Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of Drosophila mef2 allelic series reveals mef2 activity levels differentially affect gene expression during muscle development

ABSTRACT: The conserved Mef2 transcription factor is a major regulator of gene expression and differentiation. Recent genomic studies have identified a large number of mef2-regulated target genes with distinct temporal expression profiles during Drosophila myogenesis. However, the question remains as to how a single transcription factor can control such diverse patterns of gene expression. The aim of this project was to investigate whether there are genes with different mef2-requirements for their expression during muscle differentiation in vivo during the development of Drosophila melanogaster. Experiment Overall Design: We used microarrays in conjunction with a mef2 allelic series to determine the gene expression profile in developing embryos at five different levels of mef2 activity. The allelic series extends from a null mutation, mef222.21, through to the control, via three hypomorphic alleles, mef2113, mef2424, and mef265. The series corresponds to different levels of Mef2 in the following order: control > 65 > 424 > 113 > 22.21. The control stock for the microarray analysis was dp cn a px sp, the stock used for the mutagenesis that produced mef265, mef2424 and mef2113. For the microarrays, 30 minute collections of control and mutant embryos for each mef2 allele were individually staged and pools of 150 embryos were then processed at mid stage 13. This corresponds to the early differentiation phase of muscle development and the expression of multiple muscle sarcomeric protein genes. Quadruplicate samples were assayed using AffymetrixÂ® Genechips by the Flychip Drosophila microarray resource.

ORGANISM(S): Drosophila melanogaster

SUBMITTER: Michael Victor Taylor

PROVIDER: E-GEOD-9889 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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mef2 activity levels differentially affect gene expression during Drosophila muscle development.

Elgar Stuart J SJ Han Jun J Taylor Michael V MV

Proceedings of the National Academy of Sciences of the United States of America 20080115 3

Cell differentiation is controlled by key transcription factors, and a major question is how they orchestrate cell-type-specific genetic programs. Muscle differentiation is a well studied paradigm in which the conserved Mef2 transcription factor plays a pivotal role. Recent genomic studies have identified a large number of mef2-regulated target genes with distinct temporal expression profiles during Drosophila myogenesis. However, the question remains as to how a single transcription factor can ...[more]

PMID: 18198273

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Project description:Skeletal muscles are formed in a variety of shapes and sizes, and this diversity impacts muscle function and disease susceptibility. To understand how muscle diversity is generated, we performed gene expression profiling of muscle subsets from Drosophila embryos. By comparing the transcriptional profiles of two muscle subsets, we identified a core group of founder cell-enriched genes. We screened mutant embryos for muscle defects and identified Sin3A and 10 other transcription and chromatin regulators as having novel functions in the Drosophila embryonic somatic musculature. Sin3A is required for the morphogenesis of a subset of muscles, and Sin3A mutants display muscle loss and misattachment. Additionally, misexpression of identity gene transcription factors in Sin3A heterozygous embryos leads to direct transformations of one muscle into another, while overexpression of Sin3A results in the reverse transformation. Our data implicate Sin3A as a key buffer controlling muscle responsiveness to transcription factors in the formation of muscle identity, thereby generating tissue diversity. We used microarray analysis to look for changes in gene expression between different musle founder cell subsets Flies expressing GFP or RFP transgenes were maintained at 25° C on a 12-hour light/12-hour dark cycle in an incubator and fed with yeast paste spread on apple juice agar plates. After a 2-hour pre-lay, flies were allowed to lay for 2 hours; these plates were removed and aged for an additional 8 hours. Embryos were dissociated by dounce homogenization into a single cell suspension as previously described (Dobi et al., 2011). Cells were sorted using standard methods on a Beckton-Dickson FACS Aria Cell Sorter (Estrada and Michelson, 2008). Total cellular RNA from each sample was prepared from sorted cells using TRIzol LS Reagent without amplification (Invitrogen). Total cellular RNA was labeled and hybridized to Drosophila Affymetrix GeneChip 2.0 arrays according to the manufacturers instructions. Three independent sorts were made for each transgene, and each RNA sample was independently labeled and hybridized. For apME-GFP, we sorted 1,000,000 GFP-positive cells from 4 X 108 starting cells, a positive-rate of 0.03%, which yielded 1 μg of RNA. The purity upon resort of the cells was 74%. The purity of S59ME-RFP and twiprom-actin-GFP was 87% and 95%, respectively, with positive-rates and yields also correspondingly higher. Affymetrix microarray data was normalized and compared using Partek Genomics Suite Software. apterousME-GFP and slouchME-RFP-positive cells were compared to twiprom-actin-GFP-expressing cells to generate lists of differentially regulated genes, fold change ≥ 1.7, p-value < 0.05. The Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.7 was used to assign Gene Ontology classifications to the genes identified by the array (Huang et al., 2009a; 2009b). Default parameters were used to perform Functional Annotation of gene lists, p-value < 0.05.

Dataset Information

Transcription profiling of Drosophila mef2 allelic series reveals mef2 activity levels differentially affect gene expression during muscle development

Publications

mef2 activity levels differentially affect gene expression during Drosophila muscle development.

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