© 2014 Nature America, Inc. All rights reserved. Here we have used single-cell RNA-based dissection of the DRG DRG the of dissection RNA-based single-cell used have we Here Correspondence should Correspondence be addressed to P.E. ( School, Boston, USA. Massachusetts, Chemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden. 1 populations cell reveal to profiles expression similar of grouping unbiased single-cell sampling, RNA-seq of each cell and unsupervised Hence, cells. individual of tional identity cell-type will be in reflected the gene expression profile classification and discovery type cell high at for strategy a regulation suggested have others and we In particular, resolution. gene studying for opportunity unprecedented into physiology. sensory insight new lend could types neuronal of assignment subtype types different of neurons responsive broadly of ensembles from qualitative by in and the of quantitative summation integrated differences activities neuronal and/or of sensation of dedication modalities the specific for by subtypes both arise could stimuli different between discrimination as Furthermore, identification. their mark for ers of absence and types of classification unbiased an of lack of itch, touch, pain, temperature underlying and proprioception remains largely elusive stimuli because chemical and physical the to types neuronal of relation The states. transcriptional unique on founded unbiased an by classification objective an than allow would that rather strategy comprehensive aspects functional to relate markers established of Currently, handful a how on stimuli. based classified are specific types to neuronal respond to tuned neurons sensory (DRG) ganglion root dorsal specialized of existence the by reflected is sensation of types diverse discriminate and perceive to ability The somatic sensation. distinct neuronal types. The resulting catalog illustrates the diversity of sensory types and the cellular complexity underlying type. itch-generating We demonstrate single-cell RNA-seq as an effective strategy for dissecting sensory responsive cells into For example, our results suggest that itching during inflammatory skin diseases such as atopic dermatitis is linked to a distinct anticipated major neuronal types, our results also classify and provide markers for new, functionally distinct subtypes. and mechanosensitive nociceptive neurons with markedly different molecular and operational properties. Confirming previously neurons, mechanoreceptive two and proprioceptive, six principal types of itch thermosensitive, sensitive, type C low-threshold independent of any a priori knowledge of sensory subtypes. Our results reveal eleven types: three distinct low-threshold unclear. We used analysis transcriptome comprehensive of 622 single mouse neurons to classify them in an unbiased manner, The primary sensory system requires the integrated function of multiple cell types, although its full complexity remains Patrik Ernfors Jens Hjerling-Leffler UsoskinDmitry large-scale single-cell RNA sequencing Unbiased of classification sensory neuron types by nature nature Received 6 June; accepted 22 October; published online 24 November 2014; Division Division of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden. Recent advances in single-cell transcriptomics have opened an an opened have transcriptomics single-cell in advances Recent NEUR OSCI 1 , EN 5 1 , , Alessandro Furlan C E

1 advance online publication online advance , , Jesper Haeggström de novo de 4 Division Division of Hematology/Oncology, Children’s Hospital, Boston, USA. Massachusetts, cell type discovery is possible by possible is discovery type cell 3– [email protected] 5 based on the idea that func that idea the on based 1 , Saiful , IslamSaiful 2 , , Olga Kharchenko ) ) or S.L. (

1 [email protected] , , Hind Abdo 1 6 doi:10.1038/nn.388 , , 2 7 - - . ,

heavy chain ( chain heavy neurofilament expressing as cluster, NF the first, the identified we ( clusters neuronal four the in markers of known expression the Examining non-neuronal. were example β markers non-neuronal the expressed and markers pan-neuronal of clusters ( main distinct five revealed genes and cells all across tudes 1 Fig. to 3,574 mapped exons gene On models. using average, RefSeq reads 1.14 were million known to unambiguously aligned and barcoded properly were lion bil 1.3 which of generated, were reads billion 2.76 of A total DRGs. lumbar mouse the from cells single 799 on RNA-seq We performed Identification of discrete sensory neuron populations RESULTS itch. related of a distinct class of neurons involved in transduction of inflammatory- tion. The utility of the data is by exemplified a more analysis in-depth serve as a catalog of the cellular and molecular basis for somatic sensa will gene expression, on specific information full with supplemented Thus, the establishment of an unbiased classification of types, sensory sensation. somatic underlying system sensory of complexity primary and diversity the reveals which types, neuronal of classification for Fig. Fig. 2 microglobulin ( 2 microglobulin 1 1 , , Peter V Kharchenko ). Principal component analysis (PCA) of expression magni expression of (PCA) analysis component Principal ). and and 1 , , Peter Lönnerberg Col6a2 ). Supplementary Video 1 Video Supplementary 1 Nefh ), on the basis of which we concluded that these cells cells these that we concluded of ), which on basis the ± ) and parvalbumin ( parvalbumin ) and 2,010 distinct genes in each cell ( cell in each genes distinct 2,010 3 B2m Center Center for Biomedical Informatics, Harvard Medical ), vimentin ( vimentin ), Fig. 1 Fig. 1 3 5 , , Daohua Lou These These authors jointly directed this work. , 4 b , , Sten Linnarsson and and ). One cluster lacked expression expression lacked cluster ). One Vim Pvalb 2 ) and several collagens (for (for collagens several ) and Supplementary Video 2 Video Supplementary Division Division of Physiological ), previously associated associated previously ), e r 1 , Supplementary Supplementary

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© 2014 Nature America, Inc. All rights reserved. uniquely characterizing each of the eleven groups, we compared the the compared we groups, eleven the of each characterizing uniquely genes To identify function. neuron sensory of knowledge prior with consistent were they whether examined and clusters neuronal ferent classification. the to support further ( soma sizes and characteristic neurons distinct with of consist classes neuronal identified the that show furthermore and and TRKA TRKB large, be to shown were of TRKC somata For example, markers. molecular cells dark and small light large initially, identified were that categories main Two sizes. soma types neuron sensory of origin developmental known the with relationship of the eleven different neuronal types is largely consistent to NP3, PEP1, PEP2 and TH. Notably, the identified basic hierarchical neuronal classes in this data set, which we designate NF1 to NF5, NP1 analysis ( iterative Further types. cell hierar the a among in relationship resulting chical groups, the of one only within subdivisions to led manner this in groups comparing instance, every in Notably, the within groups. subtypes represent that cells also those distinguish results show that the groups expressed between genes ofdifferentially These of neurons. cluster TH the in any heterogeneity to find unable same subclusters were identified with the different outgroups. We were ( outgroups as used were clusters TH and NP as into split outgroup, when and two groups used PEP cluster the was TH cluster the when and NP2/3, NP1 groups, rons into two split either the PEP or NP clusters were used as the outgroup. The NP neu when appeared groups three same the finding, of this robustness the the when TH cluster was as used an outgroup ( cluster NF the from appeared NF4/5, and NF2/3 NF1, clusters, new Three ‘outgroup’; (the Methods). subtype Online see another against ing only those genes that were differentially expressed when compared each of the four principal neuronal types was subjected to PCA includ ( non-neuronal were cells 109 and assignment neurons ambiguous had cells 68 neurons, as unmyelinated classified of were cells subclass distinct a in ( described hydroxylase showed tyrosine of cluster, TH expression the distinct fourth, The nociceptors. nonpeptidergic of expression cluster, showed NP the third, The nociceptors. peptidergic with associated (CGRP peptide gene-related ( P substance of expression cluster, PEP showed the second, The DRG neurons. myelinated with 1. variance and 0 mean with expression gene normalized represents key Color hydroxylase. P2X3; P; substance (TH). containing hydroxylase tyrosine and (NF) containing neurofilament (NP), nociceptors non-peptidergic (PEP), nociceptors peptidergic as defined was identity Group populations. different the to back mapped genes representative of expression relative to according colored cells with plot PCA same the show Insets types. neuronal principal ( cells. non-neuronal to back mapped genes representative of expression relative to according colored cells with plot PCA same the show (top) Insets cells. non-neuronal contained which of one clusters, five to them of 731 assigned cells ( 1 Figure e r  types identified other all to type neuronal each within expression a Fig. 2a, Fig.

) Bottom: PCA of single-cell gene expression patterns for 799 individual individual 799 for patterns expression gene single-cell of PCA ) Bottom: We next determined the patterns of expression distinguishing dif distinguishing of expression patterns the We determined next by distinct characterized are historically types cell neuron Sensory classes, main the within neurons of subtypes further Todiscover O S Nefh

Single-cell RNA-seq of DRG cells reveals cellular heterogeneity. heterogeneity. cellular reveals cells DRG of RNA-seq Single-cell b , PCAs 4–6) led to the identification of a total of eleven eleven of total a of identification the to led 4–6) PCAs , + r u , neurofilament heavy chain; chain; heavy , neurofilament nociceptors small Ntrk1 c , TRKA; , TRKA; b e ) PCA of 622 neuronal cells comprising the four four the comprising cells neuronal 622 of ) PCA Mrgprd 11 Calca , 1 2 , were later extended by introduction of by introduction , extended were later Tac1 8 and and , , CGRP; , CGRP; , 1 also known as as known also + 3 mechanoreceptors of medium size size medium of mechanoreceptors . . Our results agree with these studies , RA ( TRKA ), P2rx3 Fig. 2 Fig. Pvalb Mrgprd , previously associated with with associated , previously a Fig. 2a, Fig. ). , parvalbumin; , parvalbumin; , MGRPRD; , MGRPRD; Ntrk1 Fig. Fig. 2a, Th Calca b ), which has been been has which ), + ). In all cases, the the cases, all In ). ) and calcitonin calcitonin and ) muscle afferents afferents muscle Fig. Fig. 2 Tac1 b ), previously previously ), ). Supporting ). Supporting P2rx3 Th c encodes encodes , tyrosine , tyrosine ), lending ), lending 8 , ,

9 8 . 622 622 . , 1 0 . - - - -

CACNA1H (calcium channel, voltage-dependent, T type, alpha 1H) 1H) alpha type, T voltage-dependent, channel, (calcium CACNA1H RNA-seq data. LDHB (lactate dehydrogenase B) marked all NF classes. and staining showing mutually exclusive expression patterns ( patterns expression exclusive mutually showing staining immunohistochemical triple and double by the types of neuronal discrete existence the confirm to able were with we markers, Nevertheless, selected the identified. be can markers that better and/or possibility the other exclude not does and set data large this in sive ( antibodies available with analyses immunohistochemical binatorial selective expression, as well as a high fraction of positive cells, for com and abundant with lists 50 top the from genes some chose we genes, at visualized be can gene cell for all cells across all neuronal ( types byvalidated scatter plot of analyses absolute gene expression level per list of all genes, see top 50 of genes, merged (list subtypes ronal neu identified the of one to expression of fidelity high show genes 452 population-specific genes, this measure confirmed that most such of list thresholding to merged the applied When Methods). a Online (see on procedure based measure cells’ positive of ‘fraction a used we populations, to of specific a expression to gene is which restricted and manipulating identifying, Tostudying the neuronal types. for the evaluate extent tools provides also and function into insight lends types cell different the in expressed uniquely transcripts Identifying Interrogation of identified populations by gene expression ( population non-neuronal different of characteristics ( types functional distinct predicted analysis ( types neuronal between separation clear a The genes relative displayed genes. expression levels of population-specific these unique 452 of set common a into pooled were list each from genes fifty top the and estimate), likelihood (maximal change expressed ( tial expression (SCDE) differen approach to single-cell Bayesian developed a using recently Supplementary Table 5 Supplementary B2m a To validate Tovalidate Non-neuronal upeetr Fg 2 Fig. Supplementary Fig. Fig. 2 Col6a2 advance online publication publication online advance –2.0 0 2.0 P < 0.05) genes in each comparison were ranked by the fold e Vi and m in vivo in Supplementary TableSupplementary 2 http://lin relevance of the identified population-specific population-specific identified the of relevance 1 b Tac1 4 ( Nefh ). The selection is by comprehen no means ). selection The http://linnarsson Supplementary TableSupplementary 1 Supplementary Table 3 Supplementary narssonlab.org/drg ), in complete agreement with the the with agreement complete in ), NF cluster PEP cluster Ntrk Pval 1 Fig. Fig. 3 b ), ), as well the as confirming l a Supplementary Table 4 Supplementary nature nature Fig. 2 Fig. b . o a / r ; ; a scatter plot for any ). ). These were further g 3 Calc ). ). The differentially / d d 3 ). ). Gene ontology ontology Gene ). r TH cluste g NEUR a NP cluster P2rx3 / Mrgprd Th ) . OSCI r Fig. 3b– Fig. 3 EN C z E - - - - ;

© 2014 Nature America, Inc. All rights reserved. ter glutamate, neuropeptides and other signaling molecules onto the molecules signaling and other neuropeptides glutamate, ter channels (Ca Ca voltage-gated by conduction potential action and depolarization receptors, sensory of activation as such components, Transduction of on relies of sensation the presence many operational Operational components and functional assignment of neurons markers. protein corresponding by the distinguished be can they RNA profiling also exist by neuronal identified types our single-cell whole-genome, unbiased, was by identified expression of TH. Hence, these results show that the by their unique expression of TAC1 and FAM19A1 while the TH class 1), type and CGRP PEP SST neurons were (somatostatin); confirmed by (encoded TRKA by encoded 3, channel, ion ligand-gated P2X, receptor ergic by confirmed were (purin P2X3 C1), (plexin of PLXNC1 of expression patterns classes unique NP1–NP3 classes. these in expression myelinated sensory neurons, revealed unique complements of receptor to have used receptor,identify been previously 3), which kinase, type receptor, type 2) and TRKC (encoded by by (encoded TRKB receptors by encoded min, 2), phosphoprotein 1) protein-like SPP1 and (secreted PV (parvalbu by associated (contactin CNTNAP2 by NF5 and NF4 and A1) member identified uniquely FAM19A1 was (family with sequence similarity 19, NF3 binding tively. calcium respec 1), (calbindin CALB1 and protein 2) EF-hand NECAB2 by (N-terminal identified be uniquely could turn in which classes, NF2 and NF1 marked also (see functions or properties sensory reflecting terms specific significant most the for corrected) shows graph The ( panel. this in presented genes for profile expression population neuronal of summary See population. assigned to according grouped neurons 622 all in redundancy) excluding total, in genes (452 population each for genes enriched significantly most 50 the of expression of map ( procedure. picking the during cells (mean ( × 10 2.33 PCA4, 0.005; PCA3, 0.02; PCA2, 0.0017; PCA1, follows: as set were rates discovery false The outgroups. indicated the against tested when expressed differentially genes on based was PCA Each (arrows). bottom at indicated clusters cell generating stably outgroups with ( in shown are and splitting population corresponding for used were which PCAs, representative of plots to references provide we nodes, graph some In indicated. are cells of numbers with definitions type Cell outgroups. as types cell other all with cross-examined were identified types cell All types. neuron sensory ( PCA. unbiased iterative by neurons sensory of types 2 Figure nature nature neurons sensory second-order cord spinal c b e ) Cell diameters of identified neuronal types types neuronal identified of diameters ) Cell ) Gene ontology analysis of neuronal types. types. neuronal of analysis ontology ) Gene ) PCA plots showing identified cell clusters clusters cell identified showing plots ) PCA a ± ) Outline of iterative analyses defining defining analyses iterative of ) Outline s.e.m.) as measured on dissociated dissociated on measured as s.e.m.)

NEUR Supplementary Table 2 Supplementary Identification of eleven principal principal eleven of Identification −6 ; PCA5, 9 × 10 ; PCA5, v , Na OSCI Supplementary Table 4 Supplementary P values (Benjamini-Hochberg (Benjamini-Hochberg values v Pvalb and K EN Ntrk1 C ). Double staining for these markers and the the and markers these for staining Double ). E in vivo, v

channels), and release of the neurotransmit −4 advance online publication online advance , neurotrophic tyrosine kinase, receptor, receptor, kinase, tyrosine neurotrophic , ; PCA6, 0.047. 0.047. ; PCA6, ). Ntrk2 representing distinct entities, and that d ) Heat ) Heat b . for for , neurotrophic tyrosine kinase, kinase, tyrosine neurotrophic ,

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NP TH PCA2 NP NF1–NF3 identify these as low threshold mechanoreceptors threshold low as these identify NF1–NF3 and sion sion as lightly myelinated A and heat) neurons marker inflammatory itch. The PEP1 population, defined by expression of the to in participate pruritus pain neuropathic and medium in to involved be appears class NP1 The neurofilaments. light, chain heavy of expression the by demonstrated as neurons as these suggests neurons limb proprioceptive NF5 and NF4 in PV and TRKC of presence The functions modality-specific known to ( types neuronal sensory us a to allows of propose relation the identified types neuron sensory identify to used previously markers known of absence or presence ( populations neuronal identified the in release transmitter neuro and propagation stimuli potential complements action underlying depolarization, properties reception, distinct cellular and revealed channels pain receptors, of and in important Analysis transduction be to pain. literature sensory the in neuropathic identified genes and expressed of inflammatory with associated excitatory local and by inhibitory circuits or descending fibers and by molecular changes presynaptically influenced be furthermore can Fig. 4 Fig. NF4, 22 The The RNA gene and analysis profiles, ontology for interrogation the NP2 NF4/5, 48 PEP Supplementary Table 6 Supplementary PCA5 PEP b Tac1 ). The expression patterns of of patterns expression The ). TH NF5, 26 PEP1 (substance P) is consistent with thermosensitive (cold (cold thermosensitive with consistent is P) (substance PCA3 –2.0 NP TH TH 22– 27– PEP2 NP1, 125 799 sequencedcells 2 0 2 4 9 . NP1, NP2 and NP3 neuronal types are likely likely are types neuronal NP3 and NP2 NP1, . and PEP2 as defined by 2.0 25 TH NF4/5 NF2 NP2/3, 44 , NP2, 32 NP, 169 2 δ 6 PCA6 PCA2 nociceptors. Finally,nociceptors. the TH population, and NP3 is likely to and sense transduce PCA4 Temperature stimulusdetection Temperature stimulusdetection ). 2 Mechanical stimulusdetection Mechanical stimulusdetection 1 . Consistently, all these . are Consistently, these myelinated all e NP3, 12 NF3 Muscle development Muscle development Muscle development Cellular respiration Cellular respiration Cellular respiration Corticospinal tract Corticospinal tract Corticospinal tract Ntrk2 Neuromuscular Neurofilament Neurofilament Neuropeptide Neuropeptide Inflammation Inflammation NF4 NF2/3 PEP NP TH PEP1, 64 PEP, 81 Outliers orunresolved PCA5 , PCA3 Ret, Calb1 Ret, Pain Pain Pain Pain Pain Pain Pain ATP Itc Itc Itc Ntrk1 h h h identity, 68 e r NF5 PEP2, 17 and 2 0 O S NP2 and and –log Nefh 4 r u PCA6 TH, 233 10 Ntrk3 1 6 ( expres P Fig. 4 Fig. 9 ) NP3 , NP1 16– 1 0 c TH in in 2 2 e 0 a  - - .

© 2014 Nature America, Inc. All rights reserved. gorized pain and itch mediators and neuropeptide ligands predicted predicted ligands neuropeptide and mediators itch and pain gorized uncate previously for of receptors expression selective the addition, touch and pain pleasant (C-LTMRs) mechanoreceptors involved in mechanical low-threshold C type as function a with sistent as of levels high containing e r  s m g b a y

400 800 PLXNC1 Vglut3 TRKA LDHB FAM19A1 0 TRKA andCGRPlabelNP2, ISL1 TH O S PEP1 andPEP2neurons FAM19A1 labelsNF3 TH neuronsstandalone PLXNC1 labelsNP1

LDHB definesNF and NP3neurons PLXNC1/TRKA/NEFH NF

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ISL1 Pe p ISL1 c TH e 400 800 t n h c SST PLXNC1 TRKA CACNA1H FAM19A1 Piezo2 0 z PLXNC1 labelssom express parvalbumin NF3 neuronsdonot TAC1 definesPEP1 CACNA1H labels NF NF1/2 neurons TAC1 PEP neurons Ntrk1 FAM19A1/TAC1 Plxnc1 neurons

and exclusive exclusive and PV

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ISL1 – Calb1 / ISL1 Nefh + Pe – / p ISL1 – TH e 1,000 2,000 3,000 u o i d PV TRKA PLXNC1 NECAB2 Slc17a8 FAM19A1 labelsPEP2 NECAB2 definesNF1

PLXNC1 doesnotlabel 0 CNTNAP2 PV andCNTNAP2 FAM19A1 PEP2 neuron define NF4/5 NF

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TRKA PLXNC1 labelssom SPP1 definesNF4/5 CALB1 definesNF2 0 As a validation of the overall analysis, we examined predicted predicted examined analysis we ontology Gene analysis, detail. greater in overall neurons itch-transducing the of validation a As A small population tuned to respond to inflammatory itch ( types responsiveness sensory across the subtype-specific different neuronal NP2 isdefinedby

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TRKB /FAM19A1 NF TAC1 Table ISL1 NP CGRP P2x3 ISL1 advance online publication publication online advance – Pe /TAC1 p 1

ISL1 e and and TH – f w q k TRKC NECAB2 CGRP PLXNC1 TRKA Supplementary Tables 4 Supplementary subset ofTRKCneurons 2,000 4,000 NF4/5 neuronsare separate populations PLXNC1 labelsNP2 NF1 andNF2are SST definesNP3 0

CNTNAP2 SST FAM19A1/TAC1

neurons neurons CALB1 NF

ISL1 NP ISL1 ISL1 Sst Pe p a TH r x l 400 800 nature nature Ntrk1 PEP1/2 Plxnc1 NP1–3 + and Ldhb NF1–5 0 PEP1 NP1 NP2 NF2 NF1 PEP2 NP3 NF3 NF NF4/5 7 ). Ntrk1 Ntrk3 Ntrk3 Fam19a1 NEUR NP Fam19a1 Tac1 Ntrk2 Sst + Ntrk2 lo Plxnc1 /Calc P2x3 high w Pe Cntnap + Nef /P2x /P + p Spp1 high /Calca /P v + OSCI lo h a high v + /Calca w + /Necab + /Fam19a – + 3 /Calb /P2x3? / / lo Fam19a1 TH 2 + w + + / + 1 / EN 2 1 – C / + E

© 2014 Nature America, Inc. All rights reserved. immunohistochemistry for TH, combined PLXNC1/TRKA/NEFH and ISLET1. Note TH Note ISLET1. and PLXNC1/TRKA/NEFH combined TH, for immunohistochemistry express them of Some ( (inset). exclusive FAM19A1 TRKA and inset) ( ( (inset). colocalization (NP1), express neurons NP All group. NP main the of subgroups three the showing CGRP all CGRP. FAM19A1/TAC1 and Virtually combined PLXNC1, for ( (inset). exclusive FAM19A1 ( (inset). Cntnap2 high- and low- by high of expression by identified are classes NF2 CNTNAP2 Note ISLET1. and TRKC CNTNAP2, for ( (inset). ( (inset). exclusive mutually is FAM19A1 expression and FAM19A1 ( (inset). exclusive mutually are CALB1 and NECAB2 ISLET1. and CALB1 ( CACNA1H LDHB Note (ISL1). ISLET1 ( PEP2 and PEP1 NP1–NP3, for taken genes the of some of 3 Figure neurons, sensory in ablation gene conditional require will causality demonstrating Although mediators. inflammatory these to response in behavior itching predict hence and ligands respective the to ness triene receptor expression in NP3 neurons indicate cellular responsive IL31RA and neurotensin (NTS) ( Sst tides natriuretic peptide, neurotensin and somatostatin ( neuropep the including markers, unique other in rich were neurons (co-receptors ( leukotrienes cysteinyl for receptors to tion ( population this in PLXNC1 marker the of levels ( neurons nonpeptidergic define to used previously markers staining, only low levels of RET and P2X3 and was negative for( isolectin diameter B4 in (IB4) small be NP3 neurons of to neurons and revealed size neuronal quantification unmyelinated of class each of identification Immunohistochemical NP1, as TH with either SST or did not ( GFRA2 colocalize TRKA from separate were neurons SST as 2, and PEP1 and NP2 from distinct negative; polypeptide) SST as PEP2, and NF1–NF5 from distinct was NP3 properties. unique its confirmed ( neuropeptides of ments PEP2. Each class of unmyelinated neurons contained distinct comple ( receptors serotonin and NP3 and NP2 in ( ( (IL)-31 NP2, interleukin NP1 class, chloroquine-responsive neurons ( neurons ( acid–responsive with lysophosphatidic of ( neurons classes three these across substances to pruritic profiles response distinct and predicted receptors of responsive ence and ( itch to NP2 neurons NP3 NP1, of responsiveness indicated Methods), Online (see genes itch-related containing category custom a with supplemented nature nature of expression by solely identified are neurons TH group. TH u e Fig. 5f– Fig. Cysltr2 ) Triple immunohistochemistry for CALB1, TRKB and ISLET1. Note CALB1 Note ISLET1. and TRKB CALB1, for ) Triple immunohistochemistry ) Triple immunohistochemistry for TRKA, FAM19A1 and NEFH. Note TRKA Note NEFH. FAM19A1 and TRKA, for ) Triple immunohistochemistry In-depth immunohistochemical analyses of the NP3 population population NP3 the of analyses immunohistochemical In-depth , respectively; respectively; , Ntrk1

and and )-responsive in NP3. Histamine receptors ( receptors in Histamine NP3. )-responsive n j ) Triple immunohistochemistry for SPP1, CNTNAP2 and ISLET1. Note SPP1 Note ISLET1. and CNTNAP2 SPP1, for ) Triple immunohistochemistry NEUR In vivo In k ) Triple immunohistochemistry for PLXNC1, combined FAM19A1/TAC1 and SST. TAC1) or FAM19A1/TAC1 and (FAM19A1 Note combined PLXNC1, for ) Triple immunohistochemistry − − + + ). Consistent with our previous results, we confirmed high high confirmed we results, previous our with Consistent ). /TAC1 /TAC1 /TRKC neurons (inset). ( (inset). neurons and and Spp1 Il31ra OSCI interrogation of identified neuronal types. ( types. neuronal identified of interrogation + − − Ntrk2 + /FAM19A1 neurons (inset and arrowheads). ( arrowheads). and (inset neurons ( (inset). neurons Calca p x neurons (inset) and FAM19A1 and (inset) neurons Table . All NF neurons express express neurons NF . All ) Scheme showing the two subgroups of the main PEP group. PEP1 neurons are characterized by their unique expression of of expression unique their by characterized are neurons PEP1 group. PEP main the of subgroups two the showing ) Scheme ) Triple immunohistochemistry for PLXNC1, TAC1CGRP. and PLXNC1 PLXNC1, Note for ) Triple immunohistochemistry and EN + Plxnc1 neurons were NEFH (neurofilament heavy heavy (neurofilament NEFH were neurons -expressing neurons. NF3 is defined by expression of of expression by defined is NF3 neurons. -expressing t (NP2) and and (NP2) ) Triple immunohistochemistry for TRKA, TAC1 and ISLET1. Note TRKA Note TAC1ISLET1. and TRKA, for ) Triple immunohistochemistry C Fig. 2 Fig. Fig. 5 Fig. 1 E Osmr

), with confirmed full colocalization of SST, of colocalization full confirmed with ), + Table Il31ra advance online publication online advance − /(TRKB m neurons (arrowheads). ( (arrowheads). neurons d ; PEP2 neurons are myelinated ( myelinated are neurons ; PEP2 ) Triple immunohistochemistry for NECAB2, TRKB and ISLET1. Note NECAB2 Note ISLET1. and TRKB NECAB2, for ) Triple immunohistochemistry e in vivo in e – ) and the serotonin receptor ( ). Further analysis confirmed the pres the confirmed analysis Further ). x ). The NP3 class of neurons contained contained neurons of class NP3 The ). ) and of TH ( TH of ) and 1 and Fig. 5l, Sst + and and o or TRKC or ) Triple immunohistochemistry for PLXNC1, FAM19A1 and CGRP. FAM19A1 and PLXNC1 expression is mutually mutually is CGRP. expression FAM19A1 and PLXNC1 FAM19A1 PLXNC1, and for ) Triple immunohistochemistry validation. ( validation. and and + Osmr neurons; distinct from TH and and TH from distinct neurons; Supplementary Table 7 Supplementary m P2x3 Ldhb ). + )- and cysteine leukotriene )- and leukotriene cysteine y ) neurons (inset). ( (inset). ) neurons Htr1f , Il31ra Mrgpra3 z Ntrk2 ) classes of sensory neurons. ( neurons. sensory of ) classes Cysltr2 (NP3). ( (NP3). . . ( − b + /TRKC – /TRKC m w z , , Lpar3 ) Triple immunohistochemistry for PLXNC1, P2X3 and SST. and SST P2X3 Note PLXNC1, for ) Triple immunohistochemistry ) Immunohistochemical identification of neuronal types. Validation of NF1–NF5 ( NF1–NF5 of Validation types. neuronal of identification ) Immunohistochemical v and cysteine leuko Fig. 5j– Fig. ) Triple immunohistochemistry for TRKA, SST and ISLET1. SST and TRKA expression is mutually mutually is expression TRKA and SST ISLET1. and SST TRKA, for ) Triple immunohistochemistry Htr2a ( ) Triple immunohistochemistry for TRKA, combined FAM19A1/TAC1 and ISLET1. Note TRKA Note ISLET1. FAM19A1/TAC1 and combined TRKA, for ) Triple immunohistochemistry Hth1 Ntrk2 i ), IL-31 receptor receptor IL-31 ), + and ) Triple immunohistochemistry for PV, CNTNAP2 and ISLET1. Note PV Note PV,ISLET1. for and CNTNAP2 ) Triple immunohistochemistry + s neurons (arrowheads). ( (arrowheads). neurons neurons (inset). ( (inset). neurons ) Triple immunohistochemistry for TRKA, CGRP and ISLET1. TRKA and CGRP show 1:1 1:1 show CGRP and TRKA ISLET1. and CGRP TRKA, for ) Triple immunohistochemistry and Nppb a Th ) in NP3 and and NP3 in ) ) ) were found ) Representative graphs of single-cell absolute expression levels (reads per million, RPM) RPM) million, per (reads levels expression absolute single-cell of graphs ) Representative high Mrgprx1 Nefh Htr1f k . Scale bars, 50 50 bars, . Scale Fig. Fig. 5a–

). In addi ). Lpar5 ), ), Table c , ) Triple immunohistochemistry for CACNA1H, TRKB and ISLET1. Note TRKB Note ISLET1. and TRKB CACNA1H, for ) Triple immunohistochemistry Nts + Necab2 ). ), NP3 ) and express express ) and + and /FAM19A1 ) ) in ) in d 1 ), ), ). ). + ------g

and low low and ) Triple immunohistochemistry for FAM19A1, TRKC and ISLET1. Note Note ISLET1. and TRKC FAM19A1, for ) Triple immunohistochemistry Plxnc1 l Ntrk3 + ) Scheme showing the five subgroups of the major NF group: NF1 and and NF1 group: NF major the of subgroups five the showing ) Scheme + /TRKB that discrete transcriptional states in different classes of unmyelinated time first for the shows but it formally nociceptors, of nature C-fiber polymodal a contradict not does classification Our types. neuronal neural tissue for unbiased molecular discovery of previously unknown demonstrate the utility of single-cell RNA sequencing from a complex were discovered and validated by neurons immunohistochemical staining. Thus, we sensory of types distinct fundamentally Eleven skin. glabrous a set of and full neurons proprioceptors include innervating to in DRGs order have lumbar from neurons on sensory our analysis We focused subtypes. determined clearly neurons sensory analyzed across heterogeneity cell representing cells. variance among gene that expression Wefound gene in variance high a detected we cells, single in states transcriptional of modeling and sampling unbiased Using schemes. classification subtype marker-based with consistent known functional heterogeneity among neuronssensory is not always Despite intensive research using marker and physiological approaches, DISCUSSION day ( 11 postnatal after appearing neurons most with development, in late unexpectedly emerged neurons of class this for ( scratching triggered reliably agents (LTD4).Both D4 leukotriene cysteine and IL-31 of injections subcutaneous following bouts scratching recorded nevertheless we receptors, is present in all three NP classes as well as the TH class. class. TH the as well as classes NP three all in present is receptors, itch and clove and ginger oil, mustard as such chemical pungent by (ref. TRPM3 heat of channel noxious the neurons; NP3 and NP2, classes the PEP1 (ref. TRPV1 channel sensitive heat well-studied the with that example, neurons for show, results of Our states. ensembles transcriptional of different integration broader a by encoded are sensation of neuro types other whereas specific selectively, to relatively types tuned nal be may pain mechanical acute probably detection cold TrpM8-dependent response. stimulus distinct underlie neurons polymodal /FAM19A1 b µ ) Triple immunohistochemistry for LDHB, combined TRKB/TRKC and and TRKB/TRKC combined LDHB, for ) Triple immunohistochemistry m; scale bars are all of the same size and apply to all micrographs. all to apply and size same the of all are bars scale m; The current scheme proposes that proprioception, C-LTMR, C-LTMR, proprioception, that proposes scheme current The − Fam19a1 h /TAC1 and and . NP subgroups are identified by combinatorial expression of of expression combinatorial by identified are subgroups . NP ) Triple immunohistochemistry for FAM19A1, PV and ISLET1. PV ISLET1. and PV FAM19A1, for ) Triple immunohistochemistry Ntrk2 low + + /TRKA /CNTNAP2 neurons (inset). ( (inset). neurons Fam19a1 − + neurons are PLXNC1 are neurons ( and NEFH and Ntrk2 . All PEP neurons express express neurons PEP . All 3 − /PLXNC1 3 + ) is present in NP1 and PEP1; and TRPA1, activated TRPA1,activated and PEP1; and NP1 in present is ) + /TAC1 low /TAC1 , NF4 and NF5 by expression of of expression by NF5 and , NF4 + neurons (inset). ( (inset). neurons ) and ) and + /FAM19A1 + + − neurons (inset). ( (inset). neurons (inset) and TRKA and (inset) /NEFH f Calb1 ) Triple immunohistochemistry for NECAB2, NECAB2, for ) Triple immunohistochemistry + /TRKB Fig. 5 Fig. − + , respectively. , respectively. neurons (inset). ( (inset). neurons (arrowheads and inset). ( inset). and (arrowheads + (PEP2 and NF3, respectively, respectively, NF3, and (PEP2 28 + k n high , /PLXNC1 ) Triple immunohistochemistry ) Triple immunohistochemistry 3 Ntrk1 ). Furthermore, the SST marker marker SST the Furthermore, ). 2 , some forms of pruritus, and and pruritus, of forms some , neurons (inset). (inset). neurons q + ) Triple immunohistochemistry ) Triple immunohistochemistry + /TAC1 and and /PLXNC1 Cacna1h + + (arrowheads) and SST and (arrowheads) /CNTNAP2 Calca − z Ntrk3 neurons (arrowheads). (arrowheads). neurons e r ) Scheme showing the the showing ) Scheme + /P2X3 . . ( is expressed expressed is b 2 O S and and Fig. 5o– Fig. – y 7

l ) ) Triple r ) is present in in present is ) ), of ), ) Scheme ) Scheme + + Pvalb neurons neurons neurons neurons r u

Tac1 + P2x3 q / , ). + c . /

+

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© 2014 Nature America, Inc. All rights reserved. ferent in each unique neuronal class. Our results thus support the the support notion thatthus sensation can arise results both through the Our selective activation of class. neuronal unique each in ferent dif be may quality signal and thresholds to regard with receptor or channel same the from integration and signal However, connectivity e r  subtypes to modality-specific functions or previous functional classification is indicated below brackets. Myel., myelinated; Unmyel., unmyelinated. Unmyel., myelinated; Myel., brackets. below indicated is classification functional previous or functions modality-specific to subtypes sensory of relation Proposed field. the in studied commonly products gene of a distribution of examples are bottom at products Gene markers). used previously selected black, markers; new (red, subtypes of identification for markers suggested are top on products Gene analyses. transcriptome RNA ( receptors. opioid R., Opioid chemokines; chemok., transporters; glutamate K channels; cation P2Y;or amiloride-sensitive Accn, See map. heat the below named are genes selected Some sensitization. in participate to known are products gene of a number pain neuropathic or inflammatory during and influences, inhibitory and excitatory both to subject presynaptically is terminal central the Furthermore, cord. spinal the in molecules signaling other and neuropeptides glutamate, of release in resulting eventually channels ion voltage-gated of presence and terminals peripheral at channels ion transducer of specificity the by encoded properties activation of combination unique the by reflected is identity Neuronal CNS. the to transferred is information where terminal a central and CNS the to periphery the from potentials action conducts that axon an stimuli, sensory to responsive is that terminal a peripheral contain neurons All neurons. of classes different in neurons sensory of components operational various the of 4 Figure b a

TH PEP NP NF O S

Unique patterns of expression of operational components in different sensory types. ( types. sensory different in components operational of expression of patterns Unique Supplementary Table 6 Supplementary Peripheral ending r u Trpc3 Trp CACNA1H TRKB NECAB2 Trpa1 Sensory perception NEF LDHB NF1 Trpv1 c P2rx6 high H

P2ry1 P2x/yr e P2rx3 Accn

CACNA1 Ramp3 TRKB LTMRs CALB1 NEF LDHB Neuro- RET RET NF peptide 2 H lo Npy2r receptors for data presented in this panel. Trp, transient receptor potential cation channels; P2x channels; cation potential receptor Trp, panel. transient this in presented data for w H

Mrgprd Mrgpra3 Mrgpr Myelinated FAM19A TRKC NEF LDHB RET RET NF Scn8a Scn1a Na 3 V H high Scn10a

1 Scn11a Kcnj12 CNTNAP2 V TRKC RUNX3 Soma -rectif., open rectifier potassium channels (Kcnk), neurofilam., neurofilaments; Glu transp., transp., Glu neurofilaments; neurofilam., (Kcnk), channels potassium rectifier open -rectif., ASIC1 NEFH LDHB SPP N PV F4 Proprioceptors 1 low Conduction Kcns1 KV CNTNAP2 TRKC RUNX3 ASIC1 NEF LDHB SPP NF PV N 5 H 1 lo w Kcnip4 -

PLXNC1 Kcnk1 b MRGPRD NAV1.8/9 GFRA play distinct patterns of expression across neuronal types, and the the and types, neuronal across expression of patterns distinct play classes. neuronal different of ensembles in or type by neuronal a as of dedicated the encoded integration activity ) Proposed new sensory neuron classification based on unbiased full full unbiased on based classification neuron sensory new ) Proposed TRPC TRPA1 K -rectif.

P2X3 V RE Cacna1h P1 Hierarchical clustering shows that a large number of genes dis genes of number large a that shows clustering Hierarchical T 3 2 high CaV Cacna1c Nonpeptidergic PLXNC1 MRGPRA advance online publication publication online advance

NAV1.8/9 Nefl Neurofilam. TRPC TRPA1 TRPV1 CGRP TRKA P2X3 Neuro-transmission RE NP Unmyelinated T 2 a 3 ) Heat map of expression (fraction of positive cells) cells) positive of (fraction expression of map ) Heat high

3 Neuro- peptides Gal

PLXNC1 Tac1

NAV1.8/9 Calca TRPC TRPA1 TRPV1 P2X3 RE SST N Glu transp.

Central synapse P3

T Chemok. 3 high Presynaptic regulation

Gabra1 GABA

PLXNC1 receptors NAV1.8/9 TRPV1 CGRP

TRKA Gabra3 PEP1 TAC1 KI T

Glrb /yr, purinergic receptor P2X P2X receptor /yr, purinergic Peptidergic

low Gly/Glu Grim8 receptors

nature nature Grik1 CNTNAP2 FAM19A NAV1.8/9 Oprm1 Opioid R CGRP NEFH TRKA PEP2 Myel. KI T Htr3a 1 Inflam- NEUR

Asic3 Chronic pain Ptgir matory Htr1f pain PIEZO2 1 0 NAV1.8/9 C-LTMRs Cysltr2 VGLUT Unmyel. GFRA TRPA1 OSCI Osmr RE TH II31ra T high 2

3 Neuro-

EN II6st pathic Lpar3 C pain E

Adcyap1 -

© 2014 Nature America, Inc. All rights reserved. recruitment recruitment of NP3 neurons, whereas S1P S1P receptor the via lipid sphingosine-1-phosphate mediator the immunomodulatory by pro-nociception that predict data our For instance, networks. nal neuro distinct possibly thereby and subtype neuronal dedicated a activate to tuned are mediators cellular unknown with stimuli acute Despite the complexity of somatic sensation, our system. data predict somatosensory the in that mechanisms many functional explaining for Our data and of have modulation properties. utility cellular extensive and maintenance of excitation, and regulation induction end organs, of of in connectivity, induction establishment genes participate likely Such release. molecule signaling or signaling receptor adhesion, cell modulation, or activity ionic in involved are products gene those all Notably,nearly class. that within exclusively expressed genes of sets However, properties. class, we for functional find also each identified combination of patterned gene expression underlies cell type–selective See The populationsizeandthefractionofthatwouldcorrespondtoonecellareshownattop. nature nature in PEP1 and PEP2 neurons; the involvement of injury–induced nerve receptors receptors and in neuropeptides small size neurons Table 1 Tac1 Nts Sst Nppb Calca Nmb Neuropeptides Htr3a Htr1f Htr2a Hrh1 Trpv1 Osm Il31ra Cysltr2 Mrgprx1 Mrgpra3 Lpar5 Lpar3 Trpa1 Mrgprd Adcyap1 Agrp Itch receptors Symbol Supplementary Table 7 r

NEUR Expression Expression profile (fraction of positive cells by thresholding method) of itch OSCI 1 1 cellrepresents: ( Ratio to Population: S1pr1 EN 11.94 forfulllistsofitch-relatedandneuropeptidegenes. C 0.19 0.35 0.03 0.39 0.30 1.97 0.21 0.31 0.02 0.02 0.02 0.09 0.09 0.06 0.12 0.05 0.11 0.05 0.07 0.04 0.06 N E : Actb

) advance online publication online advance 34– 3 6 seems to occur almost exclusively by exclusively almost occur to seems 0.01 0.03 0.01 0.01 0 0 0.20 0.94 0 0 0.07 0 0.03 0.01 0 0.03 0.07 0.01 0.24 0.66 0.51 0.84 0.008 NP1 125 3 ( S1pr3 0.09 0.09 0.03 0.13 0.03 0.03 0.88 0.75 0.12 0.09 0.03 0.09 0.28 0.38 0.03 0 0.53 0.63 0.03 0.03 0.22 0.22 0.03 NP2 32 ) ) mediates response 0 0.17 0.75 0.25 0.83 0.83 0.08 0.67 0 0.83 0.25 0.08 0.58 0.75 0.58 0.67 0.33 0.08 0 0.08 0 0.17 0 NP3 0.08 12

0 0 0.31 0.03 0 0.02 0 0 0 0 0 0 0 0.06 0.02 0.63 0.83 0.02 0.11 0.02 0.03 0.78 0.39 0 0.05 0 0.02 PEP1 0.016 64 -

ritogenic agents without affecting nociceptive behavior nociceptive affecting pru without of agents variety ritogenic a by induced itch prevents activity NPPB blocking pathway, neuronal receptor its as the of stage next the in transduction of represent Expression somatosta specific neurotensin. and expressed peptide the natriuretic selectively tin, of cluster this of activation Neurons the cluster. NP3 the by to represented neurons of population tuned small relatively and be could conditions includ diseases, dermatitis molecules itch atopic both inflammatory ing to As linked addressed. directly been have experimentally was itch induce containing class (NP3). PLC the to itch serotonin and NP3) (NP2, genes these both ing express classes to itch histamine delimiting possibly PEP1, and NP3 secondary neurons in the dorsal horn of the spinal cord, from from cord, spinal the of horn dorsal the in neurons secondary involves neurons sensory NPPB-expressing primary from pathway 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1.00 1.00 0.82 0.24 PEP2 0.06 The prediction that IL-31 and cysteine leukotrienes can can leukotrienes cysteine and IL-31 that prediction The 17 0 0.01 0 0 0 0.01 0.01 0.18 0.01 0.01 0.04 0.01 0.16 0 0 0 0.004

233 TH Npra is expressed in the spinal cord dorsal horn and and horn dorsal cord spinal the in expressed is 44 their ligands, were unique to NP1 neurons. NP1 to unique were ligands, their by Lpar5 (encoded receptors acid ( pathways itch neurons. of class TH the to exclusive by class neurons. Furthermore, VGLUT3 (coded rotensin receptor ( neu high-affinity the of activation through could potentially mediate nociception directly selective for PEP2 neurons; and neurotensin ( 2 receptor prokineticin through 2 prokineticin cytokine by pro-nociceptive the produced pain inflammatory neurons; angiotensin II type 1 receptor ( during neuropathic pain II angiotensin of effects pro-nociceptive the exclusively expressed in NP1 ( class of neurons; 5 and 3 receptors acid lysophosphatidic via occurs itch static chole and pain neuropathic of maintenance and initiation the in acid lysophosphatidic neurons, while while neurons, in NP1, NP2, NP3 and to lesser degree in PEP1 but not TRPV1 (refs. PLC requires serotonin while TRPV1 and requires both PLC itchHistamine-induced detection. unreliable itch could not assigned, be probably owing to statistically significant), and PAR2-dependent this was NP2 in only (and levels low at only detected was (HRH1) H1 receptorhistamine remain elusive because, in both NP2 and NP3, however, pathways, itch Some HTR2A. and HTR1F receptors serotonin the of presence specific the to owing NP3 to assigned be can also in the same class. Serotonin-induced itch is MRGPRC11), as known (also MRGPRX1 receptor, chloroquine Another neurons. of which is exclusively expressed in the NP2 class ionotropicTRPA1) channelthe to coupled tion with MRGPRA3 receptors interac via effects its mediates Chloroquine , 4 5 Our data also allow us to predict multiple predict to us allow also data Our , our results suggest that itch in such such in itch that suggest results our , Slc17a8 , hc cn ntae th rgee by triggered itch initiate can which ), ), involved in pleasant touch, is is touch, pleasant in involved ), Trpv1 Table β Ntsr1 3 (phospholipase C, beta 3) 42 Lpar3 is expressed in NP2, NP2, in expressed is , 4 1 e r ) ) present in some TH 3 3 ). Lysophosphatidic Lysophosphatidic ). 9 ). We observed is mediated via the and and O S 2 Agtr1a 6 (functionally 4 Lpar5 Lpar3 6 Nppb . This itch itch This . r u Prokr2 ) in NP1 ) and and Plcb3 may may 37 c β ) is is ) , β 3 4 3- 4 e 8 1 3  0 ------, ,

© 2014 Nature America, Inc. All rights reserved. in the functionality of these neurons as modulators or neurotrans or modulators as neurons these of functionality the in Sst to study. present the in addition Instead, Grp neurons sensory primary from itch of transducer a as proposed been has GRP rotransmitter, activity itch-inducing NPPB the to exert released is (GRP) peptide gastrin-releasing which ( NEFH in SST indicate Arrowheads DRG. lumbar adult and P19 11, (P) day postnatal type on wild- neurons all to quantify 3), homeobox leukemia, (T cell TLX3 and population, NP3 SST,of the for neurons to label immunohistochemistry ** * to saline, compared Significance test. Two-sided Mann-Whitney (Caps). capsaicin and (5HT) 2-methyl-5-hydroxytryptamine analog (mean ( PLXNC1 (mean subclasses different the for area soma of the ( sections. DRG lumbar on adult respectively, classes, TH neuronal and PEP1-PEP2-NP2 NP1, NF1–NF5, for markers representative 5 Figure e r  Thus, horn. dorsal the in centrally and/or periphery the in mitters n

P

a Scratching bouts SST P2X3 SST RET SST NEFH was specific to the NP3 population, suggesting their participation participation their suggesting to the NP3 population, was specific < 0.01; ns, not significant; values: IL-31, IL-31, values: significant; not ns, < 0.01; , was not found to be expressed by primary sensory neurons in in neurons sensory primary by expressed be to found not was c

(per 30 min) i f a O S , , 100 20 40 60 80 ± b a NP3 0 s.e.m.). ( s.e.m.). ,

), ), TH ( d Diversity of itch responsive neuronal subtypes. ( subtypes. neuronal responsive of itch Diversity j , , ) and for SST and IL31RA ( IL31RA and SST ) for and differsfrom f r u Saline , g and and PEP1/2 d ), IB4 ( ), IB4 n c ) Behavioral response (scratching bouts) following subcutaneous administration of compounds: saline, IL-31, LTD4, IL-31, serotonin the saline, of compounds: administration subcutaneous following bouts) (scratching response ) Behavioral i , j

e IL-31 NP2 was performed on the same sections. Scale bars are shared between these panel pairs. Specific signal emitted in far-red light from from light in far-red emitted signal Specific pairs. panel these between shared are bars Scale sections. same on the performed was ** g 4 and ) and PLXNC1 ( ) PLXNC1 and 6 . In addition to NPPB as a primary itch neu itch primary a as NPPB to addition In . 47– LTD4 * 4

9 SST GFRA2 I acr wt a eet study recent a with accord In . b l ) or NTS ) ( or NTS 5HT ** j NP3 ) is shown as green. Scale bars, 50 bars, Scale as green. ) is shown differsfrom Nppb P Caps = 0.0065; LTD4, = 0.0065; m ns ). Quantification of SST ). Quantification , expression of , expression SST PLXNC1 SST IB4 ± j g s.e.m.). ( s.e.m.).

NP1 a

– SST TLX3 d ) Double immunohistochemistry for SST and NEFH ( NEFH and SST for immunohistochemistry ) Double o f – m SST TRKA P Nts ) Double immunohistochemistry for SST and RET ( RET and SST for immunohistochemistry ) Double = 0.0105; 5HT, = 0.0105; c and NP3 5 0 + - - , differsfrom neurons coexpressing RET, IB4, P2X3 or PLXNC1 is reported in RET,is reported or PLXNC1 P2X3 IB4, coexpressing neurons

µ m. PEP1/2 map of the cellular basis of somatic sensation, and the underlying underlying the and sensation, somatic of basis cellular the of map comprehensive a provide results neu these NP3 above, engage exemplified As would rons. serotonin, and histamine inflammatory as of well states as itch, chronic to linked are which leukotrienes, cysteine and IL-31 as such mediators and neurons, would NP2 to tuned itch be acute with associated histamine and neu chloroquine NP1 to rons, tuned be would disorders cholestatic with acid associated lysophosphatidic profiles: response unique with neurons sive of respon itch classes three of at least existence the support data our + neurons. Quantification in Quantification neurons.

NP2 P = 0.008; Caps, Caps, = 0.008; and P11 advance online publication publication online advance

SST TH k h p d

+ + + + P SST /marker cells per ganglion SST /marker cells per ganglion = 0.47. Mean Mean = 0.47. NP3 10 12 10 12 0 2 4 6 8 8 0 2 4 6 differsfrom

q SST SST , mean , mean PLXNC1

a RET

), GFRA2 ( ), GFRA2 P2X3 IB4 f ±

TH ) or IB4 ) ( or IB4 s.e.m. Immunohistochemistry Immunohistochemistry s.e.m. ± s.e.m. ( s.e.m. Adult

b SST NTS SST IL31RA ), TRKA ( ), TRKA nature nature g e m l ), or P2X3 ( ), or P2X3 o Neuron area ( m2) – µ q 100 200 300 400 500 600 700 P e ) Double ) Double 0 < 0.05, < 0.05, ) Quantification ) Quantification q + + NP1 c NEUR Percentage SST /TLX3 ) ) or TH (

0 1 2 3 4 5 6 7 NP2 h i P11 ) ) or

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© 2014 Nature America, Inc. All rights reserved. 15. 14. 13. 12. 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. 1. reprints/index.htm at online available is information permissions and Reprints The authors declare no competing interests.financial consultation with all authors. analysis, including statistical analyses. D.U., S.L. and P.E. wrote the manuscript in and S.I. outcarried experiments. D.U., P.V.K., P.L., P.E. and S.L. performed data D.U., S.L. and P.E. the designed study. D.U., A.F., D.L., O.K., H.A., J.H.-L., J.H. AUTH starting grant (261063) to S.L. Council advanced grant (232675) to P.E.; and by European Research Council Torsten Foundation, Söderberg Wallenberg Scholar and European Research grants (DBRM grants), the Swedish Brain Foundation, Hållsten Foundation, Medicine and Health, the Swedish Foundation for Strategic Research and Linné Foundation. This work was supported by the Swedish Research Council for The authors thank the CLICK Imaging Facility, supported by the Wallenberg online Note: Any Supplementary Information and Source Data files are available in the any gene. for expression of plots scatter izing tool for expression level and posteriors; online search engine for visualization of scatter plots of expression (RPM) for allowing any gene; visual coding), color with cells, positive of (fraction populations full with RPM), for million, and genes profiles all neuronal the expression sample annotation; per reads as (expressed wells sequenced at plates (libraries), codes. Accession the in versio available are references associated any and Methods M organisms. even and tissues other to applicable be discov should that type More classification cell and ery types. unbiased for neuronal strategy a various demonstrate the we of broadly, roles functional delin for the strategies eating direct up open data transcriptome single-cell nature nature COM Acknowledgmen

et http://linnarsso

Woolf, C.J.&Ma,Q.Nociceptors–noxious stimulusdetectors. single-cell to approach D.T.Bayesian Scadden, P.V.,& Kharchenko, L. Silberstein, and Expression H.S. Phillips, & L.H. Ling, M.P., Armanini, S.B., McMahon, in ganglia. Sensory A.R. Lieberman, K.H. Andres, implications and diversity neuron sensory somatic of Generation Q. Ma, & Y. Liu, L. Li, of specification the underlying interactions Molecular P. Ernfors, & F. Lallemend, Treutlein,B. Jaitin, D.A. Islam, S. diversity neuronal Mapping Neuroscience. E. Mazzoni, & D. Gifford, H., Wichterle, sensations. somatic Shapiro, E., Biezuner, of T. & Linnarsson, S. Single-cell coding sequencing-based technologies population talk: and meet lines Labeled Q. Ma, PopulationQ.Ma, somaticcoding ofsensations. (2007). analysis. expression differential targets. peripheral identified to neurons projecting sensory primary adult of subpopulations in receptors Trk of coexpression 1976). London, Hall, and (Chapman 188–278 ganglia] coding. sensory on neurons. neurons. sensory RNA-seq. single-cell using types. cell into tissues of RNA-seq. multiplex time. a at cell one science.whole-organism revolutionize will Invest. Clin. J. P version of the pape h O n of the pape the of n E ods TI R R et al. et NEUR CON NG 55 Cell et al. The functional organization of cutaneous low-threshold mechanosensory low-threshold cutaneous of organization functional The et al. , 1–48 (1961). 1–48 , FI et al. et

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© 2014 Nature America, Inc. All rights reserved. newly identified subpopulations were annotated after verifying that (i) this split clearlyseparatedthreeclusters.splitorother into two the These lation), while clusters becoming compacted the of one(this in resultedpopulation this cases manyis In referredgroups. two the to between asdifference an outgroup popu all other groups by applying parametric filtering of variables contributing to the againstone by comparedone groupswas initial the of Toprocess.each so, do iterativePCA-basedunbiased an using types neuronal 11 of 1 neuronsto 622 2.55 × 10 in analysis and worked over a wide range of variance filters, the only filter applied. For the confirmationfourofthe clear distinct neuronal groups. Separation robustwas 3 Table Supplementary cells, while the remaining four clusters were neurons (see gene ontology analysis, cluster tal identified (yellow in genes that passed filtering by variance at the level of 3.85 × 10 byvariance. tioned groups was robust and observed with a wide range of settings for filtering further analysis. PCA plot density pattern with one distant and four closely posi ( normalization. 2,858 genes contributing to differences between picking sessions using Qlucore Omics Explorer 2.3. PCAPrincipal componentwas analysis (P performed using mean = 0, variance = 1 including both content and cell population assignment). at areprovideddatanormalizedmeasure.million) These per kilobase per (reads hence there is no need to normalize for length, as in the commonly used RPKM genomebuild. RNA-seqOur readsmethod only 5 the any annotated gene, using the RefSeq gene models mapped on the GRCh37/hg19number of reads mapped to all code isoforms of a accession gene divided by under all reads mapped (GEO) to GSE59 Omnibus Expression Gene at accessible are (libraries)platesindividual for data raw The cell. one thancontainedmore or verified by visual inspection under microscope. The remaining wells were empty as cellcontained singleprocessed799wells, library. a 864 of totalper a ofOut last two libraries, L281 and L282, were processed separately(L128–L228). Thus, each of these 8 librariesand contained 96 containedsequenced wells.sequencing The for cells 48 96 wellsto up included that libraries generate to pooled were the mouse genome as previously described on an Illumina Genome Analyzer IIx or HiSeq 2000, and reads were mapped to Single-cell R measurements before collection. Cells were picked randomly. 48 cells took from 40 to 60 min. Dissociated cells were photographed for cell size 18 different animals in independent experiments. Picking of one plate containingthe cell was released. In total, 18 48-well plates were used (864 wells total) from positioned in a well of a 96-well plate containing 3 vidual cells were captured. By an automated a glass capillary attached to a CellTram syringe with visual control so that indi house around an inverted Nikon microscope. Individual cells were sucked into in- constructedsetup, cell-pickingrobotic a with pickedwere Cells B27. with used. The final cell pellet was suspended in 300 and collagenase/dispase, and in some preparations a cooled microscopeprocedure samplealwaysthe waskeptapartfrom ice, onincubation papain stage in was neuronal types could vary along the rostro-caudal axis. During the preparation L4–L6 neurons are largely limb innervating, and we note that composition and to a previously published protocol (6–8 week old, both males and females) were dissected and dissociated accordingC local ethics committees of Stockholm. All animal work was performed in accordance with the national guidelines and in groups, with food and water Animals. MET ONLINE nature nature ting was stable at different levels of parametric variable filter ( q ell picking.

http://linna ≤ Starting with identification of four neuronal groups, we assigned each of the 0.05,ANOVA test) were excluded, and totalin 22,478 genes were for used 73 −4 9 C57Bl/6 mice were used for all experiments. Animals were kept in cages NEUR . The expression level in RPM (reads per million) was taken as the as taken was million) per (reads RPM in levelexpression The . , which resulted in 11,658 genes contributing to the analysis. Figure 1 Figure Figure1 N Six hindlimb-innervating lumbar DRGs (L4–L6) from adult mice rssonlab.org/drg A-seq. OSCI H ODS b a Cells were processed for RNA sequencing and sequenced ( EN ( Supplementary Video 2 Video Supplementary SupplementaryVideo1 ). Excluding the non-neuronal group, PCA led to a to led PCA group, non-neuronal the Excluding ). C E ad libitum, / Fig.1 (together with annotation of individual wells, individual of annotation with (together 5 1 C followed by Percoll gradient centrifugation A). a ) represented) mixturea of non-neuronal PCA of normalized data was performed under 12-h light-dark cycle conditions. 5 , x 5 3 - . 16 out of 18 picked 48-well plates y - µ z l of L15 medium supplemented ), the variance filter was set at set was filter variance the ), axis drive, the capillary tip was ) shows PCA based on12,750shows ) based PCA µ l of lysis buffer, into which ′ endof transcripts q −4 values) and that . The most dis 3 , and, 5 2 - - - - - .

chical categorization in hierar the in branchingpointsappropriateto referring numberingwith PCA identification,were includedanalysis.this in Briefly, errorcell-specific models expressed between picking sessions and that were excluded during neuronal type against all other clusters. Importantly, all genes, even those cell thatdifferential expressionwere (SCDE) differentiallyanalysis method single- approachBayesianpublishedrecentlyto the used we classes,neuronal formed on the basis of PCA. Afterwards, to identify genes categorizingD individual and does not result from orthogonal superimposition. hierarchically, whereby each lower level cluster is a part of an upper level cluster reveal any noticeable density patterns, suggesting that the clusters are organized genes that contributed to the separation of the the excluding fourPCA performed clusters.we this, ToThisgroups.test fouranalysis all throughdid genes not in all four categories split between dorsal and ventral clusters, sharing common cells different from the main four categories. An example of this would be if cells tions may exist—that is, that there may be ‘superimposed’ categories consisting of from cells that also were assigned to the final eleven clusters. level groups were finalized. Hence, the four main groups were constructed only is, the eleven neuronal clusters—had been identified, the contents of the highest cluster were excluded from analysis and categorized as unclear identity. used. For subgroup splitting, representative PCA charts are shown in (ii) alternative splitting was absent even when a different outgroup population is 0.1. Then these two corresponding gene lists were analyzed by the thenClueGO appby in Table3 gene lists for neuronal versus non-neuronal cells, we used of SCDE the( first (most significant) GO term representingTable 2 each generalized term. To select into the GO term statistics outcome table, generated by ClueGOin ClueGO, ( the custom ‘itch’ term (associated with this gene list) was introduced sory neurons and associated with itch. Using a calculation approacha list implementedof 40 genes ( term representing itch among ontology terms, such groupswe ( used published data properties or functions were generated and custom( terms were createdappliedwas 0.05 generalizingof levelthresholdsignificance A all. code: evidence 4%; and settings: type of analysis: single; GO terms level: 3–8; GO term restriction:GO (Biological Processes,2 genes version from 4 July 2013) was used with the following (GO) analysis. Cytoscape 3.0.1 (ref. 300 top genes for which the by upregulation fold change (maximum likelihood estimate), and not more than elevengroups (when compared to the other neuronal populations) were ranked G used, which requires no assumption apart from independence of measurements. field. For analysis of behavioral experiment, a two-sided Mann-Whitney test was sample sizes, but our sample sizes are similar to those generally employed in the package ( This batch correction procedure is implemented in the version 1.1 of the SCDE observing a particular fold expression difference relative to batch-onlydifference posterior). between the standard and batch-effect posteriors (that is, expressionprobability difference posterior was calculated as a posterior ofdistribution of the batch-adjusted final thefold group;different(iii) each pickingwithinsession) pared, maintaining the original batch composition (that is, number of cells from randomly sampling sets of cells corresponding in size to the groups being com difference posterior accounting only for potential batch effects was generated by ard procedure without any additional corrections; (ii) a separate fold expression expression difference posterior for a given gene was first estimated using stand comparingin used expression of givena gene betweenany two groups: (i)fold in three separate picking sessions, the following batch-correction procedure was were derived over the entire population of neuronal genes. As cells were collected Supplementary Table 2 ene ontology analysis.ontology ene ata handling and statistics. and handling ata We also controlled for the hypothesis that ‘orthogonal’ sets of neuronal popula When all cells that could be assigned to any of the lowest level clusters—that For behavioral experiment, no statistical methods were used to predetermine ). The final ClueGO results ( ), ranked), genes first byfold change (maximum likelihood estimate) and P value, and selected the 300 top genes, the http:/ Supplementary Table 2 /pklab.med.harvard.e Supplementary Table 2 Figure 2 ). After this, GO term groups reflecting similar sensory Lists of genes differentially expressed in each of the of each indifferentially expressedgenes of Lists P value was 0.1 and higher were used for gene ontology All assignment of cells to populations was per populationswas to cells ofassignment All a . Cells that could not be reliably assigned to a 5 Fig. 2 4 du/scde/index.htm ) with ClueGO ). Moreover, since we did not find any GO , last tab) expected to be expressed in sen e ) were obtained by plotting the P values of which did not exceed 1 5 4 5 to compare each cell class app was used. The murine l ). doi: 10.1038/nn.3881 Supplementary Supplementary 5 6 to generate Figure 2 P value b ------

© 2014 Nature America, Inc. All rights reserved. number of neurons SST neurons of number rons counted (co-stained for RET and IB4) = 36; SST SST/NEFH/TRKA = 4; SST/GFRA2/TH = 5. SST SST SST 47; = TRKA) and NEFH for (co-stained counted neurons number of ganglia analyzed per condition = 4 or 5. mals and ganglia: subsequently mounted with glycerol mounting media (Merck). were Slices 1:1,000. of dilution a at used were respectively) 706-495-148, and and Dylight 647-conjugated secondary antibody against guinea pig (706-165-148antibody against rat (Jackson ImmunoResearch 712-166-150) and donkey Cy3- 703-505-155 and 703-605-155, respectively), donkey Cy3-conjugated secondary secondary antibodies against chicken (Jackson ImmunoResearch 703-545-155, A31570 and A31571, respectively), A21202, donkey(Invitrogen 488-, mouse 549- and and 647-conjugatedrespectively) A21448, Alexa and A21436 A11015, goat (Invitrogen A11055, A21432 and A21447, respectively), sheep (Invitrogen antibodies against rabbit (Invitrogen A21206, A31572 and A31573, respectively),647-conjugatedand secondary 555- Alexa 488-, donkey For detection, 1:500). and subsequently incubated for 1–2 h in FITC-streptavidin (Jacksontin B4 Laboratoriesstaining, slides were incubated overnight in IB4-biotin (Invitrogen, 1:500) 1:100, 39.4D5-b) and rabbit anti-neurotensin (Abcam, 1:100,1:10,000), Mouse anti-ISL1ab43833). (Developmental Studies HybridomaFor Bank (DSHB), isolec Birchmeier, Max-Delbrück-Centrum for Molecular Medicine, Berlin, Germany, Systems, 1:500, AF2769), guinea pig anti-TLX3 (kind gift from T. Müller and C. Systems, 1:500, AF429), goat anti-RET (1:100, AF482), goat anti-IL31RA (R&D (R&D anti-GFRA2 goat ab4680), 1:10,000, (Abcam, anti-NF200 chicken 10), anti-TH rabbit AB1566), 1:1,000, (Pel-Freez, 1:1,000, (Millipore, P40101-0), anti-TAC1 sheep anti-TH rabbit 1:1,000, (Novus T-5027), Laboratories, Biologicals, (Peninsula 1:2,000, anti-CGRP NB300- pig guinea ab36001), 1:1,000, (Abcam,anti-CGRP goat MAB354), 1:100, (Millipore,anti-SST rat ab10269), anti-PLXNC1 (R&D Systems, 1:200, AF5375), rabbit anti-P2X3 (Abcam, 1:1,000,Systems, 1:500, AF1494), goat anti-TRKC (R&D Systems, 1:500, AF1404), sheep AF3320), goat anti-TRKA (R&D Systems, 1:200, AF1056), goat anti-TRKBanti-PV (R&D (R&D Systems, 1:500, AF5058), goat anti-CALB1sheep APZ-005), (R&D1:100, HPA013407), Labs, anti-CNTNAP2rabbit(AlomoneSystems, 1:1,000, LDHB (Abcam, 1:400, ab75167), rabbit anti-FAM19A1 (Atlas Antibodies, 1:100, 1:1,000, HPA013998), goat anti-SPP1 (R&D Systems, 1:500, AF808), rabbit anti- showing similar results. previously described −20 °C. Samples were sectioned at 14 lowed by 30% sucrose in PBS. Tissue was then embedded in OCT and frozen at fol sucrose 20% overnightin °C 4 atincubating bycryoprotected and h 1 for °C 4 at PBS in washed subsequentlywere Samples °C. 4 at h PFA1 for4% in postfixed andout dissected wereDRGs 7.4). formaldehyde(pH (PFA)PBS in Immunohistochemistry. (RPM) to be easily visualized as a scatter plot of cells grouped by population. linna in this way, as well as a macro for performing such analysis, is presentedA list atof all genes with an expression profile for neuronal populations calculated a given gene above this threshold was then determined ( level was designated as the threshold of expression. theThe average of thefraction three cells with the highest expression.of 5% of thiscells ‘maximum’ expressing procedure. The ‘maximum’ expression level for each gene was determined to be followingthresholdingthe using expressing cells of fraction the calculatedwe summarize the expression of genes within groups of misleading.highlystatistics summarybemedian Tocellscanevenormean such as (forthat means example,which negatives, false ‘cellof rate high types’),a carry and noisy are data seq Thresholding method to determine fraction of positive cells. at of population-specific GO terms were ranked according to the same way as described above for neuronal subpopulations. The resulting lists doi: SST P Immunohistochemical staining was performed on the following numbers of ani Antibodies, (Atlas anti-NECAB2 rabbit were used antibodies primary The < 0.05. No further term selection was performed. 10.1038/nn.3881 + + + /TH /TRKA /P2X3 rssonlab.org/drg + = 0. Number of animals = 2; number of ganglia analyzed per condition + + = 25; SST 25; = = 0; SST Figure 5 7 / + . At least two animals were used for each immunostaining, . The same file also allows normalized data for each gene (co-stained for GFRA2 and TH) = 44; SST + /PLXNC1 3 + Males 6–8 weeks old were perfused using 4% para 4% using perfused were old weeks 6–8 Males counted (co-stained for P2X3 and PLXNC1) = 28; 28; = PLXNC1) and P2X3 for (co-stained counted and Supplementary Figure 2 + = 28. Number of animals = 2; number of number 2; = animals of Number 28. = µ m. The sections were used and processed as Figure 5f– Figure 5a– + /RET , number of animals = 2, Supplementary Fig. 3 k : number of SST + P = 33; SST value, with a cutoff d Single-cell RNA- , number of SST + + /GFRA2 /NEFH + /IB4 http:// + + + + neu = 0; = = 1; = 0; ). + - - - - -

immunohistochemistry for TRKA and FAM19A1. Double-positive neuronsn were wereforselected analysis ( by double immunohistochemistry for TRKA and TAC1. Double-positiverons analyzed neurons immunohistochemistry for SST ( analyzed are PLXNC1 commona is CGRP marker for PEP1NP2,and PEP2,but only andNP2 PEP1 performed and PLXNC1 Alternatively, triple immunohistochemistry for PLXNC1, CGRP and TAC1 was but negative for combined TAC1/FAM19A1 staining were selected for analysis. combined antibodies against TAC1 and FAM19A1. Neurons positive for TRKA NP2 population were identified by double immunohistochemistry for TRKA( and negative for combined NEFH/TRKA/ SST/TH staining werebut PLXNC1selected for forpositive Neurons NEFH/TRKA/SST/TH. analysisagainst antibodies doubleimmunohistochemistrybycombinedidentifiedwereandPLXNC1 for population NP1 the to belonging Neurons analyzed. were subgroup that for neuronal subgroup, neurons every expressing Fora distinctive, ImageJ. unique molecular markerusing sections immunostained on calculated were classes size. cell of Quantification from one animal each. 4. Figure5o– = SST/P2X3/PLXNC1 5; = SST/RET/IB4 condition per analyzed ganglia 53. 52. 51. n litters):saline: were repeated (where capsaicin and (Sigma,pmol100 0.9%NaCl,in Tween-80).7% acid) Number of timesexperiments ascorbic 1% NaCl, 0.9% in pmol 100 Sigma, (Me-5HT, 2-methyl-5-hydroxytryptamine NaCl), 0.9% in pmol 30 Chemical, Cayman drugs were used: IL-31 (Peprotech, 60 pmol in 0.9% was deducedNaCl), from the total numberleukotriene of scratching bouts counted.D4 The following(LTD4, floor.the ForMe-5HT, numberscratchingvehicleofthe theboutsinducedby with the hindpaw followed by licking or sitebiting injecting the hindpaw the and/orof scratchingplacing repeatedit onas defined was bout scratching Each menter was not blinded to the identity of the compound used in each injection. scratchingandrecordedmin boutswerethencounted.for 30 was experi The administeredsubcutaneously into napethe of neckthe of mouse.the Behavior After the habituation period, a 50- before the experiment by placing them in transparent glass chambers for 30 min. the fur of the back was shaved and all mice were acclimated 24 h before and just animals were excluded from the analysis. Two days before the experimental day, any other prior experimentation (surgery, drug administration, behavioral). No performedunder thelight ofpart theday cycle. Animals were not subjected to Behavioral analysis. istry for TH ( Neurons belonging to the TH population were identified by immunohistochem selected for analysis ( 57. 56. 55. 54. Supplementary Fig. 2v SupplementaryFig. = 5, = 15). Neurons belonging to the PEP2 population were identified by doublepopulationbyidentifiedPEP2were Neurons the 15). belongingto= A

sa, S. Islam, of M.Emergence P.&Koltzenburg, M., Ernfors, Alqatari, J., Hjerling-Leffler, D. Usoskin, Furlan, A., Lubke, M., Adameyko, I., Lallemend, F. & Ernfors, P. The transcription P.The Ernfors, F. & Lallemend, I., Adameyko, M., itch Lubke, A., and Furlan, pain are itch: of mechanisms the into insights New R.R. Ji, & T. Liu, Bindea,G. P. Shannon, functional sensory subtypes as defined by transient receptor potential channel potential receptor transient by expression. defined as subtypes sensory functional neurons. (2010). 16336–16341 sensory of mechanosensitivity atr m1 n got fco rcpo atvte cnrl yptei neurons sympathetic control activities receptor diversification. factor growth and Hmx1 factor mechanisms? distinct by controlled annotationpathwaynetworks.ontologyand networks. interaction biomolecular sequencing. Supplementary N = 3; capsaicin: n =10). Neurons belonging to the NP3 population were identified by q : number: adultof15;ganglia 8 = P19= analyzed 9; age: = P11per + Fig. t al. et et al. et and only PEP1 is SP n J. Neurosci. J. n Nat. Protoc. Nat. = 27). Neurons belonging to the PEP1 population were identified t al. et = 6, = t al. et EMBO J. EMBO 3 ClueGO: a CytoscapeClueGO:a plug-indecipher to functionally groupedgene Highly multiplexed and strand-specific single-cell RNA 5 RNA single-cell strand-specific and multiplexed Highly and Adult males (2–4 months old) were used. Experiments were Fig. yocp: sfwr evrnet o itgae mdl of models integrated for environment software a Cytoscape: N M n En masse in vitro functional profiling of the axonal the of profiling functional vitro in masse En n is the number of animals in each group from = 3; IL-31: 3; = ethods Supplementary Fig. 2 = 5, + ; neuronsanalyzed; 3

/CGRP

27 and

32 Fig. 7 Soma area of neurons belonging to different subdifferentto belongingneurons of area Soma , 813–828 (2012). 813–828 , , 2435–2443 (2007). 2435–2443 , , 1613–1625 (2013). 1613–1625 , N Checklist is available. Supplementary Fig. 2 = 3. 3 and + Figs. 3 /SP + µ n ( Genome Res. Genome l solution of each compound or vehicle was = 6, = Fig. Pflugers Arch. Pflugers − SupplementaryFig. 2 neurons were used for analysis. Note that and 3 N and Bioinformatics = 4; LTD4:4; = rc Nt. cd Si USA Sci. Acad. Natl. Proc.

n 5 = 30). Neurons30). the belongingto= and ; neurons analyzed Supplementary Fig. 2

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465 nature nature Supplementary Fig. 2 , 2498–2504 (2003). 2498–2504 , ; neurons analyzed , 1671–1685 (2013). 1671–1685 ,

n 25 = 5, = , 1091–1093(2009)., ; neurons; analyzed NEUR N = 4; Me-5HT:4; = n = 30). OSCI N ; neurons different n

= 32). EN ; neu ′ end 107 C E - - - - ,