Neuronal cell selective proteomics in C. elegans
Ontology highlight
ABSTRACT:
ORGANISM(S): Caenorhabditis Elegans
SUBMITTER: Wataru Aoki
PROVIDER: PXD018817 | JPOST Repository | Mon Aug 10 00:00:00 BST 2020
REPOSITORIES: jPOST
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| N2_Azf_with_enrichment_N=1.raw | Raw | |||
| N2_Azf_with_enrichment_N=2.raw | Raw | |||
| N2_Azf_with_enrichment_N=3.raw | Raw | |||
| N2_Azf_without_enrichment_N=1.raw | Raw | |||
| N2_Azf_without_enrichment_N=1.xlsx | Xlsx |
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Project description:Background: With its fully sequenced genome and simple, well-defined nervous system, the nematode C. elegans offers a unique opportunity to correlate gene expression with neuronal differentiation. The lineal origin, cellular morphology and synaptic connectivity of each of the 302 neurons are known. In many instances, specific behaviors can be attributed to particular neurons or circuits. Here we describe microarray-based methods that monitor gene expression in C. elegans neurons and thereby link comprehensive profiles of neuronal transcription to key developmental and functional attributes of the nervous system. Results: We employed complementary microarray-based strategies to profile gene expression in the embryonic and larval nervous systems. In the MAPCeL (Micro-Array Profiling C. elegans Cells) method, we used Fluorescence Activated Cell Sorting (FACS) to isolate GFP-tagged embryonic neurons for microarray analysis. To profile the larval nervous system, we used the mRNA-tagging technique in which an epitope-labeled mRNA binding protein (FLAG-PAB-1) was transgenically expressed in neurons for immunoprecipitation of cell-specific transcripts. These combined approaches identified approximately 2,500 mRNAs that are highly enriched in either the embryonic or larval C. elegans nervous system. These data are validated in part by the detection of gene classes (e.g. transcription factors, ion channels, synaptic vesicle components) with established roles in neuronal development or function. In addition to utilizing these profiling approaches to define stage specific gene expression, we also applied the mRNA-tagging method to fingerprint a specific neuron type, the A-class group of cholinergic motor neurons, during early larval development. A comparison of these data to a MAPCeL profile of embryonic A-class motor neurons identified genes with common functions in both types of A-class motor neurons as well as transcripts with roles specific to each motor neuron type. Conclusion: We describe microarray-based strategies for generating expression profiles of embryonic and larval C. elegans neurons. These methods can be applied to particular neurons at specific developmental stages and therefore provide an unprecedented opportunity to obtain spatially and temporally defined snapshots of gene expression in a simple model nervous system. Experiment Overall Design: Our goal is to profile gene expression throughout the nervous system of the model organism Caenorhabditis elegans. As a first goal, we profiled the entire nervous system at two developmental timepoints. To isolate transcripts from embryonic neurons we employed the MAPCeL (Microarray Profiling C. elegans Cells) technique in which pan-neuronal GFP+ cells are captured by FACS for RNA isolation. Since postembryonic cells are unattainable via this method, we used mRNA-tagging to isolated pan-neural RNAs from larval animals. In short, an epitope tagged poly-A binding protein (FLAG-PAB-1) was driven in all neurons using the F25B3.3 promoter. Following formaldehyde cross-linking, animals were passed through a French press and Dounce homogenized to isolate a cell-free extract. Anti-FLAG antibodies were incubated with the cell-free extract to capture FLAG-PAB bound RNAs. After elution and reversal of the crosslinks, RNAs are isolated by TriZOL extraction. We also identified transcripts enriched in a subset of C. elegans larval neurons, the A-class neurons (using unc-4::3xFLAG::PAB-1). Experiment Overall Design: 3 sets of experiments normalized separately by RMA Experiment Overall Design: larval references of sets 2 and 3 originate from the same hybridizations (accompanying CEL files are identical)
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