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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.

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Related in: MedlinePlus

Differential RNA-Seq analysis detects transcripts that are highly expressed in NSM neurons.Pairwise comparisons of (A) NSM and (B) Reference data sets with expression values represented as log2(FPKM) and Spearman rank-order correlation coefficients, rs. (C) Volcano scatter plot of log10(p-value) vs log2(fold change) of transcript expression in NSM neurons relative to the reference sample derived from all L1 larval cells. Significantly enriched or depleted transcripts (≥2.4 fold, p<0.012) are indicated in red. (D) Schematic of NSM serotonergic presynaptic terminus depicting genes that are highly enriched (E) in the NSM RNA-Seq profile. See text for additional information about specific genes.
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pone-0112102-g005: Differential RNA-Seq analysis detects transcripts that are highly expressed in NSM neurons.Pairwise comparisons of (A) NSM and (B) Reference data sets with expression values represented as log2(FPKM) and Spearman rank-order correlation coefficients, rs. (C) Volcano scatter plot of log10(p-value) vs log2(fold change) of transcript expression in NSM neurons relative to the reference sample derived from all L1 larval cells. Significantly enriched or depleted transcripts (≥2.4 fold, p<0.012) are indicated in red. (D) Schematic of NSM serotonergic presynaptic terminus depicting genes that are highly enriched (E) in the NSM RNA-Seq profile. See text for additional information about specific genes.

Mentions: Duplicate RNA-Seq data sets for the NSM neurons are well correlated as are the reference samples (Fig. 5A, B) but they show significant differences when compared to each other (e.g., NSM vs reference) (Fig. 5C). We detected a total of 1,073 transcripts with significantly elevated expression (≥2.4 fold, ≤0.05 q-value) in the NSM data set vs all L1 larval cells (see File S1). To validate this result, we compared the data set of NSM-enriched transcripts to a list of genes annotated in WormBase as previously assigned to NSM by direct observation. The overlap is highly significant with 21 of 36 known NSM genes represented in the NSM-enriched data set (p = 1.6×e−19) and an additional 13 genes that are detected at ≥1 FPKM (Table S2). Of particular note in the NSM-enriched list are genes with established roles in NSM differentiation or function. For example, canonical serotonergic genes are highly expressed in NSM. These include enzymes tph-1 (tryptophan hydroxylase/TPH) and bas-1 (aromatic amino acid decarboxylase/AAADC) required for serotonin (5-HT) synthesis, a transporter for selective uptake of monamine neurotranmitters into synaptic vesicles, cat-1 (vesicular monamine transporter/VMAT) and the synaptically localized transporter, mod-5 (serotonin reuptake transporter/SERT). mRNAs for the POU and LIM homeodomain transcription factors, unc-86 and ttx-3, respectively, which are known to activate expression of these serotonergic genes [31], [35] are also enriched in the NSM profile (Fig. 5C–E). The specificity of this data set is also underscored by the depletion of transcripts that are known to be highly expressed in other tissues or in different developmental periods e.g., tnt-3 and unc-89 (body muscle), dpf-6 (pharynx, intestine) and cht-1 (embryo) (Fig. 5C) [11], [36], [37]. As an additional test of the reliability of the NSM-enriched data set, we scored in vivo expression of promoter-GFP reporters for additional genes on this list that have not been previously assigned to NSM. The transcripts selected for this experiment display a broad range of statistical ranks (42–591) and half (2/4) of the corresponding GFP reporters were positively identified as expressed in NSM (Fig. 6) (Table 1). Together, these results strongly support the conclusion that these data sets provide an accurate representation of transcripts that are highly expressed in NSM neurons in vivo.


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)

Differential RNA-Seq analysis detects transcripts that are highly expressed in NSM neurons.Pairwise comparisons of (A) NSM and (B) Reference data sets with expression values represented as log2(FPKM) and Spearman rank-order correlation coefficients, rs. (C) Volcano scatter plot of log10(p-value) vs log2(fold change) of transcript expression in NSM neurons relative to the reference sample derived from all L1 larval cells. Significantly enriched or depleted transcripts (≥2.4 fold, p<0.012) are indicated in red. (D) Schematic of NSM serotonergic presynaptic terminus depicting genes that are highly enriched (E) in the NSM RNA-Seq profile. See text for additional information about specific genes.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0112102-g005: Differential RNA-Seq analysis detects transcripts that are highly expressed in NSM neurons.Pairwise comparisons of (A) NSM and (B) Reference data sets with expression values represented as log2(FPKM) and Spearman rank-order correlation coefficients, rs. (C) Volcano scatter plot of log10(p-value) vs log2(fold change) of transcript expression in NSM neurons relative to the reference sample derived from all L1 larval cells. Significantly enriched or depleted transcripts (≥2.4 fold, p<0.012) are indicated in red. (D) Schematic of NSM serotonergic presynaptic terminus depicting genes that are highly enriched (E) in the NSM RNA-Seq profile. See text for additional information about specific genes.
Mentions: Duplicate RNA-Seq data sets for the NSM neurons are well correlated as are the reference samples (Fig. 5A, B) but they show significant differences when compared to each other (e.g., NSM vs reference) (Fig. 5C). We detected a total of 1,073 transcripts with significantly elevated expression (≥2.4 fold, ≤0.05 q-value) in the NSM data set vs all L1 larval cells (see File S1). To validate this result, we compared the data set of NSM-enriched transcripts to a list of genes annotated in WormBase as previously assigned to NSM by direct observation. The overlap is highly significant with 21 of 36 known NSM genes represented in the NSM-enriched data set (p = 1.6×e−19) and an additional 13 genes that are detected at ≥1 FPKM (Table S2). Of particular note in the NSM-enriched list are genes with established roles in NSM differentiation or function. For example, canonical serotonergic genes are highly expressed in NSM. These include enzymes tph-1 (tryptophan hydroxylase/TPH) and bas-1 (aromatic amino acid decarboxylase/AAADC) required for serotonin (5-HT) synthesis, a transporter for selective uptake of monamine neurotranmitters into synaptic vesicles, cat-1 (vesicular monamine transporter/VMAT) and the synaptically localized transporter, mod-5 (serotonin reuptake transporter/SERT). mRNAs for the POU and LIM homeodomain transcription factors, unc-86 and ttx-3, respectively, which are known to activate expression of these serotonergic genes [31], [35] are also enriched in the NSM profile (Fig. 5C–E). The specificity of this data set is also underscored by the depletion of transcripts that are known to be highly expressed in other tissues or in different developmental periods e.g., tnt-3 and unc-89 (body muscle), dpf-6 (pharynx, intestine) and cht-1 (embryo) (Fig. 5C) [11], [36], [37]. As an additional test of the reliability of the NSM-enriched data set, we scored in vivo expression of promoter-GFP reporters for additional genes on this list that have not been previously assigned to NSM. The transcripts selected for this experiment display a broad range of statistical ranks (42–591) and half (2/4) of the corresponding GFP reporters were positively identified as expressed in NSM (Fig. 6) (Table 1). Together, these results strongly support the conclusion that these data sets provide an accurate representation of transcripts that are highly expressed in NSM neurons in vivo.

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