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Isolation of specific neurons from C. elegans larvae for gene expression profiling.

Spencer WC, McWhirter R, Miller T, Strasbourger P, Thompson O, Hillier LW, Waterston RH, Miller DM - PLoS ONE (2014)

Bottom Line: Our analysis showed that diverse classes of neurons are accessible to this approach.To demonstrate the applicability of this strategy to rare neuron types, we generated RNA-Seq profiles of the NSM serotonergic neurons that occur as a single bilateral pair of cells in the C. elegans pharynx.These data detected >1,000 NSM enriched transcripts, including the majority of previously known NSM-expressed genes.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, Vanderbilt University, Nashville, Tennessee, United States of America.

ABSTRACT

Background: The simple and well-described structure of the C. elegans nervous system offers an unprecedented opportunity to identify the genetic programs that define the connectivity and function of individual neurons and their circuits. A correspondingly precise gene expression map of C. elegans neurons would facilitate the application of genetic methods toward this goal. Here we describe a powerful new approach, SeqCeL (RNA-Seq of C. elegans cells) for producing gene expression profiles of specific larval C. elegans neurons.

Methods and results: We have exploited available GFP reporter lines for FACS isolation of specific larval C. elegans neurons for RNA-Seq analysis. Our analysis showed that diverse classes of neurons are accessible to this approach. To demonstrate the applicability of this strategy to rare neuron types, we generated RNA-Seq profiles of the NSM serotonergic neurons that occur as a single bilateral pair of cells in the C. elegans pharynx. These data detected >1,000 NSM enriched transcripts, including the majority of previously known NSM-expressed genes.

Significance: This work offers a simple and robust protocol for expression profiling studies of post-embryonic C. elegans neurons and thus provides an important new method for identifying candidate genes for key roles in neuron-specific development and function.

Show MeSH

Related in: MedlinePlus

Specific sensory and motor neurons are accessible to isolation by FACS from multiple larval stages.Primary cultures of L1 stage cells 24 hr after dissociating from transgenic lines expressing (A) mec-4::mCherry to mark ALM and PLM neurons (red) and (B) srh-142p::dsRed to label ADF sensory neurons (red). (C) GABA motor neuron marked with unc-47::mCherry (red) and cultured for 24 hour after dissociating from L4 larval animals. (D) Primary culture of A-class motor neurons marked with unc-4::GFP (green) and isolated by FACS from L2 stage larvae. (E) del-1::GFP labels VB motor neurons (green) in the ventral nerve cord of an L2 stage larva. Anterior to left. (F) FACS profile of cells dissociated from del-1::GFP L2 larvae. Propidium Iodide (PI) marks dead cells (blue). Viable GFP-labeled cells (green) are outlined with the box. Scale bars are 10 microns.
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pone-0112102-g002: Specific sensory and motor neurons are accessible to isolation by FACS from multiple larval stages.Primary cultures of L1 stage cells 24 hr after dissociating from transgenic lines expressing (A) mec-4::mCherry to mark ALM and PLM neurons (red) and (B) srh-142p::dsRed to label ADF sensory neurons (red). (C) GABA motor neuron marked with unc-47::mCherry (red) and cultured for 24 hour after dissociating from L4 larval animals. (D) Primary culture of A-class motor neurons marked with unc-4::GFP (green) and isolated by FACS from L2 stage larvae. (E) del-1::GFP labels VB motor neurons (green) in the ventral nerve cord of an L2 stage larva. Anterior to left. (F) FACS profile of cells dissociated from del-1::GFP L2 larvae. Propidium Iodide (PI) marks dead cells (blue). Viable GFP-labeled cells (green) are outlined with the box. Scale bars are 10 microns.

Mentions: We generated dissociated preparations of larval cells labeled with fluorescent markers for sensory and motor neurons to determine if specific neuron types were also accessible to isolation by FACS. Touch neurons (PLM and ALM) and serotonergic sensory neurons (ADF) from L1 larvae (Fig. 2A, B) and GABA motor neurons from L4 stage animals (Fig. 2C) were readily detected in primary cultures. To confirm that specific neuron classes could be purified by FACS, we isolated A-class (Fig. 2D) and VB type motor neurons from L2 stage larvae (Fig. 2E, F). Detection of ADF neurons in culture, which comprise only 0.3% of all L1 cells (2/558), suggested that rare types of neurons could also be isolated by this approach for gene expression profiling.


Isolation of specific neurons from C. elegans larvae for gene expression profiling.

Spencer WC, McWhirter R, Miller T, Strasbourger P, Thompson O, Hillier LW, Waterston RH, Miller DM - PLoS ONE (2014)

Specific sensory and motor neurons are accessible to isolation by FACS from multiple larval stages.Primary cultures of L1 stage cells 24 hr after dissociating from transgenic lines expressing (A) mec-4::mCherry to mark ALM and PLM neurons (red) and (B) srh-142p::dsRed to label ADF sensory neurons (red). (C) GABA motor neuron marked with unc-47::mCherry (red) and cultured for 24 hour after dissociating from L4 larval animals. (D) Primary culture of A-class motor neurons marked with unc-4::GFP (green) and isolated by FACS from L2 stage larvae. (E) del-1::GFP labels VB motor neurons (green) in the ventral nerve cord of an L2 stage larva. Anterior to left. (F) FACS profile of cells dissociated from del-1::GFP L2 larvae. Propidium Iodide (PI) marks dead cells (blue). Viable GFP-labeled cells (green) are outlined with the box. Scale bars are 10 microns.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4221280&req=5

pone-0112102-g002: Specific sensory and motor neurons are accessible to isolation by FACS from multiple larval stages.Primary cultures of L1 stage cells 24 hr after dissociating from transgenic lines expressing (A) mec-4::mCherry to mark ALM and PLM neurons (red) and (B) srh-142p::dsRed to label ADF sensory neurons (red). (C) GABA motor neuron marked with unc-47::mCherry (red) and cultured for 24 hour after dissociating from L4 larval animals. (D) Primary culture of A-class motor neurons marked with unc-4::GFP (green) and isolated by FACS from L2 stage larvae. (E) del-1::GFP labels VB motor neurons (green) in the ventral nerve cord of an L2 stage larva. Anterior to left. (F) FACS profile of cells dissociated from del-1::GFP L2 larvae. Propidium Iodide (PI) marks dead cells (blue). Viable GFP-labeled cells (green) are outlined with the box. Scale bars are 10 microns.
Mentions: We generated dissociated preparations of larval cells labeled with fluorescent markers for sensory and motor neurons to determine if specific neuron types were also accessible to isolation by FACS. Touch neurons (PLM and ALM) and serotonergic sensory neurons (ADF) from L1 larvae (Fig. 2A, B) and GABA motor neurons from L4 stage animals (Fig. 2C) were readily detected in primary cultures. To confirm that specific neuron classes could be purified by FACS, we isolated A-class (Fig. 2D) and VB type motor neurons from L2 stage larvae (Fig. 2E, F). Detection of ADF neurons in culture, which comprise only 0.3% of all L1 cells (2/558), suggested that rare types of neurons could also be isolated by this approach for gene expression profiling.

Bottom Line: Our analysis showed that diverse classes of neurons are accessible to this approach.To demonstrate the applicability of this strategy to rare neuron types, we generated RNA-Seq profiles of the NSM serotonergic neurons that occur as a single bilateral pair of cells in the C. elegans pharynx.These data detected >1,000 NSM enriched transcripts, including the majority of previously known NSM-expressed genes.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, Vanderbilt University, Nashville, Tennessee, United States of America.

ABSTRACT

Background: The simple and well-described structure of the C. elegans nervous system offers an unprecedented opportunity to identify the genetic programs that define the connectivity and function of individual neurons and their circuits. A correspondingly precise gene expression map of C. elegans neurons would facilitate the application of genetic methods toward this goal. Here we describe a powerful new approach, SeqCeL (RNA-Seq of C. elegans cells) for producing gene expression profiles of specific larval C. elegans neurons.

Methods and results: We have exploited available GFP reporter lines for FACS isolation of specific larval C. elegans neurons for RNA-Seq analysis. Our analysis showed that diverse classes of neurons are accessible to this approach. To demonstrate the applicability of this strategy to rare neuron types, we generated RNA-Seq profiles of the NSM serotonergic neurons that occur as a single bilateral pair of cells in the C. elegans pharynx. These data detected >1,000 NSM enriched transcripts, including the majority of previously known NSM-expressed genes.

Significance: This work offers a simple and robust protocol for expression profiling studies of post-embryonic C. elegans neurons and thus provides an important new method for identifying candidate genes for key roles in neuron-specific development and function.

Show MeSH
Related in: MedlinePlus