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Unbiased classification of sensory neuron types by large-scale single-cell RNA sequencing

Nature Neuroscience volume 18, pages 145–153 (2015)Cite this article

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Abstract

The primary sensory system requires the integrated function of multiple cell types, although its full complexity remains unclear. We used comprehensive transcriptome analysis of 622 single mouse neurons to classify them in an unbiased manner, independent of any a priori knowledge of sensory subtypes. Our results reveal eleven types: three distinct low-threshold mechanoreceptive neurons, two proprioceptive, and six principal types of thermosensitive, itch sensitive, type C low-threshold mechanosensitive and nociceptive neurons with markedly different molecular and operational properties. Confirming previously anticipated major neuronal types, our results also classify and provide markers for new, functionally distinct subtypes. For example, our results suggest that itching during inflammatory skin diseases such as atopic dermatitis is linked to a distinct itch-generating type. We demonstrate single-cell RNA-seq as an effective strategy for dissecting sensory responsive cells into distinct neuronal types. The resulting catalog illustrates the diversity of sensory types and the cellular complexity underlying somatic sensation.

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Figure 1: Single-cell RNA-seq of DRG cells reveals cellular heterogeneity.
Figure 2: Identification of eleven principal types of sensory neurons by iterative unbiased PCA.
Figure 3: In vivo interrogation of identified neuronal types.
Figure 4: Unique patterns of expression of operational components in different sensory types.
Figure 5: Diversity of itch responsive neuronal subtypes.

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Gene Expression Omnibus

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Acknowledgements

The authors thank the CLICK Imaging Facility, supported by the Wallenberg Foundation. This work was supported by the Swedish Research Council for Medicine and Health, the Swedish Foundation for Strategic Research and Linné grants (DBRM grants), the Swedish Brain Foundation, Hållsten Foundation, Torsten Söderberg Foundation, Wallenberg Scholar and European Research Council advanced grant (232675) to P.E.; and by European Research Council starting grant (261063) to S.L.

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Author notes

  1. Sten Linnarsson and Patrik Ernfors: These authors jointly directed this work.

Authors and Affiliations

  1. Division of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden

    Dmitry Usoskin, Alessandro Furlan, Saiful Islam, Hind Abdo, Peter Lönnerberg, Daohua Lou, Jens Hjerling-Leffler, Olga Kharchenko, Sten Linnarsson & Patrik Ernfors

  2. Division of Physiological Chemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden

    Jesper Haeggström

  3. Center for Biomedical Informatics, Harvard Medical School, Boston, Massachusetts, USA

    Peter V Kharchenko

  4. Division of Hematology/Oncology, Children's Hospital, Boston, Massachusetts, USA

    Peter V Kharchenko

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Contributions

D.U., S.L. and P.E. designed the study. D.U., A.F., D.L., O.K., H.A., J.H.-L., J.H. and S.I. carried out experiments. D.U., P.V.K., P.L., P.E. and S.L. performed data analysis, including statistical analyses. D.U., S.L. and P.E. wrote the manuscript in consultation with all authors.

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Correspondence to Sten Linnarsson or Patrik Ernfors.

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Integrated supplementary information

Supplementary Figure 1 Number of genes detected.

Distribution of the number of detected genes, including only the UCSC mRNA gene models (i.e. excluding many non-coding RNAs, ribosomal and tRNAs and expressed repeat families). The average number of detected genes was 3574 (standard deviation, 2010).

Supplementary Figure 2 Immunohistochemical identification of neuronal types.

(a) Triple immunohistochemistry for LDHB, TRKB and TRKC and ISLET1. Note TRKB+-TRKC+/LDHB+ neurons (inset). All neurons belonging to the large Neurofilament, heavy polypeptide neuron class express Lactate dehydrogenase B (Ldhb). (b-e) Validation of the NF1 and NF2 TRKB+ subgroups. (b) Triple immunohistochemistry for CACNA1H, TRKB and ISLET1. Note TRKB+/CACNA1H+ neurons (inset). The Calcium channel, voltage-dependent, T type, alpha 1H subunit (CACNA1H) labels most of the neurotrophic tyrosine kinase, receptor, type 2, TRKB-expressing neurons. (c) Triple immunohistochemistry for NECAB2, TRKB and ISLET1. Note NECAB2+/TRKBhigh neurons (inset). The NF1 subgroup of neurons expresses the N-terminal EF-hand calcium binding protein 2 (NECAB2) and TRKB at high levels (TRKBhigh). (d) Triple immunohistochemistry for CALB1, TRKB and ISLET1. Note CALB1+/TRKBlow (inset). The NF2 subgroup expresses Calbindin (CALB1) and TRKB at low levels (TRKBlow, NF2). (e) Triple immunohistochemistry for NECAB2, CALB1 and ISLET1 shows NECAB2 and CALB1 being mutually exclusive (inset). (f-j) Validation of the NF3, NF4 and NF5 TRKC+ subgroups. The large, Neurotrophic tyrosine kinase, receptor, type 3 (TRKC) population is formed by 3 subgroups. (f) Triple immunohistochemistry for FAM19A1, TRKC and ISLET1. Note FAM19A1+/TRKC+ neurons (inset) and TRKC+/FAM19A1- neurons (arrowheads). NF3 is defined as a subpopulation of TRKC neurons expressing the Chemokine-like protein TAFA-1 (FAM19A1). (g) Triple immunohistochemistry for FAM19A1, Parvalbumin (PV) and ISLET1. Pvalb and Fam19a1 expression is mutually exclusive (inset). NF3 neurons are negative for Parvalbumin. (h) Triple immunohistochemistry for CNTNAP2, PV and ISLET1. Note PV+/CNTNAP2+ neurons (inset). (i) Triple immunohistochemistry for SPP1, CNTNAP2 and ISLET1. Note SPP1+/CNTNAP2+ neurons (inset) representing NF4 and NF5. NF4 and NF5 PV-positive neurons are a subpopulation of TRKC-positive neurons. (j) Triple immunohistochemistry for TRKC, CNTNAP2 and ISLET1. Note NF4 and NF5 CNTNAP2+/TRKC+ neurons (inset), subgroups of the largest TRKC population. We could not distinguish NF4 from NF5. However, Inhibin, beta B (INHBB) stood out as a good candidate to mark neurons belonging to the NF5 subgroup of TRKC+/PV+ neurons. (k) Triple immunohistochemistry for TH (sole marker defining TH population), combined TRKA/PLXNC1/NEFH and ISLET1. Note TH+/TRKA-/PLXNC1-/NEFH- neurons (inset). TH labels neither neurons belonging to the NF group (defined by Nefh expression), nor the NP group (defined by Plxnc1 expression) or the PEP group (defined by TrkA). (l-v) A combinatorial immunohistochemistry strategy was used to successfully distinguish the NP (l-p, v) and the PEP (q-u) classes of sensory neurons. Data analysis identified Plexin C1 (PLXNC1) as a common marker for all three subgroups of the NP group and, additionally, being expressed in some PEP1 neurons. (l) Triple immunohistochemistry for P2X3, PLXNC1 and SST. Note SST+/PLXNC1+/P2X3+ (inset). PLXNC1 labels all Somatostatin (SST) positive neurons belonging to NP3 and P2X3+ neurons belonging to NP1. (m) Triple immunohistochemistry for combined FAM19A1/TAC1, PLXNC1 and SST. Note (FAM19A1+ or TAC1+)/PLXNC1+/SST- (arrowheads) and (FAM19A1- and TAC1-) /PLXNC1+/SST+ neurons (inset). PLXNC1 expression is detected in NP3 and some neurons belonging to PEP groups. (n) Triple immunohistochemistry for FAM19A1, PLXNC1 and CGRP. Fam19a1 and Plxnc1 expression is mutually exclusive (inset); PEP2 neurons do not express PLXNC1 (o) Triple immunohistochemistry for TAC1, PLXNC1 and CGRP. Note TAC1+/PLXNC1+/ CGRP+ neurons (inset); expression patterns of PLXNC1 and of Tachykinin, precursor 1 (TAC1), a unique marker of PEP1 neurons, show some overlap. (p) Triple immunohistochemistry for PLXNC1, combined FAM19A1 and TAC1 and CGRP, shows CGRP+/FAM19A1-TAC1-/PLXNC1+ neurons belonging to the NP2 subgroup. (q) Triple immunohistochemistry for CGRP, TRKA and ISLET1. TRKA and CGRP show 1:1 co-localization (inset). The Neurotrophic tyrosine kinase, receptor, type 1 (TRKA) and Calcitonin receptor-like (CGRP) are expressed by all neurons belonging to NP2, PEP1 and PEP2 subgroups. (r) Triple immunohistochemistry for TAC1, TRKA and ISLET1. TAC1 defines PEP1 neurons. Note TRKA+/TAC1+ (inset) and TRKA+/TAC1- neurons (arrowheads) belonging to the other two TRKA-expressing groups, NP2 and PEP2, respectively. (s) Triple immunohistochemistry for FAM19A1, TRKA and NEFH. Note NEFH+/FAM19A1+/ TRKA+ and NEFH+/FAM19A1+/ TRKA- neurons, belonging to PEP2 and NF3, respectively (inset) and TRKA+/FAM19A1- neurons (arrowheads) belonging to the other two TRKA-expressing groups (NP2 and PEP1). (t) Triple immunohistochemistry for combined FAM19A1/TAC1, TRKA and ISLET1. Note TRKA+/FAM19A1-/TAC1- neurons (inset and arrowheads), belonging to NP2 subgroup. (u) Triple immunohistochemistry for SST, TRKA and ISLET1. Sst and TrkA expression is mutually exclusive (inset). Sst defines the NP3 subgroup. (v) Double immunohistochemistry for PLXNC1 and combined NFH/TRKA/SST/TH. Note PLXNC1+/NFH-/TRKA-/SST-/TH- neurons belonging to the NP1 subclass. Scale bar = 50 μm.

Supplementary Figure 3 Illustration of how threshold level was determined for each gene to calculate the fraction of positive cells (thresholding method).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–3 (PDF 964 kb)

Supplementary Methods Checklist (PDF 346 kb)

Supplementary Table 1

Full lists of genes showing differential expression (fold change and significance) for all eleven individual neuronal types analyzed using the SCDE method against all remaining neurons (pooled) See first worksheet (Tab1-INFO) for detailed legend. (XLSX 14786 kb)

Supplementary Table 2

GO analysis of biological processes for eleven neuronal types To Fig. 2e. See first worksheet (Tab1-INFO) for detailed legend. (XLSX 638 kb)

Supplementary Table 3

Differential expression (fold change and significance) for neuronal versus non-neuronal populations and gene ontology analysis (biological process) distinguishing these two populations See first worksheet (Tab1-INFO) for detailed legend. (XLSX 1886 kb)

Supplementary Table 4

Expression profile (fraction of positive cells, with color coding) for 452 genes (fused 11 lists with top 50 genes enriched in each of neuronal categories, by SCDE method, after redundancy removal; “top 50” list) and all neuronal types. To Fig. 2d. See first worksheet (Tab1-INFO) for detailed legend. (XLSX 67 kb)

Supplementary Table 5

Expression profile (fraction of positive cells, with color coding) of 18 genes (17 picked from “top 50” gene lists (Table S2) and Ntrk3 (TrkC) as extensively used sensory marker) used for in vivo immunohistochemical validation experiments. To Fig. 3. See first worksheet (Tab1-INFO) for detailed legend. (XLSX 17 kb)

Supplementary Table 6

Expression profile (fraction of positive cells, with color coding) for genes participating as operational components (shown in Figure 4a, in the same order) of sensory neurons in the different neuronal types. To Fig. 4a. See first worksheet (Tab1-INFO) for detailed legend. (XLSX 48 kb)

Supplementary Table 7

Expression profile (fraction of positive cells, with color coding) for itch-related and neuropeptide genes in unmyelinated neurons. To Table 1. See first worksheet (Tab1-INFO) for detailed legend. (XLSX 29 kb)

41593_2015_BFnn3881_MOESM19_ESM.avi

3D video for PCA plot of five clusters representing four neuronal and one non-neuronal cell populations shown in Figure 1a (AVI 6221 kb)

3D video for PCA plot of four principal neuronal types shown in Figure 1b (AVI 12342 kb)

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Usoskin, D., Furlan, A., Islam, S. et al. Unbiased classification of sensory neuron types by large-scale single-cell RNA sequencing. Nat Neurosci 18, 145–153 (2015). https://doi.org/10.1038/nn.3881

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