Unknown,Transcriptomics,Genomics,Proteomics

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Effects of dsRNA-GFP as exogenous control on honey bee RNAi assays: gene expression changes in workers

ABSTRACT: A green fluorescence protein (GFP)-derived dsRNA (dsRNA-GFP) has been used as an exogenous control for Apis mellifera RNAi assays by multiple research groups. Its sequence does not share any significant homology with any known honey bee genes. Although dsRNA-GFP is not expected to trigger an RNAi response in treated bees, undesirable effects on gene expression, pupal pigmentation or developmental timing have been routinely observed. To better understand the multiple molecular and phenotypic effects of dsRNA-GFP in honey bees and to evaluate its use as a control for RNAi studies, we examined the impact of dsRNA-GFP on global gene expression patterns in developing workers. We found that dsRNA-GFP causes large-scale changes in gene expression associated with multiple biological processes. Furthermore, dsRNA-GFP exposure tended to preferentially decrease, rather than increase, expression of genes compared to controls. Gene expression differences were analyzed using a dye-swap design. Second instar worker larvae received 0.5 M-NM-

ORGANISM(S): Apis mellifera

SUBMITTER: Ana Bomtorin

PROVIDER: E-GEOD-41004 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Neural basic helix-loop-helix (bHLH) transcription factors are important for the differentiation and cell type specification of neurons. They are thought to share direct downstream targets in their common role as neuronal differentiation factors, but have distinct targets with respect to their cell type specific roles. Little is known about distinct cell-type specific bHLH targets as previous work did not distinguish these from common targets. Based on previous genetic evidence, we hypothesize that bHLH transcription factors have unique targets for their function in regulating neuronal sub-type specification. Atoh1 (Math1) is a bHLH transcription factor that specifies different cell types of the proprioceptive pathway in mammals such as the dorsal interneuron 1 population of the developing neural tube. Using microarray analyses of neighboring specific bHLH sorted populations from developing mouse neural tubes, we determine transcripts unique to the Atoh1-derived population and not those common to bHLH transcription factors in related neural progenitor populations. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) experiments of native tissue followed by enhancer reporter analyses identified five direct cell-type specific targets of Atoh1 in vivo: Klf7, Rab15, Rassf4, Selm, and Smad7, along with their Atoh1-responsive enhancers. These Atoh1 targets were found from native tissue in the appropriate developmental context and have diverse functions that range from transcription factors to regulators of endocytosis and signaling pathways. Only Rab15 and Selm are expressed across several different Atoh1-specified cell types including external granule cells (EGL) in the developing cerebellum, hair cells of the inner ear, and Merkel cells, demonstrating that even within Atoh1 lineages, not all Atoh1 specific targets are shared. Our work establishes on a molecular level that the neuronal differentiation bHLH transcription factors also have distinct targets for their roles in neuronal sub-type specification. From this work, we can begin to address how bHLH transcription factors are able to specify unique cell types and initiate programs that organize neuronal diversity. Gene expression analysis: Two samples, Atoh1-GFP and dNeurog1-GFP, were analyzed. Two biological replicates of each. Atoh1-GFP transgenics are mice with GFP inserted into a BAC that drives GFP to the Atoh1-derived population of the developing neural tube marking the dorsal interneuron 1 population (Raft et al. Development 2007). dNeurog1-GFP transgenics are mice with a Neurog1 enhancer that drives GFP to the dorsal part of the developing neural tube marking the dorsal interneuron 2 population (Nakada et al. Dev Bio 2004). Chip-seq analysis: Series GSE22111

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Effects of dsRNA-GFP as exogenous control on honey bee RNAi assays: gene expression changes in workers

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