bioRxiv preprint first posted online Jan. 26, 2017; doi: http://dx.doi.org/10.1101/103408. The copyright holder for this preprint (which was
not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
Heterogeneity of hypothalamic Pro-opiomelanocortin-expressing neurons revealed by single-cell
RNAsequencing
Brian Y. H. Lam1, Irene Cimino1, Joseph Polex-Wolf1, Sara Nicole Kohnke1, Debra Rimmington1,
Valentine Iyemere1, Nicholas Heeley1, Chiara Cossetti2, Reiner Schulte2, Luis R. Saraiva3, Darren W.
Logan3,ClemenceBlouet1,StephenO’Rahilly1,AnthonyP.Coll1&GilesS.H.Yeo*1
1
MRCMetabolicDiseasesUnit,UniversityofCambridgeMetabolicResearchLaboratories,Wellcome
Trust-MRCInstituteofMetabolicScience,Addenbrooke’sHospital,CambridgeCB20QQ,UK
2
FlowCytometryCore,CambridgeInstituteofMedicalResearch,UniversityofCambridge,Cambridge
CB20QQ,UK
3
WellcomeTrustSangerInstitute,WellcomeGenomeCampus,Hinxton,CambridgeCB101SA,UK
*CorrespondingAuthor
Contact:
GilesSHYeo([email protected];@GilesYeo)
UniversityofCambridgeMetabolicResearchLaboratories,
MRCMetabolicDiseasesUnit,
WellcomeTrust-MRCInstituteofMetabolicScience,
Box289,Addenbrooke’sHospital,
CambridgeCB20QQ,UK
AccessionNumbers:GSE92707
bioRxiv preprint first posted online Jan. 26, 2017; doi: http://dx.doi.org/10.1101/103408. The copyright holder for this preprint (which was
not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
Summary
Arcuate proopiomelanocortin (POMC) neurons are critical nodes in the control of body weight.
Often characterised simply as direct targets for leptin, recent data suggest a more complex
architecture.UsingsinglecellRNAsequencing,wehavegeneratedanatlasofgeneexpressionin
murine POMC neurons. Of 163 neurons, 118 expressed high levels of Pomc with little/no Agrp
expressionandwereconsidered“canonical”POMCneurons(P+).Theother45/163expressedlow
levelsofPomcandhighlevelsofAgrp(A+P+).UnbiasedclusteringanalysisofP+neuronsrevealed
fourdifferentclasses,eachwithdistinctcellsurfacereceptorgeneexpressionprofiles.Further,only
12%(14/118)ofP+neuronsexpressedtheleptinreceptor(Lepr)comparedwith58%(26/45)ofA+P+
neurons.Incontrast,theinsulinreceptor(Insr)wasexpressedatsimilarfrequencyonP+andA+P+
neurons (64% and 55%, respectively). These data reveal arcuate POMC neurons to be a highly
heterogeneouspopulation.
Keywords
POMC,melanocortin,AGRP,leptin,insulin,hypothalamus,arcuatenucleus,geneexpression,
neuron,transcriptome
bioRxiv preprint first posted online Jan. 26, 2017; doi: http://dx.doi.org/10.1101/103408. The copyright holder for this preprint (which was
not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
Introduction
Theleptin-melanocortinpathwayplaysakeyroleinthecontroloffoodintakeandbodyweight,with
geneticdisruptionofmultiplecomponentsofthepathwayresultinginsevere,early-onsetobesityin
bothhumansandmice(reviewedby(YeoandHeisler,2012)).
In the brain, POMC expression is localized to neurons in the arcuate nucleus (ARC) of the
hypothalamusandinthenucleusofthesolitarytract(NTS)(Cone,2005).POMCisapro-peptidethat
isprocessedtoarangeofbioactivepeptideproductsincludingαandβ-MSH(Bertagna,1994;Castro
andMorrison,1997),bothofwhichactonmelanocortinreceptors3and4,whicharewidelyexpressed
inthecentralnervoussystem.TheroleofMC4RinmediatingtheeffectsofPOMConenergybalance
is well established, whereas the role of MC3R is less clear (Cone, 2005). In the ARC, anorexigenic
POMC neurons exist alongside orexigenic neurons that express the endogenous melanocortin
antagonistagouti-relatedpeptide(AgRP) (Fanetal.,1997;Ollmannetal.,1997).Thecanonicalview
of the melanocortin pathway is one where leptin, which is produced from white adipose tissue to
reflectfatmass,signalstoleptinreceptorsonthePOMCandAgRPneurons,increasingtheactivityof
POMCneuronsandreducingtheactivityofAgRPexpressingneurons(Cone,2005;YeoandHeisler,
2012).
However,thereisabodyofevidencethatindicatethisviewoftheleptinmelanocortinpathwaytobe
an over-simplistic representation of the relevant circuitry. Woods and Stock, using FOS
immunoreactivityoftheimmediateearlygenec-fosasamarkerofneuronalactivation,reportedthat
whileneuronsinthearcuatenucleusofleptindeficientob/obmicewereactivatedbyperipheralleptin
administration,theareaofthehypothalamusmoststronglyactivatedwastheparaventricularnucleus
(WoodsandStock,1996).Later,itbecameclearthatnotallPOMCneuronsexpressthereceptorsfor
leptinorinsulin,withwhole-cellpatch-clampelectrophysiologyindicatingthatasubpopulationare
depolarizedbyleptin,whileaseparateanddistinctpopulationishyperpolarizedbyinsulin(Williams
et al., 2010). POMC neurons also vary in their expression of the neurotransmitters glutamate, Ƴaminobutyricacid(GABA)(Hentgesetal.,2009;JarvieandHentges,2012),andacetylcholine(Meister
et al., 2006) and their downstream projections. For instance, 40% of POMC neurons appear to be
GABAergicandhenceinhibitory(Hentgesetal.,2009).Inaddition,onlyabout40%ofPOMCneurons
expresstheserotonin2Creceptor(5HT2C-R),thetargetfortheweight-lossdrugLorcaserin,through
whichitisthoughttomediatemuchofitsactionsonsatiety(Burkeetal.,2016;Heisleretal.,2002).
Finally,micewithatargeteddeletionoftheleptinreceptorspecificallyfromPOMCneuronsdisplaya
surprisinglymildphenotype(Balthasaretal.,2004),ascomparedtocompletePomcnullmice(Challis
etal.,2004;Yaswenetal.,1999)anddb/dbmicehomozygousforaLeprmutation(Chenetal.,1996).
A recent paper by Henry et al reported the gene expression profiles of pooled AGRP and POMC
neuronsfromfedandfooddeprivedmice,andfoundthattheglobalpatternofgeneexpressionin
responsetoanacuteenergydeficitarefarmoredramaticinAGRPexpressingthanPOMCexpressing
neurons(Henryetal.,2015).However,becauseofitsuseofpooledcells,thetranscriptomicprofiles
ofindividualneurons,andhencetheirvariabilityingeneexpression,wasnotexamined.
bioRxiv preprint first posted online Jan. 26, 2017; doi: http://dx.doi.org/10.1101/103408. The copyright holder for this preprint (which was
not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
WenowreporttheresultsofsinglecellRNAsequencinginisolatedPOMCexpressingneuronsofthe
arcuatenucleuswhichhasrevealedapreviouslyunappreciateddegreeofheterogeneitywithinthis
anatomicallyhighlylocalisedpopulation.
Results
RNA-sequencingofPOMCneurons
UsingaPOMC-eGFPmouse(Cowleyetal.,2001),wesuccessfullysequencedthetranscriptomeof163
GFP positive (MBH Pos) and 7 GFP negative cells (MBH Neg) from the hypothalamic tissue
encompassingthearcuatenucleus(Fig1A),and24cellsfromthepituitary(Pit)aspositivecontrols
knowntoexpresshighlevelsofPOMC(GEOAcc#GSE92707).Whenwevisualizedthetranscriptomic
data using t-SNE dimension reduction analysis (Maaten and Hinton, 2008), the pituitary and
hypothalamic cells segregated into two discrete clusters, with the hypothalamic neurons further
segregatingintoGFPpositiveandGFPnegativecells(Fig1B).
Allcellssequencedwereofsimilarsize,asmeasuredbydegreeofforwardscatteronthecell-sorter,
andexpressedbothActbandGapdh(Fig1C).Allthehypothalamiccellsexpressedtheneuronalmarker
Ncam,butbecausethepituitarycellscontainedbothneuronalderivedposteriorandtheectodermally
derivedanterior,therewasalargerheterogeneityofNcamexpressioninthepituitarycells(Fig1C).
Incontrast,theGFPsignal,whichreflectsthedegreeofPomcexpression,wasconsistentlyhighwith
verylittleheterogeneityinthepituitaryPOMCcells,whereashypothalamicGFPneuronsshowamuch
larger variation in signal. Cartpt, which is canonically expressed together with Pomc in the same
neurons,alsoshowedvariableexpressioninhypothalamicGFPneuronsandwasnotdetectedinthe
pituitary(Fig1C).POMCisprocessedbytheproproteinconvertases,PC1(PCSK1)andPC2(PCSK2).
Usingthedetectioncut-off≥5linearRPM,mostcells(156/163)expressedeitherorbothPcsk1and
Pcsk2(Fig1D).Onaverage,6882differenttranscriptsweredetectedfromeachindividualneuron(≥5
linearreadspermillionorRPM).However,acrossall163POMCneurons,12,187differenttranscripts
wereexpressed(Fig1E),indicatingsignificantheterogeneityingeneexpressionrepertoireamongthe
neurons.
AgRP/NPYexpressingPOMCneurons
Unexpectedly,wefoundthat27%(45/163)ofPomc-expressingneuronsalsoexpressedhighlevelsof
theendogenousmelanocortinantagonistAgrp(Fig2A).Inaddition,theseneuronsexpressedahigh
levelofNpy,whilePomcexpressionitselfwasamagnitude(>5logFC)lowerthanthatseeninPOMC
neuronsnotexpressingorexpressinglowlevelsofAgrp(Fig2B).Comparingourdatawithapublished
transcriptomeofpooledAgRPneurons(Henryetal.,2015),wefoundasignificantsimilarityingene
expressionprofiles(R2=0.71p<0.00001),withhighexpression,forinstance,oftheghrelinreceptor
Ghsr(Fig2B).AnalysisoftherawdatafromHenryetal2015alsoindicatedthatPomcandCartptwere
expressedintheAgRPneuronsthattheyhadanalysed(Henryetal.,2015),albeitatlowlevels(Fig
2D).
bioRxiv preprint first posted online Jan. 26, 2017; doi: http://dx.doi.org/10.1101/103408. The copyright holder for this preprint (which was
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Takentogether,these45highAgrpandlowPomcexpressingneurons(hereafterreferredtoasA+P+
neurons) showed a striking similarity to canonical AgRP/NPY neurons, likely reflecting their
developmentalorigins,withupto25%ofAgRPneuronssharingthesamehypothalamicprogenitors
as POMC neurons (Padilla et al., 2010). We defined the rest of the 118 neurons, which expressed
higherlevelsofPomc,ascanonicalPOMCexpressingneurons(P+)andconductedourfurtherstudies
onthispopulation.
ExpressionofhypothalamicneuroendocrinegenesinPOMCneurons
Weproducedamatrixshowingtheexpressiondistributionof23wellcharacterisedneuroendocrine
genes,allknowntoplayaroleintheregulationofenergyhomeostasis,ontheindividualPOMC(P+)
neurons (Fig 3A). We found, as has been previously reported, that a large percentage of POMC
neuronsexpressedMc3r,Cnr1,Htr2candtheNpy-y1,y2andy5receptors(YeoandHeisler,2012).Of
note,thepercentageofHtr2cexpressingneurons(34/118;29%)wascomparabletopreviousstudies
showingthat5HT2CRs(theproteinproductofHtr2c)areexpressedonapproximately40%ofPOMC
neurons(Burkeetal.,2016).Wealsofoundthatasmallerpercentageofneurons,12/118,expressed
Mc4r(Fig3B),with11/118expressingGlp1r.ARCPOMCneuronsexpressingGLP1Rarethoughtto
mediate,inpart,theweight-losseffectsoftheGLP1analogueliraglutide(Secheretal.,2014).
Whileweexcludedthe45A+P+neuronsfromthismatrix,alargenumberoftheremainingP+neurons
stillexpressedlowlevelsofAgrp(66/118)andNpy(98/118).Ourowndata(Fig3A)andre-analysisof
therawdatafrompreviouslypublishedworkindicatedthattheconversewasalsolikelytobetrue,
withapercentage‘bonafide’AgRPneuronsalsoexpressinglowlevelsofPomc(Fig2D),
Unbiasedclusteringrevealsfour‘classes’ofPOMCneurons
InordertodeterminewhetherP+neuronssegregateintodistinctclasses,wefirstadjustedforbatch
effects and then performedunbiased clustering of the 118 cells based on the consensus matrix of
Euclidean,PearsonandSpearmancorrelations-usingthe‘SC3’Rpackage(Kiselevetal.,2016).We
obtained 4 main clusters (Fig 4A), which could also be visualized using t-SNE (Maaten and Hinton,
2008)(Fig4B).Cluster1wasthelargestwith69cells,followedbycluster4(27)andcluster2(15).
Cluster3hadtheleastnumberofcells(7).Cluster2showedresemblancetocluster1(Fig4A,B).
Weidentified2773candidategenesthatweredifferentiallyexpressedamongthe4clusters,witha
falsediscoveryrate(FDR)cut-offof5%,thatdrovethesegregationofcellsintothedifferentgroups
(Fig4C).Interestingly,therewasover-representationofmembrane(530)andsecretorygenes(191)
withinthedrivergenelist(Fig4C,D).Thesemembranegenesencodeionchannels,G-proteincoupled
receptors(GPCRs)andothertransmembranereceptors,andtransporters(Fig4D).
Figure5Aliststhetop20genes,rankedbydifferentialexpressionthatdrovetheclusteringofthefour
groups (with the exception of group 2, which was a small group and only has 3 significantly
differentiallyexpressedgenes).Shownaretheexpressionlevelforeachofthegenesandhowunique
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each gene was to each group. Additionally, Table 1 contained genes that are not in the top 20,
encodingselectedextracellularsecretedhormones,GPCRsandionchannels,indicatingwhichgroups
hadthehighestexpressionofanygivengene.Forexample,whiletheneuronsincluster4expressed
thehighestlevelsofPomcandCartpt,bothgeneswere,asexpected,alsoexpressedinClusters1-3.
Cluster4neuronshoweverwerecharacterisedbyexpressionofgenesencodingtransthyretin(Ttr),
thecholesystekininAreceptor(Cckar),neuromedinUreceptor2(Nmur2)andnascenthelixloophelix
2 (Nhlh2) (Fig 5A), as well as the GPCRs corticosterone releasing hormone receptor 1 (Crhr1),
hypocretinreceptor1(Hcrtr1),neuropeptideY2-receptor(Npy2r)andprolactinreleasinghormone
receptor (Prlhr) (Table 1) at far higher levels than in the other clusters. Cluster 1, in contrast, was
characterizedbyhighexpressionoftheserotonin-2areceptor(Htr2a),themelanocortin-3receptor
(Mc3r)andalargenumberofionchannels(Table1).
Thus, no single given ‘driver’ gene is responsible for these clusters, but rather the combined
expressionofarepertoireofgenesdrovethesegregation.Theentirerepertoiresofgenesforeach
clusterarelistedinsupplementarytable1.
Theseclustersare,ofcourse,notfixed.Forexample,Figure5Bshowsthet-SNEplotwiththecluster
4neuronshighlightedanddividingclearlyintotwogroups;onelargergroupof22andasmallerone
of 5 neurons. Comparing the transcriptomes of the two groups, we could identify differentially
expressed genes that defined the two groups (Fig 5C). The four clusters, as we report here, were
achievedbyusingaspecificstatisticalthreshold.Ifweraisedthisthreshold,andthereforestatistical
rigour,thenallclusters,couldbefurthersegregatedand,iftakentoitsextreme,couldsegregateinto
theoriginal163individualneuronssequenced.
LeptinreceptorandinsulinreceptorexpressingPOMCneurons
NextweexaminedthetwomostwellstudiedtypesofPOMCneurons,thoseexpressingtheleptin
(Lepr)andinsulinreceptors(Insr).Lookingatall163neurons,oursequencingdatashowedthat40/163
(25%)neuronsexpressedtheleptinreceptorand94/163(57%)expressedtheinsulinreceptor.11(7%)
expressonlyLeprandnotInsr,65(40%)expressonlyInsrandnotLepr,while29(18%)expressboth
receptors.58(36%)donotexpresseitherofthereceptors(Fig6A).
Whenwesplitthe163cellsintothehighAgrpexpressingA+P+neuronsandhighPomcexpressingP+
neurons,andthenlookedatthedistributionofLeprinthesetwogroupsseparately,wesawthat26/45
(58%)ofA+P+neuronsexpressedLeprascomparedtoonly14/118(12%)ofP+neurons,almosteight
foldmore.Incontrast,theInsrisexpressedin29/45(64%)ofA+P+neurons,whichiscomparableto
itsexpressionin65/118(55%)ofP+neurons.
Todetermineifwehadintroducedanyselectionbiasduringtheflowsorting,wesoughttodefinethe
number Lepr and Insr expressing POMC neurons there were in the intact mouse hypothalamus.
Attemptstouseimmunocytochemistrytodetecteithertheleptinortheinsulinreceptoronwhole
brainslicesprovedtobetechnicallyunreliable(datanotshown).Wethereforemeasuredlevelsof
Stat3phosphorylation(pStat3)afterleptinadministrationandlevelsofAktphosphorylation(pAkt)
afterinsulinadministrationusingimmunohistochemistry,asaproxyforexpressionoftherespective
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receptors.WeperformedtheseexperimentsonPOMC-GFPmice,allowingustoco-localizepStat3or
pAktimmunofluorescenceinneuronsexpressingGFP(Fig6B,C).
Afterleptinadministration,pStat3positivecellswerelargelyconcentratedtothebaseofthearcuate
nucleus surrounding the median eminence (Fig 6B). Cell counting across multiple brain slices from
threemiceindicatedthat23.8%ofPOMC-GFPneuronswerepositiveforpStat3inresponsetoleptin,
whichwasconsistentwithourRNAseqdata(Fig6C).Insulinadministrationhoweverresultedina
different pattern of activation, with pAkt immunofluorescence evident more broadly through the
arcuate nucleus (Fig 6B). We found that 41.1% of POMC-GFP neurons were positive for pAkt in
responsetoinsulin,whichwasnotsignificantlydifferent(p=0.357)tothepercentageofGFPneurons
expressingInsrasdeterminedbyoursequencingdata(Fig2C).Thesefindingsincreasedconfidence
thattheFACSsortinghascapturedarepresentativesnapshotofthePOMCneuronalpopulation.
Discussion
We describe here the use of single cell RNA sequencing to determine the transcriptomes of 163
individual murine POMC neurons. Unexpectedly, 25% of POMC positive neurons also express high
levels of Agrp. The transcriptomes of these neurons are comparable with those of previously
publishedpooledAgRPneurons(Henryetal.,2015),indicatingthatthesearenotanartefactbutlikely
tobearealsubpopulationofcanonicalAgRPneurons.Additionally,whenweexaminetherawdata
fromHenryetal,wefindthatAgRP/NPYGFPneuronsalsoexpresslowlevelsofPOMC(Henryetal.,
2015). This phenomenon likely reflects the shared developmental origins of POMC and AgRP cells
(Padillaetal.,2010).POMChasbeenshowntobebroadlyexpressedinhypothalamicprogenitors,
withupto25%ofthematureAgRP/NPYpopulationsharingacommonprogenitorwithPOMCneurons
(Padillaetal.,2010).TheA+P+populationwedescribeherecouldbecellswherePOMCtranscription
isneverfullysilenced,evenafterthedifferentiationintoAgRP/NPYneurons.Anotherpossibilityisthat
the high Agrp/Npy expressing neurons could be newly committed AgRP/NPY neurons from these
progenitorcells,wherePomcexpressionisyettobefullysuppressed.Regardlessoftheexplanation,
giventhatPomcandAgRPgenetictoolsareinwideuse,itisimportantforthefieldtoknowthata
‘Pomc-specific’deletionofagivengenewouldalsoberemovingthegenefromasignificantproportion
of AgRP neurons. It is possible for example, the mild phenotype seen in the mice where Lepr is
‘specifically’deletedfromPOMCneurons(Balthasaretal.,2004)couldbeinpartduetoLeprdeletion
fromaproportionofAgRPneurons.
The remaining 118 P+ neurons display striking heterogeneity and fall into four clusters. A few
importantpointstokeepinmindwhileinterpretingthesedata.First,thesegroupshaveemergedin
an unbiased fashion after achieving a certain statistical threshold, which means that they are not
invariant. Thus increasing the threshold would invariably result in more clusters. Second, these
groupingsarenotsolelycharacterisedbysmallnumbersofuniquegenes;rathertheyaredrivenbya
‘signature’expressionprofileofawholerepertoireofgenes.Thuswhilethedrivergenes(seeFigure
5)areonaveragethehighestexpressedintheirparticulargroup,manytranscriptsarenotuniqueto
asinglegroupandarenotnecessarilyexpressedinalloftheneuronsinanygivengroup.Third,these
differentclustersarelikelytobedynamic,dependingonthenutritionalstateoftheanimalatthetime
thebrainwasobtained,withthemiceinthisparticularexperimentbeingadlibitumfed.
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ThegroupwiththehighestexpressionofPomcandCartptiscluster4,andbasedonitsexpression
profileofGPCRs,appearstoberesponsivetocorticotrophinreleasinghormone,hypocretin,prolactin,
Npy,CCKandneuromedinU.Thelargestgroupiscluster1,whichbasedonitsrepertoireofGPCR
expression, responds to vasopressin, melanocortin peptides (via the MC3R), serotonin and
somatostatin.Cluster1isnotableinthat, comparedtotheother3groups,itexpressesthehighest
levelsofawidevarietyofplasmamembraneionchannels.Weacknowledgethatfurtherexperiments
will be required to functionally characterize these neurons. However, we speculate that the
enrichmentofdistinctclassesofsurfacereceptorsonthedifferentneuronalclusterspossiblyspeaks
to the temporal nature of the downstream effector function, with signalling through ion channels
typicallybringingaboutrapidchanges,andcirculatinghumouralfactorsprovidinglongertermsignals.
Twodrugsthatareusedforweight-losscurrentlyonthemarketaretheserotoninreceptoragonist
Locaserin(Smithetal.,2010)andtheGLP1analogueLiraglutide(Astrupetal.,2009).Thereisevidence
thatbothmediatetheireffectsonfoodintakeviaARCPOMCneuronsexpressing5HT2CR(Burkeetal.,
2016; Heisler et al., 2002) and GLP1R (Secher et al., 2014), respectively. Our data provides two
additionalpiecesofinformation(Figure3).First,thatlessthan10%(11/118)ofARCPOMCneurons
expresstheGLP1R.Second,ofthePOMCneuronsexpressingeither5HT2CRandGLP1R,only3neurons
expressbothreceptors.ThusitislikelythatdifferentsubgroupsofPOMCneuronsmediatethesatiety
effects of each compound, and poses the question of whether a potential combinatorial therapy,
withoutincreasingindividualdrug-dose,mightpossiblyresultinincreasedweight-loss.
As previously mentioned, the 45 A+P+ neurons are very distinct from the 118 P+ neurons, clearly
segregatingintoaseparategroup.Thisisparticularlytruewhenoneexaminestheexpressionofthe
leptinandinsulinreceptorsinthesegroups.Thelowpercentage(12%)ofcanonicalPOMCneurons
thatexpresstheleptinreceptorisconsistentwiththeactionsofleptinontheseneuronsbeinglargely
indirect(Cowleyetal.,2001;Vongetal.,2011).Althoughwedidnotspecificallyorcomprehensively
evaluateanAgrppositivepopulationofneurons,themuchhigherpercentageofLepRpositivityinthe
cellswherewedocumentedhighlevelsofAgrpexpression(57%)supporttheideathatAgRPneurons
aremorelikelytobedirecttargetsofleptin(Cowleyetal.,2001).Ourdataalsoprovidesomeevidence
ofautocrine/paracrineregulationbyPOMCneuronsthemselves,with10%ofP+neuronsexpressing
Mc4randnearly40%expressingtheMc3r.Onecouldspeculatethattheremaybeamplificationof
the melanocortin system, whereby P+ neurons that respond directly to leptin go on to signal to
Mc3r/Mc4rexpressingneurons,butthisremainstobedetermined.
In contrast, the expression of insulin receptors is comparable between the A+P+ and P+ neurons,
indicatingthatinsulinsactionsonboththesepopulationsofneuronsarelikelytobedirect(Hilletal.,
2010;Konneretal.,2007).
In conclusion, single cell RNAseq has revealed previously unappreciated heterogeneity in POMC
neurons. We have provided a comprehensive atlas, which we hope will be of utility to basic and
translationalresearchersattemptingtobetterunderstandthefundamentalnatureofregulationof
energybalancebythehypothalamusandtomanipulatethesesystemsfortherapeuticbenefit.
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MaterialandMethods
Animals
7-9weekoldmalePomc-eGFPtransgenicmice(Cowleyetal.,2001)(kindgiftfromProf.MalcolmLow)
were housed under a standard 12hr light-dark cycle and were fed ad libitum. All studies were
approved by the local Ethics Committee and conducted according to the UK Home Office Animals
(ScientificProcedures)Act1986.
Isolation of single hypothalamic POMC-eGFP neurons using Fluorescence-activated Cell Sorting
(FACS)
AdlibitumfedPomc-eGFPmiceweresacrificedbycervicaldislocationandtissuefromthemedialbasal
hypothalamus (MBH) region was micro-dissected into Hibernate A medium without calcium and
magnesium (BrainBits, Springfield, IL, USA). The tissue was then digested with Papain (20U/ml,
Worthington,Lakewood,NJ,USA)for30minat37 °Cwithmildagitation,followedbytriturationin
HibernateAmediumcontainingDNaseI(0.005%,Worthington).Thecellsuspensionwasthenfiltered
througha40 μmcellstrainerintoanewfalcontube.
Fluorescence-activating cell sorting was performed using an Influx Cell Sorter (BD Biosciences, San
Jose,CA,USA)utilisingaprotocolthatwaspreviouslyusedforsingle-cellisolation(Macaulayetal.,
2016;Schulteetal.,2015).Thecellgatingwassetaccordingtocellsize(FSC),cellgranularity(SSC),
FSCpulse-widthforsinglets,fluorescenceat488nm/532nmforGFPand647/670nmfornuclearstain
with DraQ5 (Biostatus, Shepshed, Leicester, UK). Cells were sorted direct into a 96 well plate
containinglysisbufferwithRNaseinhibitor.
Whole-transcriptomeamplificationandsingle-cellRNAsequencing
RNA from the single cell lysate was reverse transcribed and PCR amplified (19 cycles) into double
strandedcDNAusingClontechSMART-Seqv4UltraLowInputRNAKit(TakaraClontech,Mountain
View,CA,USA).150pgofamplifiedcDNAwasthenusedtogeneratebarcodedsequencinglibraries
usingIlluminaNexteraXTlibrarypreparationkit(SanDiego,CA,USA)accordingtomanufacturer’s
manual.Samplesfailingtoshowamplificationwereremovedfromtheexperiment.
Thesequencinglibrarieswerenormalizedto10nMconcentrationandcombinedintopoolsof96.The
pooledlibrariesweresequencedon4lanesofanIlluminaHiSeq4000instrumentatsingle-end50bp
(SE50),yieldinganaverageof15.7millionreadspercell.Librarypreparationwasperformedbythe
Genomics and Transcriptomic Core at the Institute of Metabolic Science. The sequencing was
performedattheGenomicsCore,CancerResearchUKCambridgeInstitute.Alltherawdatainthis
manuscripthasbeensubmittedtoGEO,accessionnumberGSE92707.
LeptinandInsulinadministrations
ForpSTAT3immuno-labelling,micewerefastedovernightandleptin(3mg/kg,PeproTech,RockyHill,
NJ,USA)orsalinewasinjectedintraperitoneally.45minutesafterleptinadministration,theanimals
werethensacrificedbyalethaloverdoseofpentobarbital(60mg/kg)thenperfusedtranscardiallywith
PBSfollowedby4%paraformaldehyde.ForpAKTimmunofluorescenceovernightfastedanimalswere
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administeredwithActrapid(2mU/kg,NovoNordisk,Bagsværd,Denmark)intravenously.Animalswere
sacrificed15minutesafteradministrationandperfusedasdescribedbefore.
ImmunohistochemistryforPOMC-eGFP,pStat3andpAkt
Brainswerecollectedimmediatelyafterperfusionsandfixedfor2furtherhoursina15%sucrose-4%
paraformaldehydesolution.Thebrainswerecryo-preservedin30%sucrosePBSovernightandthen
embedded in OCT (Bright, Huntingdon, UK) and stored at -80°C until further processing. Frozen
coronal sections were sliced at 30 µm and processed for immunofluoresence. For pSTAT3
immunostaining,sectionswerepre-treatedfor20minin1%NaOHandthenplacedovernightwitha
rabbitanti-pSTAT3antibody(1:200,Tyr-705CellSignalling).ForpAKTimmunostaining,sectionswere
incubatedwithanti-pAKTantibody(1:100,Ser-473CellSignalling)overnight.Theprimaryantibodies
were localized with Dylight 594 Goat anti-Rabbit IgGs (Vector; 1:500). Coronal images of Arcuate
nucleus were taken using a Laser-scanning confocal microscope (LSM 510, Zeiss, Oberkochen,
Germany) and eGFP positive cells and double labelled GFP-pSTAT3- or GFP-pAKT immune-reactive
cellswerecountedusingImageJ.
ImmunohistochemistryforMc4r-MAPT,andPomc
B6.Cg-Tg(Mc4r-MAPT/Sapphire)21Rck/Jmalemicewereperfusedtranscardiallyviaa23gaugeneedle
placedintheleftventriclewith100mlof0.1MheparinizedPBS,pH7.4,followedby100mlof4%
paraformaldehyde in PBS, and the fixed brains were cryoprotected in 30% sucrose for 48h at 4C.
Coronal hypothalamic sections of 30 μm thickness were prepared on a freezing microtome. Free
floatingsectionswereincubatedfor15minin1%hydrogenperoxide,washed2timesinPBS,blocked
2hin0.3%TritonX-100and5%NGSinPBS,beforebeingincubatedovernightat4°Cinrabbitantiproopiomelanocortinprecursor(1:200,PhoenixPharmaceuticals)in0.3%TritonX-100and5%NGS.
Sectionswerethenwashedandincubatedfor2hwithAlexaFluor594goatanti-rabbitIgG.Sections
were washed and incubated overnight at 4° C in chicken anti-GFP (1:500, Abcam). Sections were
washed and incubated in Alex Fluor 488 goat anti-chicken IgG for 2h. Sections were floated onto
superfrostPlusmicroscopeslides(Fisher),andcoverslippedwithVectashield(Vector).Tissuesections
were digitized using a Zeiss Axioplan fluorescent microscope, and areas of interest were outlined
basedoncellularmorphology.
Bioinformatics
ThequalityofsequencereadswasexaminedusingFastQCandMultipleGenomealignment(MGA)
was used to rule out contamination from other DNA sources throughout the wet experimental
procedures.Readswerealignedtothemousegenome(GRCm38,Ensemblv74)usingTophatversion
2.0.11(Kimetal.,2013)andtherawcountsatthegenelevelweredeterminedusingCufflinksversion
2.2.1(Trapnelletal.,2010).TheaboveanalyseswereperformedattheHigh-performancecomputing
clusterattheResearchComputingService,UniversityofCambridge.
The raw counts from cells were normalised and batch correction was performed using edgeR
(Robinsonetal.,2010)andlimma(Ritchieetal.,2015)packages.Rtsnepackagewasusedtogenerate
t-SNEplots(MaatenandHinton,2008)andSC3packagewasusedtoperformtheclusteringanalysis
(Kiselevetal.,2016).GeneSpringGXsoftware(Agilent)wasusedtogeneratetheheatmaps.Gene
abundanceisrepresentedinlogRPM,whichstandsforthebase-2logarithmofReadsperMillion.The
bioRxiv preprint first posted online Jan. 26, 2017; doi: http://dx.doi.org/10.1101/103408. The copyright holder for this preprint (which was
not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
plotsweregeneratedusingggplot2package,MicrosoftExcelandGeneSpringGX.Geneannotations
andpathwayanalysiswereperformusingIngenuityPathwayAnalysis(Qiagen,RedwoodCity,CA,USA)
Acknowledgements
This work was supported by the UK Medical Research Council (MRC) Metabolic Disease Unit
(MRC_MC_UU_12012/1 & MRC_MC_UU_12012/5), a Wellcome Trust Strategic Award
(100574/Z/12/Z)andtheHelmholtzAllianceICEMED.
FigureLegends
Figure1FACSsortingandsingle-cellRNAsequencingofPOMC-eGFPneurons.
(A)TheFACSgatingforGFP(530nm)andreferencechannel(460nm).TheGFPpositivecellsfromthe
mediobasalhypothalamus(MBHPos)arelabelledblueandnegativecells(MBHNeg)arecollected
from the orange square within the negative population (purple). The cells were further gated for
nuclearstain(DraQ5),cellsize(FSC)andgranularity(SSC),datanotshown.(B)t-SNEwasusedtoshow
thesegregationofcells(MBHPosandNeg,aswellasGFPpositivecellscollectedfromthepituitary)
basedontheirtranscriptomicprofiles.(C)QualitychecksforFACSshowingtheGFPintensityandcell
sizemeasurementamongstthe3celltypes.WealsoexaminedforRNAexpressionofhousekeeping
genesActb,Gapdh,neuronalmarkersMap2andNcam1,andalsoexpressionofPomcandCartpt.(D)
Pcsk1andPcsk2arewidelyexpressedinthecellssequenced.(E)Wedetectedanaverageof6882
genespercell(≥5RPM),addingallthegenesfromallthe163cellstogetherresultedinatotalof12187
detectedgenes,indicatingheterogeneityingeneexpression.
Figure227.6%ofPOMCcellsalsoexpresshighlevelsofAgrpandNpy
(A)Wedetected45cellsthatexpressedhighlevelsofAgrpandNpy(A+P+),interestinglymanyofthese
cellsalsoexpressLepr.(B)DifferentialexpressionanalysisoftheA+P+vsrest(P+)cellsshowssimilar
foldenrichmentscomparedtoAgRPvsPOMCcomparisonfrom(Henryetal.,2015).Theexpression
ofthetop200enrichedgenes(fromthepublisheddata)areplottedinthegraph.(C)TheAgrp/Npy
cellsformastrongclusterinat-SNEplotshowingonlythe163MBHPoscells.(D)Expressionlevelsof
Pomc,Cartpt,Agrp,andNpyinpublishedAgRPandPOMCneurons.TheAgRPneuronsexpresslow
butdetectablelevelsofPomcandCartpt,ontheotherhandNpyandAgrparealsodetectableinPOMC
neurons.
Figure3Theexpressionmatrixofselectedneuroendocrinegenes
(A) An expression matrix showing the number of cells expressing ≥2.32 log RPM of a selection of
neuroendocrinegenes,theAgrp/Npycellswereexcludedfromthematrix.Onecanlookuptocoexpressionofupto2genesbylookingattheintersectionsbetweenrowsandcolumns,e.g.thereare
43 Mc3r expressing cells (9th row) and 25 of them also expressed Insr (7th column). The average
abundanceofgeneexpressionisalsoshowninbluebars(averageofallcellsinthecohort)andgreen
(expressingcellsonly)(B)Wefound12cellsexpressingMc4rwithinourcohortandthiswasconfirmed
byimmunostainingforPOMCthearcuatenucleusofMC4R-GFPmice.
bioRxiv preprint first posted online Jan. 26, 2017; doi: http://dx.doi.org/10.1101/103408. The copyright holder for this preprint (which was
not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
Figure4Clusteringanalysisandidentificationofdrivergenesforheterogeneity
(A)Clusteringanalysiswasperformedusingthe‘SC3’packageinordertoidentifyclustersofcellsthat
showsimilarityintheirtranscriptomicprofiles.Usingk-meansclustering,wefound4clusterswithan
averagesilhouettewidthof0.64.(B)At-SNEplotshowingthe4clustersfromtheanalysis.(C)Using
theclusteringresultsweidentified2773candidategenesthatdroveheterogeneity.(D)Thereare
over-representationsofgenesfromtheplasmamembrane(1.50-fold)andextracellularspace(1.28fold) within the list of driver gene candidates. This enrichment is mainly driven by overrepresentations of plasma membrane protein encoding genes including ion channels, G-protein
coupled receptors, transporters and other transmembrane receptors. There was also a 1.8-fold
enrichmentofgrowthfactorsinthedrivergenelist.
Figure5Topcandidatedrivergenesfortheresultingclusters
(A)Thetop25genes‘markers’wererankedbymaximumfolddifferenceandadifferentialexpression
FDRcutoffof0.05,andthelistsforeachoftheclustersareshown.Thenumberofcellsexpressing
thegeneisalsoincludedintherightmostcolumn(green).PomcandCartptareexpressedinall4
clustersbuttheexpressionofthetwogenesarehighestinCluster4cells(>1logFCfromcluster2
whichissecondhighest).(B)Theclusterscanbefurthersubdivided,e.g.cluster4showedhere.(C)
Topdifferentialexpressedgenesbetweenthetwosubgroupsofcluster4cells.Alargerversionofthe
heatmapisavailableinthesupplementarymaterial.
Figure6LeprandInsrexpressioninhypothalamicPOMCneurons
(A)Wedetected30Leprand83Insrexpressingcellsoutofthe163cellsinoursequencingcohort.(B)
The percentages were compared to the number of cells that exhibited pStat3 and pAkt
immunoreactivitypost-administrationofleptinandinsulin(respectively).(C)Representativeimages
showingPOMC-eGFP,immunoreactivityofpStat3(upperpanel)andpAkt(lowerpanel).Cellsthat
were double-stained were marked with white arrows. (D) The t-SNE plot shows a lack of clear
clusteringforLeprandInsrexpressingcells,indicatingtheexpressionsofthe2receptorsalonewere
notsufficienttodrivesegregationatthewholetranscriptomelevel.
References
Astrup,A.,Rossner,S.,VanGaal,L.,Rissanen,A.,Niskanen,L.,AlHakim,M.,Madsen,J.,Rasmussen,
M.F., Lean, M.E., and Group, N.N.S. (2009). Effects of liraglutide in the treatment of obesity: a
randomised,double-blind,placebo-controlledstudy.Lancet374,1606-1616.
Balthasar,N.,Coppari,R.,McMinn,J.,Liu,S.M.,Lee,C.E.,Tang,V.,Kenny,C.D.,McGovern,R.A.,Chua,
S.C.,Jr.,Elmquist,J.K.,etal.(2004).LeptinreceptorsignalinginPOMCneuronsisrequiredfornormal
bodyweighthomeostasis.Neuron42,983-991.
Bertagna,X.(1994).Proopiomelanocortin-derivedpeptides.EndocrinolMetabClinNorthAm23,467485.
Burke,L.K.,Doslikova,B.,D'Agostino,G.,Greenwald-Yarnell,M.,Georgescu,T.,Chianese,R.,Martinez
deMorentin,P.B.,Ogunnowo-Bada,E.,Cansell,C.,Valencia-Torres,L.,etal.(2016).Sexdifference
bioRxiv preprint first posted online Jan. 26, 2017; doi: http://dx.doi.org/10.1101/103408. The copyright holder for this preprint (which was
not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
in physical activity, energy expenditure and obesity driven by a subpopulation of hypothalamic
POMCneurons.MolMetab5,245-252.
Castro, M.G., and Morrison, E. (1997). Post-translational processing of proopiomelanocortin in the
pituitaryandinthebrain.CritRevNeurobiol11,35-57.
Challis, B.G., Coll, A.P., Yeo, G.S., Pinnock, S.B., Dickson, S.L., Thresher, R.R., Dixon, J., Zahn, D.,
Rochford,J.J.,White,A.,etal.(2004).Micelackingpro-opiomelanocortinaresensitivetohigh-fat
feedingbutrespondnormallytotheacuteanorecticeffectsofpeptide-YY(3-36).ProcNatlAcadSci
USA101,4695-4700.
Chen,H.,Charlat,O.,Tartaglia,L.A.,Woolf,E.A.,Weng,X.,Ellis,S.J.,Lakey,N.D.,Culpepper,J.,Moore,
K.J., Breitbart, R.E., et al. (1996). Evidence that the diabetes gene encodes the leptin receptor:
identificationofamutationintheleptinreceptorgeneindb/dbmice.Cell84,491-495.
Cone,R.D.(2005).Anatomyandregulationofthecentralmelanocortinsystem.NatNeurosci8,571578.
Cowley,M.A.,Smart,J.L.,Rubinstein,M.,Cerdan,M.G.,Diano,S.,Horvath,T.L.,Cone,R.D.,andLow,
M.J.(2001).LeptinactivatesanorexigenicPOMCneuronsthroughaneuralnetworkinthearcuate
nucleus.Nature411,480-484.
Fan,W.,Boston,B.A.,Kesterson,R.A.,Hruby,V.J.,andCone,R.D.(1997).Roleofmelanocortinergic
neuronsinfeedingandtheagoutiobesitysyndrome.Nature385,165-168.
Heisler, L.K., Cowley, M.A., Tecott, L.H., Fan, W., Low, M.J., Smart, J.L., Rubinstein, M., Tatro, J.B.,
Marcus, J.N., Holstege, H., et al. (2002). Activation of central melanocortin pathways by
fenfluramine.Science297,609-611.
Henry, F.E., Sugino, K., Tozer, A., Branco, T., and Sternson, S.M. (2015). Cell type-specific
transcriptomicsofhypothalamicenergy-sensingneuronresponsestoweight-loss.Elife4.
Hentges,S.T.,Otero-Corchon,V.,Pennock,R.L.,King,C.M.,andLow,M.J.(2009).Proopiomelanocortin
expressioninbothGABAandglutamateneurons.JNeurosci29,13684-13690.
Hill,J.W.,Elias,C.F.,Fukuda,M.,Williams,K.W.,Berglund,E.D.,Holland,W.L.,Cho,Y.R.,Chuang,J.C.,
Xu,Y.,Choi,M.,etal.(2010).Directinsulinandleptinactiononpro-opiomelanocortinneuronsis
requiredfornormalglucosehomeostasisandfertility.CellMetab11,286-297.
Jarvie,B.C.,andHentges,S.T.(2012).ExpressionofGABAergicandglutamatergicphenotypicmarkers
inhypothalamicproopiomelanocortinneurons.JCompNeurol520,3863-3876.
Kim,D.,Pertea,G.,Trapnell,C.,Pimentel,H.,Kelley,R.,andSalzberg,S.L.(2013).TopHat2:accurate
alignmentoftranscriptomesinthepresenceofinsertions,deletionsandgenefusions.GenomeBiol
14,R36.
Kiselev,V.Y.,Kirschner,K.,Schaub,M.T.,Andrews,T.,Chandra,T.,Natarajan,K.N.,Reik,W.,Barahona,
M.,Green,A.R.,andHemberg,M.(2016).SC3 -consensusclusteringofsingle-cellRNA-Seqdata.
bioRxiv.
Konner,A.C.,Janoschek,R.,Plum,L.,Jordan,S.D.,Rother,E.,Ma,X.,Xu,C.,Enriori,P.,Hampel,B.,
Barsh,G.S.,etal.(2007).InsulinactioninAgRP-expressingneuronsisrequiredforsuppressionof
hepaticglucoseproduction.CellMetab5,438-449.
Maaten, L.v.d., and Hinton, G. (2008). Visualizing data using t-SNE. Journal of Machine Learning
Research9,2579-2605.
Macaulay,I.C.,Svensson,V.,Labalette,C.,Ferreira,L.,Hamey,F.,Voet,T.,Teichmann,S.A.,andCvejic,
A. (2016). Single-Cell RNA-Sequencing Reveals a Continuous Spectrum of Differentiation in
HematopoieticCells.CellRep14,966-977.
Meister, B., Gomuc, B., Suarez, E., Ishii, Y., Durr, K., and Gillberg, L. (2006). Hypothalamic
proopiomelanocortin(POMC)neuronshaveacholinergicphenotype.EurJNeurosci24,2731-2740.
Ollmann, M.M., Wilson, B.D., Yang, Y.K., Kerns, J.A., Chen, Y., Gantz, I., and Barsh, G.S. (1997).
Antagonismofcentralmelanocortinreceptorsinvitroandinvivobyagouti-relatedprotein.Science
278,135-138.
Padilla, S.L., Carmody, J.S., and Zeltser, L.M. (2010). Pomc-expressing progenitors give rise to
antagonisticneuronalpopulationsinhypothalamicfeedingcircuits.NatMed16,403-405.
bioRxiv preprint first posted online Jan. 26, 2017; doi: http://dx.doi.org/10.1101/103408. The copyright holder for this preprint (which was
not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
Ritchie,M.E.,Phipson,B.,Wu,D.,Hu,Y.,Law,C.W.,Shi,W.,andSmyth,G.K.(2015).limmapowers
differentialexpressionanalysesforRNA-sequencingandmicroarraystudies.NucleicAcidsRes43,
e47.
Robinson, M.D., McCarthy, D.J., and Smyth, G.K. (2010). edgeR: a Bioconductor package for
differentialexpressionanalysisofdigitalgeneexpressiondata.Bioinformatics26,139-140.
Schulte,R.,Wilson,N.K.,Prick,J.C.,Cossetti,C.,Maj,M.K.,Gottgens,B.,andKent,D.G.(2015).Index
sortingresolvesheterogeneousmurinehematopoieticstemcellpopulations.ExpHematol43,803811.
Secher,A.,Jelsing,J.,Baquero,A.F.,Hecksher-Sorensen,J.,Cowley,M.A.,Dalboge,L.S.,Hansen,G.,
Grove,K.L.,Pyke,C.,Raun,K.,etal.(2014).ThearcuatenucleusmediatesGLP-1receptoragonist
liraglutide-dependentweightloss.JClinInvest124,4473-4488.
Smith,S.R.,Weissman,N.J.,Anderson,C.M.,Sanchez,M.,Chuang,E.,Stubbe,S.,Bays,H.,Shanahan,
W.R., Behavioral, M., Lorcaserin for, O., et al. (2010). Multicenter, placebo-controlled trial of
lorcaserinforweightmanagement.NEnglJMed363,245-256.
Trapnell,C.,Williams,B.A.,Pertea,G.,Mortazavi,A.,Kwan,G.,vanBaren,M.J.,Salzberg,S.L.,Wold,
B.J.,andPachter,L.(2010).TranscriptassemblyandquantificationbyRNA-Seqrevealsunannotated
transcriptsandisoformswitchingduringcelldifferentiation.NatBiotechnol28,511-515.
Vong,L.,Ye,C.,Yang,Z.,Choi,B.,Chua,S.,Jr.,andLowell,B.B.(2011).LeptinactiononGABAergic
neuronspreventsobesityandreducesinhibitorytonetoPOMCneurons.Neuron71,142-154.
Williams,K.W.,Margatho,L.O.,Lee,C.E.,Choi,M.,Lee,S.,Scott,M.M.,Elias,C.F.,andElmquist,J.K.
(2010). Segregation of acute leptin and insulin effects in distinct populations of arcuate
proopiomelanocortinneurons.JNeurosci30,2472-2479.
Woods,A.J.,andStock,M.J.(1996).Leptinactivationinhypothalamus.Nature381,745.
Yaswen,L.,Diehl,N.,Brennan,M.B.,andHochgeschwender,U.(1999).Obesityinthemousemodel
ofpro-opiomelanocortindeficiencyrespondstoperipheralmelanocortin.NatMed5,1066-1070.
Yeo,G.S.,andHeisler,L.K.(2012).Unravelingthebrainregulationofappetite:lessonsfromgenetics.
NatNeurosci15,1343-1349.
B
10
10000
100
t−SNE Dimension 2
−5
0
5
1000
MBH Neg
10
MBH Neg
MBH Pos
Pit
MBH Pos
0
10
100
1000
GFP Intensity 530nm (FU)
−10
Reference Channel 460nm (FU)
A
bioRxiv preprint first posted online Jan. 26, 2017; doi: http://dx.doi.org/10.1101/103408. The copyright holder for this preprint (which was
not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
10000
−15
−10
−5
0
5
t−SNE Dimension 1
10
15
D
C
Pcsk1
Pcsk2
Ncam1
Map2
FSC (Cell Size)
Log RPM
LogFU
Log RPM
GFP
Gapdh
MBH MBH Pit
Neg Pos
MBH MBH Pit
Neg Pos
5
10
E
Pomc
Cartpt
+
Log RPM
Log RPM
Actb
0
MBH MBH Pit
Neg Pos
MBH MBH Pit
Neg Pos
...
Average detected genes per cell : 6882
= 12187
genes
bioRxiv preprint first posted online Jan. 26, 2017; doi: http://dx.doi.org/10.1101/103408. The copyright holder for this preprint (which was
not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
A
Lepr
Insr
Pomc
Cartpt
Npy
Agrp
Agrp/Npy Expressing Cells
Log RPM
0
5
10
Agrp
5
Fold-enrichment Agrp/Npy cells vs rest
10
B
Npy
Ghsr
0
5
5
10
5
10 Cartpt
Pomc
2
R =0.71
10
p < 0.00001
Published fold-enrichment AgRP vs POMC Neurons
6
C
−6
−4
t−SNE Dimension 2
−2
0
2
4
Agrp/Npy cells
−6
−4
−2
0
2
t−SNE Dimension 1
4
D
Pomc
Cartpt
Normalised Abundance (Log RPM)
15
10
5
0
AgRP
POMC
AgRP
Agrp
POMC
Npy
15
10
5
0
AgRP
POMC
AgRP
POMC
6
bioRxiv preprint first posted online Jan. 26, 2017; doi: http://dx.doi.org/10.1101/103408. The copyright holder for this preprint (which was
not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
bioRxiv preprint first posted online Jan. 26, 2017; doi: http://dx.doi.org/10.1101/103408. The copyright holder for this preprint (which was
not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
A
B
t−SNE Dimension 2
−2
0
2
4
6
1
0.5
0
−4
Agrp/Npy Cells
Cluster 1
Cluster 2
−6
Cluster 3
Cluster
C
1
2
3
4
Cluster 4
−6
−4
D
Compartment
−2
0
2
t−SNE Dimension 1
4
Fold over-representation
1
Gene Type
1
p-value
bioRxiv preprint first posted online Jan. 26, 2017; doi: http://dx.doi.org/10.1101/103408. The copyright holder for this preprint (which was
not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
A
15
5
0
log RPM
100%
50%
0%
% cells
C
−6
−4
t−SNE Dimension 2
−2
0
2
4
6
B
−6
−4
−2
0
2
t−SNE Dimension 1
4
log RPM
0
5
10
bioRxiv preprint first posted online Jan. 26, 2017; doi: http://dx.doi.org/10.1101/103408. The copyright holder for this preprint (which was
not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.