Project description:We use mRNA-seq to transcriptionally profile larval fat body and midgut tissues from Drosophila third instar larvae. These data provide insights into tissue physiology and can be used to identify tissue specific transcripts. Fat bodies from wandering third instar larvae were dissected from ~50 male larvae and gonads were removed to eliminate contaminating transctips from the gonads. Larval midguts were dissected from ~50 wandering third instar larvae. Larval tissues were removed to Graces unsupplemented medium on ice prior to RNA extraction with TRIzol reagent. mRNA-seq samples were prepared from 5ug of total RNA and subject to Illumina based sequencing.
Project description:We use mRNA-seq to transcriptionally profile larval fat body and midgut tissues from Drosophila third instar larvae. These data provide insights into tissue physiology and can be used to identify tissue specific transcripts.
Project description:We used RNA-seq in a derived European Drosophila melanogaster population from Germany (MU) to examine coding gene expression variation in the larval fat body during the late wandering third instar stage.
Project description:We analyzed Origin Recognition Complex Subunit 2 (ORC2) ChIP-seq from hand-dissected fat body tissue from 68hr (after egg laying, AEL), 92hr AEL, and late-third wandering Drosophila melanogaster larvae. Fat body was dissected from wild-type (OrR) males and testes were removed. We examined ORC2 binding genome-wide with particular focus on the underreplicated regions in the fat body.
Project description: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.
Project description:The lysosomal function is down-regulated in the white prepupal fat body, resulting in the enlargement of lysosomes in the tissue. The enlargement is blocked by the forced activation of lysosomes by the overexpression of mitf, a sole homolog of the MiTF/TFE family transcription factors. Thus, it is possible to speculate that mitf participates in the down-regulation of lysosomes in the fat body. To test this possibility, we performed a comparative mRNA-seq of (1) wild-type white prepupal fat body, (2) mitf overexpressed white prepupal fat body, and (3) wild-type third instar larval fat body. First, a comparison of (1) and (2) showed that the overexpression of mitf upregulated transcription of most of the lysosome-related genes in the fat body, consistent with previous studies. Next, a comparison of (1) and (3) indicated that the transcription level of several lysosome-related genes was decreased in (1) compared to (3). However, most of the genes regulated by Mitf were not transcriptionally affected. These results suggest that mitf is dispensable for the downregulation of lysosomes in the white prepupal fat body.
Project description:The role of circadian clocks in regulating metabolic processes has been studied extensively. Yet, the physiological impacts of the circadian system on metabolic states across species and life stages remain to be explored. This study investigates the relationship between circadian rhythms and metabolic regulation in the fat body of Drosophila larva, an organ crucial for maintaining metabolic homeostasis, growth and developmental timing. Larval fat body is analogous to the mammalian liver and adipose tissue but lacks a canonical circadian clock. Around-the-clock RNA-sequencing analysis on the fat bodies of wild-type and period clock gene null mutant larvae revealed circadian rhythms in the transcriptome of wild-type larvae. Surprisingly, period mutant exhibited 12-h rhythms in the expression of numerous genes, particularly those involved in peroxisome function, lipid metabolism, and oxidative stress response. Consistent with these transcriptomic data, peroxisome biogenesis and activity demonstrated 12-h rhythms in period mutant fat bodies. Furthermore, levels of reactive oxygen species displayed inverse-phased rhythms to that of lipid peroxidation, with 24-h rhythms in wild-type and 12-h rhythms in period mutant fat bodies. Moreover, while daily fat storage levels in wild-type larvae remained constant, period mutants exhibited fluctuations with a 12-h period and a net reduction in body fat storage. Collectively, our results identified a clock-independent ultradian rhythms in lipid metabolism, which may contribute to maintaining the metabolic, energetic, and redox homeostasis essential for larval survival and development.
Project description:We performed mRNA-seq from hand-dissected fat body tissue from 68hr (after egg laying, AEL) and 92hr AEL Drosophila melanogaster larvae. Fat body was dissected from wild-type (OrR) males and testes were removed. We examined gene expression genome-wide with particular focus on genes in the underreplicated regions in the fat body.
Project description:Activation of innate immune responses in the Drosophila larval fat body affects the physiological host responses. In order to characterize the effect of the activated immune responses in the fat body on the Drosophila, we used whole-genome microarray analysis and found that activation of the immune deficieny pathway (Imd) in the fat body alters the transcriptional profiles of Drosophila larvae. As we expected many of genes involved in regulation of antimicrobial peptides were upregulated in the larvae with elevated Imd activity in the fat body. Notably, we found activatioan of Imd in the fat body negatively affects expression of genes involved in glycolysis, energy production and insulin signaling pathway. Overall, our analysis showed that activation of innate immune signaling in the larval fat body significantly affects cellular pathways that regulate metabolism.