat bodies were isolated 6 hours after a heat shock, and RNA was extracted to determine the effects of transiently increased InR activation on gene expression using Illumina RNA-seq.
Project description:Mitochondrial dysfunction causes biophysical, metabolic and signalling changes that alter homeostasis and reprogram cells. We used a Drosophila model in which individual subunits of 4 OXPHOS complexes are knocked-down in neurons in the larval CNS. We used microarray analysis to investigate gene expression changes caused by knock down of OXPHOS subunits of complexes I, III, IV and V in neurons. Overall design: RNA was purified from the late third instar larval CNS from control larvae (nSyb-Gal4/w), or larvae expressing RNAi transgenes targeting ND-75 (complex I, RNAi line #33910), UQCR-14 (complex III, RNAi line #109542), COX5B (complex IV, RNAi line #105769) and ATPsynCf6 (complex V, RNAi line #107826) in neurons using nSyb-Gal4. Three replicates are included for the each condition.
Project description:Expression of mutant lamins in human muscle causes muscular dystrophy. We have generated a drosophila model that expresses mutant lamins, modeled after those that cause disease in humans. We used affymetrix microarrays to determine the global changes in gene expression caused by mutant lamins. Dissected 3rd instar larval body wall from transgenic Drosophila expressing either a full length wildtype Lamin C, an N-terminal Lamin C truncation of the first 42 amino acids or a single amino acid substitution in Lamin C at position 489 from a G residue to a V residue under control of the Gal4-UAS. RNA was extracted from 3rd instar larval body wall expressing these mutant Lamin C transgenes and microarray analysis using Affymetrix microarrays.
Project description:OBJECTIVES:The evolutionary conserved JNK pathway plays crucial role in cell death, yet factors that modulate this signalling have not been fully disclosed. In this study, we aim to identify additional factors that regulate JNK signalling in cell death, and characterize the underlying mechanisms. MATERIALS AND METHODS:Drosophila were raised on standard media, and cross was carried out at 25°C. The Gal4/UAS system was used to express proteins or RNAi in a specific temporal and spatial pattern. Gene expression was revealed by GFP fluorescence, X-gal staining or immunostaining of 3rd instar larval eye and wing discs. Cell death was visualized by acridine orange (AO) staining. Images of fly eyes and wings were taken by OLYMPUS microscopes. RESULTS:We found that licorne (lic) encoding the Drosophila MKK3 is an essential regulator of JNK-mediated cell death. Firstly, loss of lic suppressed ectopic Egr-triggered JNK activation and cell death in eye and wing development. Secondary, lic is necessary for loss-of-cell polarity-induced, physiological JNK-dependent cell death in wing development. Thirdly, Lic overexpression is sufficient to initiate JNK-mediated cell death in developing eyes and wings. Furthermore, ectopic Lic activates JNK signalling by promoting JNK phosphorylation. Finally, genetic epistatic analysis confirmed that Lic acts in parallel with Hep in the Egr-JNK pathway. CONCLUSIONS:This study not only identified Lic as a novel component of the JNK signalling, but also disclosed the crucial roles and mechanism of Lic in cell death.
Project description:Chemosensory proteins (CSPs) have been predicted to be involved in development; however, direct evidence for their involvement is lacking, and genetic basis is largely unknown. To determine the function of the chemosensory protein 9 (Si-CSP9) gene in Solenopsis invicta, we used RNA interference to silence Si-CSP9 in 3rd-instar larvae. The 3rd-instar larvae failed to shed their cuticle after being fed Si-CSP9-directed siRNA, and expression profiling of RNAi-treated and untreated control larvae showed that 375 genes were differentially expressed. Pathway enrichment analysis revealed that 4 pathways associated with larval development were significantly enriched. Blast analysis revealed that one fatty acid amide hydrolase (FAAH) gene was up-regulated and 4 fatty acid synthase (FAT) genes and one protein kinase DC2 gene (PKA) were down-regulated in the enriched pathways. Significantly higher expression of these genes was found in 4th-instar larvae, and Pearson correlation analysis of the expression patterns revealed significant relationships among Si-CSP9, PKA, FAAH, and FAT1-4. Moreover, we confirmed that expression levels of Si-CSP9, FAAH, and FAT1-4 were significantly reduced and that the development of 3rd-instar larvae was halted with PKA silencing. These results suggest that Si-CSP9 and PKA may be involved in the network that contributes to development of 3rd-instar larvae.