Miller DM III. Cell-specific microarray profiling experiments reveal a comprehensive picture of gene expression in the C. elegans nervous system

Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN 37232-8240, USA.
Genome biology (Impact Factor: 10.81). 02/2007; 8(7):R135. DOI: 10.1186/gb-2007-8-7-r135
Source: PubMed


With its fully sequenced genome and simple, well-defined nervous system, the nematode Caenorhabditis 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 properties of the nervous system.
We employed complementary microarray-based strategies to profile gene expression in the embryonic and larval nervous systems. In the MAPCeL (Microarray 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 (for example, transcription factors, ion channels, synaptic vesicle components) with established roles in neuronal development or function. Of particular interest are 19 conserved transcripts of unknown function that are also expressed in the mammalian brain. 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.
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.

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    • "Synchronized L1 worms of control (N2) and experimental strains huIs1;deIs10 (for activation of Wnt signaling) and deIs26;deIs10 (for inhibition of Wnt signaling) were heat shocked at the L2/L3 molt as described previously (Gleason et al., 2002; Gleason and Eisenmann, 2010). mRNA enrichment by PAB-1 affinity purification was performed as described previously (Von Stetina et al., 2007). mRNA (50 ng) from three biological replicates of the three strains was linearly amplified using the NuGen WT-Ovation Pico System as per manufacturer's instructions and 3-6 μg of the sample was used for microarray analysis on Affymetrix C. elegans genome microarrays. "
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    • "Heat shock and mRNA tagging protocol Mixed-stage embryos from gravid adults of experimental strains (deIs10; huIs1 and deIs10; deIs26) and control strains (N2 and deIs10; scm::gfp) were hatched overnight in 50 ml of M9. The synchronized L1s obtained were grown for 26 hr (L2/L3 molt) at 20°, heat shocked at 37° for 30 min, and then recovered for 1 hr at 20°. mRNA tagging was performed as previously described with a few modifications (Von Stetina et al. 2007b). Briefly, heat shocked and recovered animals were treated with 20% paraformaldehyde for 1 hr at 4°, sonicated utilizing a Branson 450 Sonifier (2 W output, 20% duty cycle, 6 pulses for 6 rounds), homogenized for 5 min using a 7-ml Dounce homogenizer, and centrifuged at 11,750 rpm for 20 min at 4° to pellet worm debris; 1 ml of worm lysate supplemented with 10 ml rRNAsin (Promega #N-2515) and 8 ml 200 mM ribonucleoside vanadyl complex (Sigma #R-3380) was added to 100 ml prewashed anti-FLAG beads (Sigma #F-2426) and rocked at 4° for 12–16 hr. "
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