Purpose: Communication between growth cones and their environment plays a central role in assembling neural circuits. We use Tandemly-Tagged Ribosome Affinity Purification (T-TRAP) of mRNA from R cells followed by RNA-seq for multiple time points during development to follow gene expression during target selection and synapse formation. Methods: We chose a ribosome trap method by modifying the N-terminus of the Drosophila ribosomal protein RpL10 with two tandemly arranged epitopes, 3X FLAG and GFP, separated by the Tobacco Etch Virus (TEV) protease site and expressed this in specific cell types using the GAL4/UAS system. cDNA libraries were prepared from mRNA associated with the affinity purified ribosomes and sequenced using an Illumina HiSeq 2000. We mapped raw reads to the D. melanogaster reference genome (release FB2013_01) with the gapped aligner Tophat. Only reads uniquely aligned were collected.Transcript expression levels were quantified using RPKM units using customized scripts written in Perl. Results: In this study, we observed massive changes in expression of cell surface proteins over short time scales (i.e. 5 fold differences in the expression of many hundreds of genes over 5 hr intervals) as R cell growth cones encounter the processes of many different neurons during their conversion from growth cones to synaptic terminals. In addition, to changes in transcripts encoding cell surface proteins, other mRNAs changed significantly as did non-coding RNAs (lincRNAs) associated with ribosomes. Although dramatic changes in transcript levels of presynaptic proteins were not observed preceding the onset of synapse formation, marked changes in the 3''-untranslated regions of these transcripts were seen. Conclusions: These studies provide a step towards merging traditional genetic and global genomic approaches to understanding cellular recognition underlying the assembly of neural circuits. Overall design: We chose 7 time points for RNA-seq analysis of R cells during pupal development corresponding to 24, 35, 40, 45, 53, 65 and 96 hrs after pupal formation (APF).
Rapid Changes in the Translatome during the Conversion of Growth Cones to Synaptic Terminals.
Age, Specimen part, Subject
View SamplesThe formation of neuronal connections requires the precise guidance of developing axons towards their targets. In the Drosophila visual system, photoreceptor neurons (R cells) project from the eye into the brain. These cells are grouped into some 750 clusters comprised of eight photoreceptors or R-cells each. R cells fall into three classes, R1-R6, R7 and R8. Posterior R8 cells are the first to project axons into the brain. How these axons select a specific pathway is not known.
Robo-3--mediated repulsive interactions guide R8 axons during Drosophila visual system development.
Specimen part
View SamplesPurpose: Information processing in the brain relies on precise patterns of synapses between neurons. The molecular mechanisms by which this specificity is achieved remains elusive. In the medulla of the Drosophila visual system, different neurons form synaptic connections in different layers. Methods: we developed methods to purify seven neuronal cell types (R7, R8 and L1-L5 neurons) using Fluorescence Activated Cell Sorting. Results: we show that neurons with different synaptic specificities express unique combinations of mRNAs encoding hundreds of cell surface and secreted proteins. Using RNA sequencing and MiMIC-based protein tagging, we demonstrate that 21 paralogs of the Dpr family, a subclass of Immunoglobulin (Ig)-domain containing proteins, are expressed in unique combinations in homologous neurons with different layer-specific synaptic connections. Dpr interacting proteins (DIPs), comprising nine paralogs of another subclass of Ig superfamily proteins, are expressed in a complementary layer-specific fashion in a subset of synaptic partners. We propose that pairs of Dpr/DIP paralogs contribute to layer-specific patterns of synaptic connectivity. Conclusions: This complexity is mirrored by the complexity of the cell surface and secreted molecules expressed by each of the R cell and lamina neurons profiled in this study. How this complexity contributes to specificity remains elusive, but the convergence of improved histological, genetic and molecular tools promises to provide important insights into the molecular recognition strategies controlling synaptic specificity. Overall design: We chose 7 time points for RNA-seq analysis of R cells during pupal development corresponding to 24, 35, 40, 45, 53, 65 and 96 hrs after pupal formation (APF).
Ig Superfamily Ligand and Receptor Pairs Expressed in Synaptic Partners in Drosophila.
Age, Specimen part, Subject
View SamplesThis SuperSeries is composed of the SubSeries listed below.
Systematic classification of melanoma cells by phenotype-specific gene expression mapping.
Cell line
View SamplesRecent trials with MAPK inhibitors have shown promising results in many patients with metastatic melanoma; however, nearly all responding patients experience disease relapse. We describe here how melanoma cells respond to MAPK inhibition in a phenotype-specific manner, suggesting that slow cycling invasive phenotype cells provide a treatment-resistant pool from which disease relapse may be derived. The implication is that while MAPK inhibition may successfully treat proliferating cells, another cell population needs to be addressed at the same time.
A proliferative melanoma cell phenotype is responsive to RAF/MEK inhibition independent of BRAF mutation status.
Cell line
View SamplesThe plasma protein FHR1 induces release of inflammatory cytokines IL-1ß, IL-6, IL-18 or TNFa from blood-derived human monocytes. RNA sequencing was performed from RNA of BSA- or FHR1-treated monocytes from 4 different donors. In response to FHR1, 522 monocytic genes were upregulated (gene ontology enrichment analysis), including 35 inflammation related genes, e.g. TNF. Also, G protein-coupled receptors such as EMR2/ADGRE2 were upregulated in response to FHR1. Overall design: Blood-derived monocytes were treated with BSA or FHR1, after 4h RNA was isolated. RNA of 4 donors were combined and sequenced.
Serum FHR1 binding to necrotic-type cells activates monocytic inflammasome and marks necrotic sites in vasculopathies.
Specimen part, Treatment, Subject
View SamplesThis SuperSeries is composed of the SubSeries listed below.
Cellulose-Derived Oligomers Act as Damage-Associated Molecular Patterns and Trigger Defense-Like Responses.
Specimen part, Treatment, Time
View SamplesPlants can perceive the presence of pathogens at the cell surface and plant damage-derived molecules via recognition of conserved microbial molecules, named pathogen- or microbe-associated molecular patterns (PAMPs) and damage associated molecular patterns (DAMPs). Well-studied examples of PAMPs are chito-oligomers, breakdown products of fungal cell walls and insect exoskeletons. Pectin-derived oligogalacturonides (OGs) are well-characterized DAMPs. Both PAMPs nd DAMPs are capable of activating plant immunity, generating changes in gene expression that lead to increased production of defense compounds and proteins; thus, equipping the plant cell to defend itself.
Cellulose-Derived Oligomers Act as Damage-Associated Molecular Patterns and Trigger Defense-Like Responses.
Specimen part, Treatment, Time
View SamplesPlants can perceive the presence of pathogens at the cell surface and plant damage-derived molecules via recognition of conserved microbial molecules, named pathogen- or microbe-associated molecular patterns (PAMPs) and damage associated molecular patterns (DAMPs). Well-studied examples of PAMPs are chito-oligomers, breakdown products of fungal cell walls and insect exoskeletons. Pectin-derived oligogalacturonides (OGs) are well-characterized DAMPs. Both PAMPs nd DAMPs are capable of activating plant immunity, generating changes in gene expression that lead to increased production of defense compounds and proteins; thus, equipping the plant cell to defend itself.
Cellulose-Derived Oligomers Act as Damage-Associated Molecular Patterns and Trigger Defense-Like Responses.
Specimen part, Treatment, Time
View SamplesTranscriptional changes upon elicitor treatment over time (0, 30, 60 min) have been analysed with the A.thaliana Landsberg (wt) and fls2-17 (flagellin receptor mutant).
Perception of the bacterial PAMP EF-Tu by the receptor EFR restricts Agrobacterium-mediated transformation.
Age, Compound, Time
View Samples