Unknown,Transcriptomics,Genomics,Proteomics

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Molecular profiling of hypothalamic leucine-sensing neurons via PhosphoTRAP

ABSTRACT: To identify the molecular signature of leucine-activated neurons in the MBH, we adopted the PhosphoTRAP assay following parenchymal injection of leucine into the MBH. Neurons activated following MBH leucine injection (and expressing the neuronal activation marker cFos) co-express Ser240/244-phosphorylated ribosomal S6 protein , allowing selective immunoprecipitation-based capture (TRAP) and RNA sequencing of polysomes from leucine-activated neurons. Differential expression analysis between IP and input samples generated a list of enriched transcripts in leucine-activated neurons

INSTRUMENT(S): Illumina HiSeq 2000

ORGANISM(S): Mus musculus

SUBMITTER: Brian Lam

PROVIDER: E-MTAB-16665 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Adult reproductive behaviors in Drosophila melanogaster males and females are vastly different. Yet, neurons that express sex-specifically spliced fruitless transcripts (fru P1) underlie these behaviors in both sexes. How the same set of neurons can drive such different behaviors is an important and unresolved question in developmental genetics. A particular challenge is that fru P1-expressing neurons are only 2-5% of the adult nervous system, making studies of adult head tissue, or even the whole brain, unlikely to yield informative inferences. Translating Ribosome Affinity Purification (TRAP) identifies the actively translated pool of mRNAs from fru P1-expressing neurons and allowed us to conduct a sensitive, cell-specific assay of gene expression. The male and female fru P1-expressing neurons have a shared set of 1,642 genes with enriched TRAP transcripts that form a distinct repertoire, relative to TRAP analyses of all neurons in the adult head. Further, there are a striking number of genes (3,147) that have sex-biased TRAP enrichment in fru P1-expressing neurons. Yet, most of these genes (3,107) have only male-biased TRAP enrichment. This suggests an underlying mechanism to generate dimorphism in behavior, with the transcript repertoire that specifies female behaviors present in both sexes and a large additional set of genes with expression in the male dramatically altering the pattern. Thus, these additional genes invoke the male-specific behaviors by establishing cell fate in the same context of gene expression observed in females. These results suggest a possible global mechanism for how distinct behaviors can arise in different environments, from a shared set of neurons. Libraries were prepared from five independent biological replicates, from TRAP adult heads samples from males and females, and the mRNA input from adult heads from males and females. For each experimental condition, approximately 1,000 flies that were 8 to 24 hours post-eclosion were used.

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Molecular profiling of hypothalamic leucine-sensing neurons via PhosphoTRAP

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