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Transcriptional profiling at whole population and single cell levels reveals somatosensory neuron molecular diversity

  1. Isaac M Chiu  Is a corresponding author
  2. Lee B Barrett
  3. Erika K Williams
  4. David E Strochlic
  5. Seungkyu Lee
  6. Andy D Weyer
  7. Shan Lou
  8. Gregory S Bryman
  9. David P Roberson
  10. Nader Ghasemlou
  11. Cara Piccoli
  12. Ezgi Ahat
  13. Victor Wang
  14. Enrique J Cobos
  15. Cheryl L Stucky
  16. Qiufu Ma
  17. Stephen D Liberles
  18. Clifford J Woolf  Is a corresponding author
  1. Boston Children's Hospital, United States
  2. Harvard Medical School, United States
  3. Medical College of Wisconsin, United States
  4. Dana-Farber Cancer Institute, Harvard Medical School, United States
  5. University of Granada, Spain
Research Article
Cite this article as: eLife 2014;3:e04660 doi: 10.7554/eLife.04660
15 figures, 3 tables, 2 data sets and 2 additional files

Figures

Figure 1 with 1 supplement
Fluorescent characterization of SNS-Cre/TdTomato and Parv-Cre/TdTomato DRG populations.

(A) SNS-Cre/TdTomato and Parv-Cre/TdTomato lumbar DRG sections imaged for TdTomato (red), IB4-FITC, anti-CGRP, or anti-Parvalbumin (green). Scale bars, 50 μm. (BC) Proportions of IB4+, CGRP+, NF200+, Parvalbumin+ populations expressing SNS-Cre/TdTomato or Parv-Cre/TdTomato, and converse TdTomato proportions expressing each co-stained marker (mean ± s.e.m., n = 8–20 fields from 3 animals). (D) Venn diagram depicting distinct DRG populations as labeled by Isolectin B4, NF200, and TdTomato populations. (E) For transcriptional profiling, three non-overlapping DRG populations were FACS purified: IB4+SNS-Cre/TdTomato+, IB4SNS-Cre/TdTomato+, and Parv-Cre/TdTomato+ cells.

https://doi.org/10.7554/eLife.04660.003
Figure 1—figure supplement 1
SNS-Cre/TdTomato and Parv-Cre/TdTomato DRG and spinal cord characterization.

(A) SNS-Cre and Parv-Cre mice were bred with Rosa26-TdTomato mice to generate lineage reporter progeny. (B) Confocal microscopy images of whole mount L4 DRG from TdTomato progeny. Scale bars, 50 μm. (C) Lumbar spinal cord sections were stained with Isolectin B4-FITC (green) and anti-CGRP (blue). SNS-Cre/TdTomato fibers overlapped with CGRP and IB4 staining in dorsal horn laminae I–II. By contrast, Parv-Cre/TdTomato fibers extended to lamina III, Clark Nucleus (C.N.) and ventral horns. (D) Lumbar sections show SNS-Cre/TdTomato fibers in lamina II (colocalized with IB4), but not lamina III stained by anti-PKC-γ. Parv-Cre/TdTomato does not innervate superficial laminae. Scale bars, 100 μm.

https://doi.org/10.7554/eLife.04660.004
Electrophysiological properties of SNS-Cre/TdTomato and Parv-Cre/TdTomato neurons.

Whole cell current clamp recordings were conducted on SNS-Cre/TdTomato and Parv-Cre/TdTomato neurons in response to 200 pA injection. (A) Representative action potential waveforms before and after application of 500 nM TTX. (B–C) Statistical comparisons of action potential (AP) half-widths and capacitances between sensory populations (SNS-Cre/TdT+, n = 13; Parv-Cre/TdT+, n = 9; p-values by Student's t test).

https://doi.org/10.7554/eLife.04660.005
Figure 3 with 2 supplements
FACS purification of distinct somatosensory neuron populations.

(A) Mouse DRG cells were stained with DAPI and subjected to flow cytometry. After gating on large cells by forward and side scatter (R1), dead cells were excluded by gating on the DAPI events; Next, TdTomato (hi) events were purified. Following purification, fluorescence and DIC microscopy show that the majority of sorted neurons are TdTomato+ (images on right). (B) Representative FACS plots of Parv-Cre/TdTomato+ and SNS-Cre/TdTomato+ DRG populations. Right, quantification of proportions of DAPI events in the DRG constituting each neuron population (n = 5 SNS-Cre/TdTomato mice, n = 4 Parv-Cre/TdTomato mice; p-values, Student's t test; Error bars, mean ± s.e.m.). (C) Representative FACS plot shows relative percentages of IB4-FITC surface stained and IB4 neuronal populations among the total SNS-Cre/TdTomato (hi) gate.

https://doi.org/10.7554/eLife.04660.006
Figure 3—figure supplement 1
Flow cytometric sorting and analysis of TdTomato+ neurons.

(A) By FACS analysis, TdTomato labeled both ‘high’ and ‘low’ fluorescence populations (see gates). Purified high-expressing populations corresponded to neuronal cell bodies, while the lower fluorescence consisted of fluorescent axonal debris, as shown by microscopy images post-sorting (right). (B) TdTomato neurons purified and plated onto glass slides. After 24 hr, post-sorted SNS-Cre/TdT+ neurons showed neurite outgrowth and relatively pure populations compared to unsorted SNS-Cre/TdT+ neurons. (C) Representative FACS plot overlay of light scattering properties for Parv-Cre/TdT+ vs SNS-Cre/TdT+ populations. Comparison of forward and side scatter properties on left (SNS-Cre/TdT, n = 4; Parv-Cre/TdT, n = 4; error bars, s.e.m).

https://doi.org/10.7554/eLife.04660.007
Figure 3—figure supplement 2
Transcriptome analysis of purified neuronal samples relative to whole DRG tissues.

(A) Individual expression profile comparisons of sorted neuron samples (Red and green show numbers of trancripts >twofold differential expression). (B) Plots of absolute RMA normalized transcript levels for myelin associated, nociceptor associated, and proprioceptor associated genes in FACS purified SNS-Cre/TdT+ and Parv-Cre/TdT+ samples vs whole DRG samples. p-values by One-way ANOVA: ***p < 0.001. (C) Fold-change vs fold-change comparison of sorted neurons vs whole DRG datasets (red transcripts are >twofold enriched in whole DRG; blue transcripts are >twofold enriched in both sorted subsets).

https://doi.org/10.7554/eLife.04660.008
Hierarchical clustering and principal components analysis of transcriptomes.

(A) Hierarchical clustering of sorted neuron molecular profiles (top 15% probesets by coefficient of variation), showing distinct groups of transcripts enriched in IB4+SNS-Cre/TdT+, IB4SNS-Cre/TdT+, and Parv-Cre/TdT+ neuron populations. (B) Principal component analysis shows distinct transcriptome segregation for the purified populations along three principal components axes.

https://doi.org/10.7554/eLife.04660.010
Functional somatosensory mediators show clustered gene expression across purified DRG populations.

Heat-map showing relative transcript levels for known somatosensory mediators plotted across IB4+SNS-Cre/TdTomato+, IB4SNS-Cre/TdTomato+, and Parv-Cre/TdTomato+ purified neuron transcriptomes (rows show individual samples; columns are specific transcripts). Genes were grouped based on known roles linked to thermosensation/nociception, pruriception, tactile function, neurotrophic receptors, and proprioception.

https://doi.org/10.7554/eLife.04660.011
Heat-map distribution of voltage-gated and TRP channels across neuronal subsets.

Expression patterns of different sub-types of voltage-gated ion channels and transient receptor potential (TRP) channels were hierarchically clustered and analyzed across IB4+SNS-Cre/TdT+, IB4SNS-Cre/TdT+ and Parv-Cre/TdT+ purified neuron samples (columns are individual samples, heat-maps). (A) Sodium channel levels, (B) calcium channel levels, (C) potassium channel levels (top 60 differentially expressed transcripts by CoV), (D) chloride channel levels, and (E) TRP channel levels are plotted as heat-maps. For AE, plotted transcripts show minimum expression >100 in at least one neuronal subgroup.

https://doi.org/10.7554/eLife.04660.012
Heat-map distribution of ligand-gated ion channels, G-protein coupled receptors, and transcription factors across neuronal subsets.

(A) Expression patterns of ligand-gated ion channels, including glutamatergic, chlorinergic, HCN, P2X channels, were analyzed by hierarchical clustering (columns are individual samples). (B) Differentially expressed G-protein coupled receptors (GPCRs) were clustered and plotted across sensory subsets (Top 60 by CoV are shown). (C) Differentially expressed transcription factors were clustered and plotted across sensory subsets as a heat-map (Top 60 by CoV are shown). For AC, plotted transcripts show minimum expression >100 in at least one neuronal subgroup.

https://doi.org/10.7554/eLife.04660.013
Differential volcano plot analysis of SNS-Cre/TdTomato vs Parv-Cre/TdTomato transcriptomes.

(A) Pairwise comparison of SNS-Cre/TdT+ vs Parv-Cre/TdT+ profiles showing differentially expressed (DE) transcripts as a volcano plot (blue transcripts, Parv-Cre/TdT enriched; red, SNS-Cre/TdT enriched, twofold, p < 0.05). (B) Most enriched Gene ontology (GO) categories and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways in SNS-Cre/TdT vs Parv-Cre/TdT enriched transcripts, plotted as heat-map of −log (p-value). (C) Volcano plots depicting (i) calcium channels, (ii) potassium channels, and (iii) TRP channels expression differences between populations. Individual transcripts highlighted (red, SNS-Cre/TdT+ enriched; green, Parv-Cre/TdT+ enriched; blue, not significantly different: twofold, p < 0.01).

https://doi.org/10.7554/eLife.04660.014
Differential volcano plot analysis of IB4+ and IB4 SNS-Cre/TdTomato subset transcriptomes.

(A) Pairwise comparison of IB4+SNS-Cre/TdT+ vs IB4SNS-Cre/TdT+ neuronal profiles show differentially expressed (DE) genes by volcano plot (blue, IB4+ enriched; red, IB4enriched, twofold, p < 0.05). (B) Top Gene ontology (GO) categories of biological processes (BP) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways for IB4+SNS-Cre/TdT+ and IB4SNS-Cre/TdT+ enriched transcripts, plotted as heat-maps of −log (p-value). (C) Volcano plots showing differential expression of (i) ion channels, (ii) cell adhesion molecules, and (iii) G-protein coupled receptors between neuronal populations (red, IB4+ enriched transcripts; green, IB4 enriched; blue, not significantly different: twofold, p < 0.01).

https://doi.org/10.7554/eLife.04660.015
Figure 10 with 1 supplement
Analysis of most enriched marker expression by IB4+, IB4 SNS-Cre/TdTomato and Parv-Cre/TdTomato+ populations.

(AC) Fold-change/fold-change comparisons illustrate most differentially enriched genes in each subset (highlighted in color are threefold and twofold enriched numbers). (D) Heat-maps showing relative expression of the top 40 transcripts enriched in each of the three neuronal subsets (>threefold), ranked by product of fold-change differences.

https://doi.org/10.7554/eLife.04660.016
Figure 10—figure supplement 1
Fluidigm analysis of 100 and 10 cell-samples.

FACS sorted 100 cell or 10 cell samples consisting of IB4+SNS-Cre/TdT+, IB4SNS-Cre/TdT+, and Parv-Cre/TdT+ neurons were analyzed by Fluidigm for 80 different transcript levels chosen based on microarray results, and normalized to Gapdh expression. Hierarchical clustering of transcript levels is shown for 100 cell and 10 cell groups as heat-maps.

https://doi.org/10.7554/eLife.04660.017
Single cell transcript levels show log-scale distribution across neuronal populations.

Normalized transcript levels in single cells determined by parallel qRT-PCR are plotted on a log-scale comparing IB4+SNS-Cre/TdT+, IB4SNS-Cre/TdT+, and Parv-Cre/TdT+ cells. (A) Nociceptor related transcript levels (Trpv1, Trpa1, Mrgprd, P2rx3, Nppb, Ptgir), (B) Proprioception related transcript levels (Pvalb, Runx3, Cdh12). Individual neurons are shown as dots in plots.

https://doi.org/10.7554/eLife.04660.019
Figure 12 with 2 supplements
Hierarchical clustering analysis of single cell qRT-PCR data reveals distinct neuronal subgroups.

Heat-map of 334 single neurons and 80 genes after spearman-rank hierarchical analysis of RT-PCR data (relative gene expression normalized to gapdh). Each column represents a single sorted cell, and each transcript is shown per row. Clustering analysis finds seven distinct subgroups (I, II, III, IV, V, VI, VII). Characteristic transcript expression patterns that delineate each somatosensory subset are written below.

https://doi.org/10.7554/eLife.04660.020
Figure 12—figure supplement 1
Expression of neuronal-associated transcripts across purified single cell samples by qRT-PCR.

Heat-map showing expression levels of neuron-associated transcripts across single cells (from 1 to 334) by qRT-PCR.

https://doi.org/10.7554/eLife.04660.021
Figure 12—figure supplement 2
Transcript expression levels for characteristic marker genes in single cell neuron Group I and Group VII.

Plotted are normalized transcript levels of Group I and Group VII transcripts, ordered from highest to lowest expression (i.e., Grik1 to Wnt2b for Group I, Pvalb to Cdh12 for Group VII).

https://doi.org/10.7554/eLife.04660.022
Single cell subgroups distribute differentially across originally purified populations.

(A) Principal Components Analysis of single cell transcriptional data shows distinct segregation of Groups I, V, and VII neurons. (B) Proportions of each neuronal subgroup relative to original labeled IB4+SNS-Cre/TdTomato+, IB4SNS-Cre/TdTomato+, and Parv-Cre/TdTomato+ neurons.

https://doi.org/10.7554/eLife.04660.023
Figure 14 with 2 supplements
Focused analysis of single cell heterogeneity and transcript enrichment in neuronal subgroups.

(A) Relative expression levels of subgroup specific transcripts in single cells across each neuronal subgroup (each bar = 1 cell). Group I (Lpar3, Mrgprd), group VI (Il31ra, Nppb), and group VII markers (Gpcr5b) show subset enrichment and highly heterogeneous expression at the single cell level. (BC) Nearest neighbor analysis by pearson correlation of Mrgprd and Pvalb transcript levels to all 80 probes across the single cell expression dataset was generated. Correlation levels go from left to right.

https://doi.org/10.7554/eLife.04660.024
Figure 14—figure supplement 1
Defining the transcriptional characteristics of Group I, II, and IV neurons.

Transcript levels for selected genes that define the characteristics of specific neuronal subgroups Group I, II, and IV neurons were plotted across all 334 individual neurons. (A) Group 1 neurons were found with high levels of P2rx3, Lpar3. (B) Group II neurons show high levels of Ntrk1 and Kcnv1. (C) Group IV are characterized by Trpv1 expression but lack of Scn10a expression.

https://doi.org/10.7554/eLife.04660.025
Figure 14—figure supplement 2
Expression plots of nociceptor-associated transcripts across single cell transcriptional data.

Transcript levels for nociceptor associated genes (A) Trpa1, (B) Scn11a, and (C) Aqp1 were plotted across all individual neurons.

https://doi.org/10.7554/eLife.04660.026
Figure 15 with 2 supplements
DRG subgroups I, VI, and VII characteristics defined by double RNA in situ hybridization.

(A) Double RNA in situ hybridization in SNS-Cre/TdTomato and Parv-Cre/TdTomato lumbar DRG sections for TdTomato (red) with Lpar3, Il31ra, or Gpcr5b (green), which are Group I, VI, and VII markers respectively. Lpar3 and IL31ra expression colocalize with SNS-Cre/TdTomato but not Parv-TdTomato, while Gpcr5b colocalizes with Parv-Cre/TdTomato but not SNS-Cre/TdTomato. (B) Double in situ hybridization in lumbar DRG sections for group VI marker IL31ra vs Group I marker Lpar3, Group VI marker Gpcr5b, or Group VI marker Nppb. Il31ra and Nppb in shown in a distinct subset of DRG neurons. Scale bars, 100 μm.

https://doi.org/10.7554/eLife.04660.028
Figure 15—figure supplement 1
Immunofluorescence characteristics of DRG subgroup V.

(A) Expression plot shows enrichment of Th expression in Group V neurons. (B) SNS-Cre/TdTomato lumbar DRG sections were imaged for TdTomato (red), anti-TH (blue), and IB4-FITC (green). (C) Quantification of neuronal proportions TH+ neurons that are IB4SNS-Cre/TdT+, IB4SNS-Cre/TdT+, or Parv-Cre/TdT+ neurons expressing TH. Statistical analysis by Student's t test (n = 8–10 fields from 3 mice each). Scale bars, 100 μm.

https://doi.org/10.7554/eLife.04660.029
Figure 15—figure supplement 2
Group I marker Prkcq is in a distinct subset of DRG neurons.

(A) Transcript levels for Prkcq plotted across all individual neuron subgroups. (B) Double in situ hybridization (ISH) of lumbar DRG sections for TdTomato (red) and for Lpar (green) shows that Prkcq+ neurons showed SNS-Cre/TdTomato expression whereas they were did not express SNS-Cre/TdTomato. Scale bars, 100 μm. (C) Double ISH of lumbar DRG sections shows that Prkcq does not colocalize with Group VI marker IL31ra.

https://doi.org/10.7554/eLife.04660.030

Tables

Table 1

Transcriptional samples analyzed in this study

https://doi.org/10.7554/eLife.04660.009
Sample nameSample descriptionTypen
SNS-Cre/TdT+SNS-Cre/TdTomato+ FACS purified neuronsNeuron population4
Parv-Cre/TdT+Parv-Cre/TdTomato+ FACS purified neuronsNeuron population4
IB4+SNS-Cre/TdT+IB4+SNS-Cre/TdT+ FACS purified neuronsNeuron population3
IB4SNS-Cre/TdT+IB4SNS-Cre/TdT+ FACS purified neuronsNeuron population3
Whole DRGHomogenized DRG tissueWhole tissue3
IB4+SNS-Cre/TdT+ (individual neurons)IB4+SNS-Cre/TdT+ FACS purified single cellsSingle cells132
IB4SNS-Cre/TdT+ (individual neurons)IB4SNS-Cre/TdT+ FACS purified single cellsSingle cells110
Parv-Cre/TdT+ (individual neurons)Parv-Cre/TdT+ FACS purified single cellsSingle cells92
  1. In this study, we performed microarray profiling of FACS purified neuron populations, DRG tissue, and single neuron samples. This table summarizes the sample names, descriptions, types, and numbers of samples analyzed. For neuron populations and whole DRG tissue, each biological replicate consisted of pooled total DRG cells from n = 3 animals.

Table 2

Taqman assays used for single cell transcriptional profiling

https://doi.org/10.7554/eLife.04660.018
SNS-Cre/TdT+ enriched (vs Parv-Cre/TdT)IB4+ SNS-Cre/TdT+ enrichedIB4 SNS-Cre/TdT+ enrichedParv-Cre/TdT+ enriched
Trpv1MrgprdSmr2Pvalb
Trpa1P2rx3Npy2rRunx3
Scn10aAgtr1aNppbCalb2
Scn11aPrkcqKcnv1Slit2
Isl2Wnt2bProkr2Spp1
Kcnc2Slc16a12PtgirAno1
Galr1Lpar3ThStxbp6
Car8Lpar5Il31raSt8sia5
Chrna3Trpc3Ntrk1Ndst4
Atp2b4Trpc6BvesEsrrb
Aqp1Moxd1Kcnq4Esrrg
Chrna6A3galt2Htr3aGprc5b
Pde11aSt6gal2S100a16Car2
MrgprC11Mrgprb4Pou4f3Pth1r
Syt5Mrgprb5Cgnl1Wnt7b
Gfra3PtgdrKcnc1
Klf7Ggta1Etv1
Cysltr2Grik1Pln
Irf6Mmp25Cdh12
Prdm8Casz1
Etv5Bnc2
StacKlf5
Lypd1
Housekeeping genes
 GapdhActb
  1. To perform Fluidigm single cell analysis, Taqman assays were chosen to cover four categories of population-enriched transcripts first identified by microarray whole transcriptome analysis: (1) SNS-Cre/TdT+ (total population) enriched markers (vs Parv-Cre/TdT+ neurons), (2) IB4+SNS-Cre/TdT+ enriched markers (vs other 2 groups), (3) IB4SNS-Cre/TdT+ markers (vs other 2 groups), and (4) Parv-Cre/TdT+ markers (vs other 2 groups). Taqman assays for housekeeping genes Gapdh and Actb were also included.

Table 3

RNA in situ hybridization probes

https://doi.org/10.7554/eLife.04660.027
GeneForward primerReverse primerProbe length (bp)
Gpcr5b5′-ATGTTCCTGGT5′-TCACCAATGGTG1233
Lpar35′-TTGTGATCGTCCTGTGCGTG5′-GCCTCTCGGTATTGCTGTCC870
TdTomato5′-ATCAAAGAGTTCATGCGCTTC5′-GTTCCACGATGGTGTAGTCCTC615
Prkcq5′-TCTTGCTGGGTCAGAAGTACAA5′-TCTGTGGTTGAGTGGAATTGAC919
Nppb5′-TGAAGGTGCTGTCCCAGATGATTC5′-GTTGTGGCAAGTTTGTGCTCCAAG545
Il31ra5′-CTCCCCTGTGTTGTCCTGAT5′-TTCATGCCATAGCAGCACTC559
  1. Probesets used for RNA in situ hybridization analysis. Listed are gene symbols, sequences for forward and reverse primers, and resulting probe lengths.

Data availability

The following data sets were generated
  1. 1
  2. 2

Additional files

Supplementary file 1

Comparison of SNS-Cre/TdT vs Parv-Cre/TdT neuron expression profiles. Differential expression analysis of microarray data from SNS-Cre/TdTomato+ neurons (n = 4) vs Parv-Cre/TdTomato+ neurons (n = 4). Transcripts are ranked by fold-change, with the following information given: Affymetrix ID, genebank accession number, gene symbol, description, average RMA normalized levels, standard deviation, fold-change, p-value and FDR.

https://doi.org/10.7554/eLife.04660.031
Supplementary file 2

Comparison of IB4 positive vs IB4 negative SNS-Cre/TdT neuron profiles. Differential expression analysis of microarray data from IB4+SNS-Cre/TdTomato+ neurons (n = 3) vs IB4SNS-Cre/TdTomato+ neurons (n = 3). These cells were sorted from the same animals. Transcripts are ranked by fold-change, with the following information given: Affymetrix ID, genebank accession number, gene symbol, description, average RMA normalized levels, standard deviation, fold-change, p-value and FDR.

https://doi.org/10.7554/eLife.04660.032

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