Investigation of a non-invasive method of assessing the equine circadian clock using hair follicle cells

Watts, Browne, Murphy

Description

A comprehensive understanding of the equine circadian clock involves the evaluation of circadian clock gene expression. A non-invasive and effective method for detecting equine clock gene expression has yet to be established. Currently, research surrounding this area has relied on collecting tissue biopsies or blood samples that can often be costly, time consuming and uncomfortable for the animal. Methods Five mares were individually stabled under a light–dark (LD) cycle that mimicked the external environmental photoperiod during a time of year corresponding with the vernal equinox. Hair follicles were collected every 4 h over a 24-h period by plucking hairs from the mane. RNA was extracted and quantitative (q) PCR assays were performed to determine temporal expression patterns for the core clock genes; ARNTL, CRY1, PER1, PER2, NR1D2 and the clock controlled gene, DBP . Results Repeated measures ANOVA for the clock gene transcripts PER1 and PER2 and the clock controlled gene, DBP , revealed significant variation in expression over time ( p < .05 , respectively). Cosinor analysis confirmed a significant 24-h temporal component for PER1 ( p = .002 ) and DBP (p = .0033) and also detected rhythmicity for NR1D2 (p = .0331) . Conclusions We show that the extraction of RNA from equine hair follicle cells can identify the circadian 24 h oscillations of specific clock genes and a clock-controlled gene and therefore provide a valuable non-invasive method for evaluating the equine peripheral circadian clock. This method will serve as a useful tool for future evaluations of equine circadian rhythms and their response to environmental changes.

Sujets

• Equus (genus)
• Horse
• CLOCK
• PCR quantitative
• Hair follicle
• PER1
• PER2
• DBP
• Rev-ErbA alpha
• Circadian rhythm

Informations

• Publié par: biomed
• Publié le: 01 janvier 2012
• Nombre de lectures: 8
• Langue: English

Extrait

Watts
etal.JournalofCircadianRhythms
2012,
10
:7
http://www.jcircadianrhythms.com/content/10/1/7

RESEARCH

OpenAccess

Investigationofanon-invasivemethodof
assessingtheequinecircadianclockusinghair
folliclecells
LisaMWatts,JohnABrowneandBarbaraAMurphy
*

Abstract
Background:
Acomprehensiveunderstandingoftheequinecircadianclockinvolvestheevaluationofcircadian
clockgeneexpression.Anon-invasiveandeffectivemethodfordetectingequineclockgeneexpressionhasyetto
beestablished.Currently,researchsurroundingthisareahasreliedoncollectingtissuebiopsiesorbloodsamples
thatcanoftenbecostly,timeconsuminganduncomfortablefortheanimal.
Methods:
Fivemareswereindividuallystabledunderalight
–
dark(LD)cyclethatmimickedtheexternal
environmentalphotoperiodduringatimeofyearcorrespondingwiththevernalequinox.Hairfollicleswere
collectedevery4hovera24-hperiodbypluckinghairsfromthemane.RNAwasextractedandquantitative(q)
PCRassayswereperformedtodeterminetemporalexpressionpatternsforthecoreclockgenes;
ARNTL,CRY1,PER1,
PER2,NR1D2
andtheclockcontrolledgene,
DBP
.
Results:
RepeatedmeasuresANOVAfortheclockgenetranscripts
PER1
and
PER2
andtheclockcontrolledgene,
DBP
,revealedsignificantvariationinexpressionovertime(
p<.05
,respectively).Cosinoranalysisconfirmeda
significant24-htemporalcomponentfor
PER1
(
p=.002
)and
DBP(p=.0033)
andalsodetectedrhythmicityfor
NR1D2(p=.0331)
.
Conclusions:
WeshowthattheextractionofRNAfromequinehairfolliclecellscanidentifythecircadian24h
oscillationsofspecificclockgenesandaclock-controlledgeneandthereforeprovideavaluablenon-invasive
methodforevaluatingtheequineperipheralcircadianclock.Thismethodwillserveasausefultoolforfuture
evaluationsofequinecircadianrhythmsandtheirresponsetoenvironmentalchanges.
Keywords:
Equine,Horse,Clock,qPCR,Hairfollicle,PER1,PER2,DBP,NR1D1,Circadian

Background
feedbackloops[3]thatultimatelygiveriseto24-haltera-
Thecircadiansystemsuppliesorganismswithameanstotionsingeneexpressionandbehaviouraloutputs.
adapttheirinternalphysiologytothecontinuouslychan-Circadianclockgeneexpressionisdetectablenotonly
gingenvironmentalstimulithatexistonarotatingplanetintheSCNbutinalmostallperipheraltissues.Themo-
[1].Thecentralpacemakerislocatedinthesuprachias-lecularclockworkmechanismcomprisesaseriesoffeed-
maticnucleus(SCN)ofthehypothalamusandcoordi-backloopsthatencompassasetofcoreclockgenes:
nates,vianeuralandhumoralsignals,multipleperipheral
ARNTL
(arylhydrocarbonreceptornucleartranslocator-
clockssituatedintissuesthroughouttheanimal[2].Theselike),
CLOCK
(circadianlocomotoroutputcontrol
peripheralclocksconsistofagroupofhighlyconservedkaput),
PER1
(periodhomolog1),
PER2
(periodhomolog
‘
clock
’
genesandtheirproteinproductsfunctioningwithin2),
PER3
(periodhomolog3),
CRY1
[cryptochrome1
tightlycontrolledautoregulatorytranscription-translation(photolyase-like)],and
CRY2
[cryptochrome2(photo-
lyase-like)][4].
Thepositiveaxisoftheloopiscreatedbytranscription
*Correspondence:[e-mail]
factors
CLOCK
and
ARNTL
astheyundergotranscrip-
SchoolofAgricultureandFoodScience,UniversityCollegeDublin,Belfield,
tionandtranslation[3].
CLOCK
and
ARNTL
proteins
Dublin4,Ireland

©2012Wattsetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative
CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginalworkisproperlycited.

Watts
etal.JournalofCircadianRhythms
2012,
10
:7
http://www.jcircadianrhythms.com/content/10/1/7

heterodimerizeandbindtoE-boxenhancersupstream
of
PER
and
CRY
genesinordertotriggertranscription
[5].Followingthis,acomplexisformedby
PER
and
CRY
proteinsthatrelocatestothenucleustoinhibit
CLOCK/ARNTL
activity.Thisleadstotherepressionof
theirowntranscriptioncompletingthenegativeaxisof
thefeedbackloop[2].
RORA
,(RAR-relatedorphanre-
ceptorA)
NR1D1
(nuclearreceptorsubfamily1,group
D,member1),and
NR1D2
(nuclearreceptorsubfamily
1,groupD,member2)areorphannuclearreceptorsthat
makeupasecondaryfeedbackloop.
RORA
instigates
ARNTL
transcriptionwhilst
NR1D1
and
NR1D2
repress
itsexpression[6].Eachcycleofthemolecularclock
withinatissuegivesrisetothesimultaneousupregula-
tionofasubsetofclock-controlledgenes[7]activated
bythetranscriptionalactivityoftheARNTL/CLOCK
heterodimer.
Markersofcircadianphaseinhumansincludemela-
tonin[8]andbodytemperature[9];howevermelatonin
hasbeenestablishedasnotcircadianinthehorse[10].
Previousstudiesinhumanshaveusedwhitebloodcells
ororalmucosaasamethodofdetectinghumanclock
geneexpression[11,12].Thesemethodshaveseveral
reporteddrawbacks[11,13].Physicalstimuliandtime
delaysduetotheprocessingofcellseparationmayaffect
levelsofexpressionofclockgenesandtheoverallquality
oftheisolatedmRNA.Forinstancewithwhiteblood
cells,theissuerelatestotimedelaysduetotheproces-
singofcellspriortotranscriptionalinactivationwhichin
turnmayaffectthelevelsofmRNAofclockgenes.A
similarconcerncanbeseenwiththecollectionoforal
mucosacells.RNAsampleswereshowntobeseverely
fragmentedandthustheresultswerediscarded[12].
Thereareadditionalimpracticalitiesincollectingoral
mucosalcellsfromhorses.Thesecouldbeavoided
throughthecollectionofhairfolliclesasanalternative
samplingmethod[13].Themainadvantagesofusing
hairfolliclecellsarethattheycanbeobtainednoninva-
sivelyandcellscanbecollectedandtheRNAstabilised
simplybypluckinghairsandaddingthemdirectlytoan
RNAstabilisationbuffer.
Anon-invasiveandeffectivemethodfordetectingper-
ipheralequineclockgeneexpressionhasyettobeestab-
lished.Thishindersprogressinareasofequinecircadian
research.Currently,researchinvestigatingequineper-
ipheralclockshasreliedoncollectingmusclebiopsiesor
bloodsamples.Thesemethodscanoftenbetimecon-
suming,costlyandcanoccasionallyleadtoanimalwel-
fareconcernsasthisapproachtocollectingsamplescan
beinvasive.
Inthisreportweexamineaconvenientandnoninva-
sivemethodfordetectingequineclockgeneexpression
throughtheuseofhairfolliclecellscollectedfromthe
maneofthehorse.

Page2of6

Materialsandmethods
Samplecollection
Fivehealthy,non-pregnantmares(
Equuscaballus
)of
variouslightweightbreedswereindividuallyhousedin
standard12ftx12ftstallsfor24-hunderalight
–
dark(LD)cyclethatmimickedtheenvironmental
photoperiodforthattimeofyear.Mareswerechosen
fortheiravailabilityonUCD
’
sLyonsResearchFarm.
Stallionswerenotavailableforthestudyand
castratedmales(geldings)areconsideredunsuitableas
theirneuroendocrinesystemiscompromised.Theex-
perimentwasconductedinMarchwherethetimesof
dawnandduskwere06:00and18:00respectively,
correspondingtoa12hLight:12hDarkLDcycle
atlongitude138W6.8,latitudeN53.2(CountyKildare,
Ireland).Whilestabled,horseshadaccesstohayand
water
adlibitum.
Wecollectedmanehairsamplesat
4-hintervalsfrom16:00[ZeitgeberTime(ZT)9,
wheretimeoflightsondefinesZT0]foraperiodof
4h2.Eachhairsampleconsistedof10
–
20hairfolliclesthat
weretrimmedtoremoveexcesshairandcarefullyplaced
ina2mLscrew-captubecontaining400ulofbinding
bufferfromtheHighPureRNAIsolationKit(Roche,
Indianapolis,Indiana).
Quantitativepolymerasechainreaction(qPCR)
TotalRNAwasisolatedusingtheHighPureRNA
IsolationKit(Roche,Indianapolis,INdiana)according
tothemanufacturer'sinstructionswithminormodifi-
cation:Thehairfollicleswereplacedina2.0mL
screwcaptube,containing400uLofbindingbuffer
fromtheHighPureRNAIsolationKit(Roche)and
200uLofPBS.Asingle5mmstainlesssteelbead
(Qiagen)wasaddedtoeachtubeandthesamples
werehomogenisedatmaximumspeed(30Hertz)for
2minusingtheQiagenTissueLysersystem.Following
homogenisationthesampleswerespunfor1minat
maximumspeedtoreducefoaming,thehomogenate
wasthenappliedtothefiltercolumnandRNAwas
extractedasperinstructionsfortheHighPureIsola-
tionKit.RNAwaselutedin50uLandstoredat-80
gC.deRNAquantitywasmeasuredusingtheNanoDrop
ND1000spectrophotometerV3.5.2(NanoDropTech-
nologies,Wilmington,DE).RNAqualitywasassessed
usingtheAgilentBioanalyserRNAChip(SantaClara,
California).AllsampleswereshowntohaveaRINvalue
inexcessof7.5.TheRNAwasconvertedtocomplemen-
tary(c)DNAandacDNApoolcontaining3.5ulfrom
eachsamplewaspreparedandusedtogeneratea7
point,1in4serialdilution.Thisserialdilutionwasused
totesttheefficiencyofeachprimerpairusedinthe
study.TheremainingcDNAwasdilutedto2.0ng/ulof

Watts
etal.JournalofCircadianRhythms
2012,
10
:7
http://www.jcircadianrhythms.com/content/10/1/7

RNAequivalentsandstoredat
−
20°C.Anumberof
minusreversetranscription(RT)controlswereincluded
duringthecDNApreparation.
QuantitativePCRassayswereperformedusingBiosys-
tems7500SequenceDetectionSystemandtheSensi
MixSYBRKit(Bioline,Taunton,Massachusetts).A
panelofeightputativereferencegeneswasassessedfor
stabilityusingtheGeNormalgorithmwiththeqBase
PairSoftwearpackage.Resultsshowedthat
ARTB
and
HPRT