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Diurnal expression of the thrombopoietin gene is regulated by CLOCK

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Summary.  Background:  Most physiologic processes exhibit diurnal fluctuations controlled by the circadian regulation of sleep-wake behavior and feeding cycles. In addition, many cell types express endogenous circadian rhythms that affect
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  ORIGINAL ARTICLE Diurnal expression of the thrombopoietin gene is regulated byCLOCK C. J. TRACEY,* X. PAN,  J. H. CATTERSON,* A. J. HARMAR,* M. M. HUSSAIN  and P. S. HARTLEY* * University/BHF Centre for Cardiovascular Science, Queen Õ  s Medical Research Institute, University of Edinburgh, Edinburgh, UK;and   Departments of Cell Biology and Pediatrics, Suny Downstate Medical Centre, Brooklyn, NY, USA To cite this article: Tracey CJ, Pan X, Catterson JH, Harmar AJ, Hussain MM, Hartley PS. Diurnal expression of the thrombopoietin gene isregulated by CLOCK. J Thromb Haemost  2012; 10: 662–9. Summary. Background: Most physiologic processes exhibitdiurnal fluctuations controlled by the circadian regulation of sleep–wake behavior and feeding cycles. In addition, manycell types express endogenous circadian rhythms that affectcell-specific processes. Independent reports support thehypothesis that thrombopoietin (TPO) is under circadiancontrol. Objectives: The current study tested the hypothesisthat CLOCK, a circadian transcription factor, may regulate Thpo , the gene encoding TPO. Methods: Circadian geneexpression patterns were analyzed in mice and in human celllines,SmallinterferingRNAwasusedtoknockdown CLOCK  expression in cell lines, and gene expression was also examinedin Clock D 19/ D 19 mutant mice. Results: It was found that therewasa diurnal rhythm in the expression of  Thpo in vivo in mice,and that this was associated with concomitant rhythms of proteinabundance. Thpo wasrhythmicallyexpressedinhumancell lines, consistent with the gene being directly or indirectlyregulated by the circadian clock. Silencing of  CLOCK  in theHuh7humanhepatomacelllineledtoasignificantreductioninthe rhythmicity of  Thpo expression. The expression of  Mpl  inmurine marrow also displayed diurnal rhythmicity in vivo . In Clock D 19/ D 19 mutant mice, Thpo and Mpl  expression wasdisrupted and there was an increase in the number of maturemegakaryocytes,butnochangeintheploidydistributionwithinthe megakaryocyte population. Conclusions: These findingsestablishthatClockregulates Thpo and Mpl  expression in vivo, and demonstrate an important link between the body Õ scircadian timing mechanisms and megakaryopoiesis. Keywords : circadian, clock, megakaryocytes, Mpl, platelets,thrombopoietin. Introduction Physiologic processes exhibit diurnal fluctuations that reflectdaily cycles of sleep, wakefulness, and feeding. Many diurnalrhythms persist in the absence of environmental cues, such asthe light–dark cycle, indicating that an endogenous circadianclock drives them [1]. A master oscillator within the hypothal-amus, the suprachiasmatic nuclei (SCN), synchronizes physi-ologic rhythms with the light–dark cycle. The coordination of the SCN and physiologic rhythms is dependent on a transcrip-tion–translation feedback loop controlled by a small family of circadian Ô clock Õ transcription factors. The genetic clock isexpressed in most cell types, and synchrony between physiol-ogy, metabolism and behavior is maintained by complex cell– cell interactions and higher-order communication systemsinvolving systemic hormone signaling and the central nervoussystem. Disruption of diurnal rhythms and circadian synchro-nicity by shift-work, lifestyle choices and senescence is associ-atedwithanincreasedriskofdevelopingcardiovasculardiseaseand cancer [2,3].Megakaryocytes are derived from hematopoietic stem cells,andplayacriticalroleinhemostasisbygeneratingplatelets,thecells in the circulation that initiate thrombus formation. Therecruitment and development of megakaryocytes from hema-topoietic stem cells is primarily regulated by the cytokinethrombopoietin (TPO), which activates its cognate receptor,MPL, on megakaryocyte progenitors and developing megak-aryocytes. TPO is therefore linked with megakaryocyte devel-opment, the production of platelets, and the maintenance of platelet numbers [4–6]. Experimental modulation of  Thpo (thegene encoding TPO) in mice leads to a significant reduction inthenumberofmaturemegakaryocyteswithinthebonemarrowand a massive reduction in circulating platelet numbers, whichhas a significant effect on bleeding times [7,8].We have demonstrated that megakaryopoiesis is regulatedbylight-entrained signals emanating from the master oscillatorwithin the SCN of the hypothalamus [9,10]. We have alsodemonstrated that the concentration of plasma TPO exhibits asmall diurnal fluctuation. Thpo expression is detectable in thebone marrow, kidney, and liver, and is thought to beconstitutive, whereas plasma TPO levels fluctuate accordingto circulating platelet abundance [11–13]. Microarray studies Correspondence: Paul S. Hartley, University/BHF Centre forCardiovascular Science, Queen Õ s Medical Research Institute,University of Edinburgh, Little France, Edinburgh, EH16 4TJ, UK.Tel.: +44 131 242 6691; fax: +44 131 242 6779.E-mail: p.s.hartley@ed.ac.ukReceived 26 September 2011, accepted 15 January 2012 Journal of Thrombosis and Haemostasis , 10 : 662–669 DOI: 10.1111/j.1538-7836.2012.04643.x Ó 2012 International Society on Thrombosis and Haemostasis  have demonstrated a diurnal rhythm in the expression of  Thpo in murine liver [14,15] that may be regulated by the circadianclock [16]. Furthermore, the expression of the TPO receptorgene, Mpl  , is also rhythmic in murine bone marrow [17]. Thesedata support the notion that the circadian clock may regulate Thpo expression and megakaryopoiesis.In the current study, we tested the hypothesis that thecircadian clock may control Thpo expression by examiningthe gene Õ s expression both in vivo and in vitro . In addition,we characterized Thpo expression in mice expressing adominant-negative form of Clock ( Clock D 19/ D 19 ) [18]. Wefound that Thpo was rhythmically expressed in vivo and in vitro , consistent with the gene being directly or indirectlyregulated by the circadian clock. In support of this conclu-sion, silencing of the circadian transcription factor CLOCK  led to a significant reduction in both the rhythmicity andoverall expression of  Thpo in vitro . In Clock D 19/ D 19 mutantmice, Thpo expression was temporally disrupted, and this wasassociated with an increase in TPO and a significant increasein megakaryocyte numbers, but not in the ploidy distributionof mature megakaryocytes. These findings establish a linkbetween the body Õ s circadian timing mechanism and megak-aryopoiesis. Materials and methods Stock chemicals, RPMI-1640 medium and CelLytic were fromSigma (Poole, UK). Rabbit anti-TPO was from Abcam(Cambridge, UK). Anti-rabbit horseradish peroxidase wasfrom Invitrogen (Paisley, UK). Amersham ECL reagent wasfrom GE Healthcare (Chalfont St. Giles, UK).  Mice All experimental procedures were carried out in accordancewiththeUKAnimals(ScientificProcedures)Act,1986.C57Bl/6and Clock D 19/ D 19 micewerehousedingroupsofnomorethansix per cage, on a 12 : 12-h light–dark cycle, with ad libitumaccess to food and water. Mice were between 12 and 20 weeksof age when used for experiments or tissue collection. Datafrom Clock D 19/ D 19 mice were compared with those from wild-type and heterozygous littermates. Sampling times are alldescribed relative to the time-cue of lights going on (zeitgebertime [ZT]). Thus, samples taken 4 h after the lights switch onare referred to as ZT4. HepG2 cell culture HepG2 human hepatoma cells were cultured to confluence insix-well culture plates in DMEM supplemented with 10% (v/v) fetal bovine serum (FBS), penicillin and streptomycin at100 U mL ) 1 and Glutamax (1 : 100 of commercial stock).The cells were synchroniszed by a standard circadian protocolwith 100 n M dexamethasone for 1 h [19], after which thedexamethasone was washed from the cells by three rinses inHanks buffered saline solution. The cells were then culturedin the medium described above for the times indicated in thetext. This synchronization protocol is necessary because thecircadian clocks within different individual cells have slightlydiffering period lengths, so the population loses synchronyover time [20]. Dexamethasone resets clock gene expression inall cells in culture, so rhythms of gene expression can bemeasured in the synchronized cell population. The time whencells in vitro are synchroniszed is referred to as circadian time(CT)0; thus, samples taken 4, 8 or 12 h after this point arereferred to as CT4, CT8, CT12, and so on. HepG2 cellcultures were lysed in the dish with RLT buffer for RNAisolation, at 4-hourly intervals starting 1 h after synchroniza-tion. Quantification of gene expression RNA was extracted from liver, marrow and cultured cells witha commercial kit (Qiagen Rneasy Mini kit; Qiagen, Crawley,UK), and cDNA synthesis was with SuperScript II (RocheApplied Science, Burgess Hill, UK), according to the manu-facturer Õ sinstructions.QuantitativePCRwasperformedwithaLightCycler 480 (Roche Applied Science) with 10- l L reactionvolumes (1 l L of cDNA as template, diluted 1 : 5; 5 l LMaster Mix [Roche Applied Science]; 0.6 l L of forward andreverse primers; 0.2 l L of probe; and 2.6 l L of nuclease-freewater). Standard curves were created for each gene, andsamples were amplified in triplicate. Data were normalizedagainsttheexpressionlevelsoftheTATA-bindingproteingene( Tbp ).  Measurement of plasma TPO PlasmaTPOconcentrationwasquantifiedwiththeQuantikineMouse TPO Immunoassay kit (R&D Systems Europe,Abingdon, UK), according to the manufacturer Õ s instructions.  Small interfering RNA and serum shock  SmallinterferingRNA(siRNA)directedagainst CLOCK  ,andnon-specific control siRNA (Santa Cruz Biotechnology, SantaCruz,CA,USA),wereintroducedintoHuh7humanhepatomacells plated in 12-well plates by the use of siRNA transfectionreagent. After 72 h, cells were starved in DMEM containing0% FBS for 18 h. On the day of serum shock, 50% horseserum was added for 2 h, and the medium was then changedback to starvation medium after; cells were harvested everyfour hours for 48 hours and gene expression quantified usingreal-time PCR. Histology Femurs were fixed in 4% paraformaldehyde for 48 h, decal-cified in 400 m M EDTA for 1 week, and dehydrated in gradedethanols (70%, 90%, and 100%, each for 24 h). Femurs werethen embedded in wax, and 5-mm sections were cut, rehydrat-ed, and stained with hematoxylin and eosin. Stained sections Clock regulates thrombopoietin 663 Ó 2012 International Society on Thrombosis and Haemostasis  were then dehydrated, mounted for microscopy, and imagedwith phase-contrast optics on a Zeiss Axioskop MOT IIwidefield microscope. Isolation and quantification of megakaryocytes Megakaryocytes were isolated from femurs and tibiae byresecting the epipheses of the bones and flushing out themarrow with ice-cold phosphate-buffered saline (PBS) supple-mented with 2 m M EDTA (PBS-EDTA). The marrow wascentrifuged at 4 ° C and resuspended in cold PBS-EDTAbuffer,andthisstepwasrepeatedonce.Erythrocyteswerelysedon ice as described previously [10], and the marrow suspensionwas then centrifuged and resuspended as described above.Megakaryocytes were quantified with a hemocytometer, andtheir yield was expressed as the total number of megakaryo-cytes isolated from two femurs and two tibiae. Analysis of megakaryocyte ploidy by flow cytometry Whole marrow cell suspension was stained with anti-CD41antibodies conjugated to fluorescein isothiocyanate, fixed withformaldehyde, and permeabilized with 70% ice-cold methanolfor 10 min. Cells were then treated with RNase for 30 min atroom temperature, and stained for 30 min with propidiumiodide (25 l g mL ) 1 ) in PBS. Cells were then analyzed with aBeckton Dickinson FACscan (Beckton Dickinson, Oxford,UK). Cells in the FL-1 channel (green fluorescence, CD41-positive cells) that were two log orders brighter than themajority of marrow cells were analyzed fortheirDNA contentin the FL-2 channel (red fluorescence, propidium iodide).DistinctpopulationsofcellswithDNAploidyfrom2Nto64Nwere resolved and regarded as megakaryocytes on the basis of their CD41-positive staining and high DNA content (seeFig. S1 for examples of cytometry plots). The relative frequen-cies of different ploidy populations were then quantified withF LOW J O (Tree Star, Ashland, OR, USA). Platelet counts Bloodwasdrawnfromalateraltailvein,approximately1.0 cmfrom the base of the tail, by nicking the tail with a fresh, sterilescalpel. The first 10–20 l L of blood was discarded, and 10 l Lwas then collected with a pipette and immediately diluted in90 l L calcium/magnesium-free Hanks buffered saline contain-ing 0.38% sodium citrate and 2% formaldehydye at roomtemperature. Platelet numbers were then quantified by auto-mated counting (Coulter AcT; Miami, Coulter Corporation,FL, USA). Reticulated platelet counts Whole blood (1 mL) was collected by cardiac puncture into100 l L of 3.8% sodium citrate and platelet-rich plasma (PRP)prepared by centrifugation (180 · g for 5 min at roomtemperature). PRP was removed to a fresh tube, and 100 l Lwas fixed by adding 900 l L of 4% formaldehyde (made inPBS) for 20 min at room temperature. Fixed platelets werethen stained with thiazole orange (100 ng mL ) 1 ) for a further30 min at room temperature, and analyzed by flow cytometrywithaBecktonDickinsonFACscan.Reticulatedplateletswereidentified as a discrete subpopulation of platelets exhibitingbright fluorescence in the FL-1 channel. This subpopulation isnot present after treatment with RNase A (see [9]), indicatingthat thiazole orange labeled RNA rather than dense granules[22]. Results Thpo expression is rhythmic and in antiphase to TPO plasmaconcentration in vivo Toinvestigatediurnalvariationintheexpressionof  Thpo ,liversandbonemarrowwereharvestedfromgroupsofmaleC57Bl/6miceevery4 hovera24-hperiod.Themicewereentrainedtoa12 : 12-h light–dark schedule, and given free access to bothwater and food. Under these conditions, the expression of thecircadian gene Per2 was highly rhythmic (Fig. S2), as was thatof  Thpo in both liverand marrow (Fig. 1A; P < 0.01forbothliver and marrow), reaching a nadir just prior to lights-on andpeakingduringthesubjectivenight(thedarkphaseofthelight– darkcycle).Toestablishtherelationshipbetweentherhythmof  Thpo expression and plasma TPO levels, the concentration of free TPO within the plasma was quantified with a commercialELISA kit. As previously reported, the plasma concentrationof free TPO was rhythmic (Fig. 1B; P < 0.01), with peak andnadir levels during the subjective daytime and night-time,respectively (and thus having an antiphasic relationship withthe rhythm of  Thpo expression). 2.0 A B 280 P  < 0.01 P  < 0.01240200160120804001.61.20.80.40.0     T    h   p   o    /    T    b   p    T   P   O   (  p  g  m   L   –   1    ) 4 8 12 16ZT20 24 4 8 12 16ZT20 24 Fig. 1. Thrombopoietin (TPO) gene ( Thpo ) expression is rhythmic in -vivo. Mice were entrained to a 12 : 12-h light–dark cycle, and samples of liver, marrow and plasma were collected at 4-hourly intervals across thediurnalcycle.ZT,zeitgebertime(thetimeinhoursafterlights-on).(A)Theexpression of  Thpo relative to that of the TATA-binding protein gene( Tbp ) in the bone marrow (dark triangles) and liver (gray squares) wasmeasured by quantitative PCR, and found to be rhythmic ( P < 0.01 byone-way ANOVA ). (B) The concentration of TPO (pg mL ) 1 ) in plasma wasmeasured by ELISA, and was also rhythmic ( P < 0.01, by one-way ANOVA ), but in antiphase to the expression of  Thpo in the liver and mar-row. The black bar denotes the times of lights-off (from ZT12 to ZT24). N  = 4 mice per time point. 664 C. J. Tracey et al Ó 2012 International Society on Thrombosis and Haemostasis  Thpo expression is rhythmic  in vitro and regulated byendogenous Clock Our findings suggested that Thpo expression in vivo may beregulated by the endogenous circadian clock. To test thishypothesis, the expression of  Thpo was recorded in twoindependent human hepatocyte cell lines, HepG2 and Huh7,over one or two circadian cycles (Fig. 2). In both cell lines, Thpo expression was rhythmic ( P < 0.01 by one-way ANOVA ), indicating that Thpo expression is sustained in theabsence of external, physiologic cues, and is thereforeregulated by the circadian clock. In accordance withthis conclusion, the silencing of CLOCK with siRNA ledto abolition of rhythmic Thpo expression and a significantreduction in Thpo expression (Fig. 2C; P < 0.001, relativeto siRNA control), consistent with the significant reductionin Clock gene and Clock protein expression (Fig. 2D,E). Clock  D 19/ D 19 mutation disrupts Thpo expression The finding that Thpo is diurnally expressed in vivo and that itexhibits a circadian pattern of expression in vitro indicates thatthe endogenous circadian clock regulates this rhythm. Wepredicted that mice expressing a dominant-negative form of Clock ( Clock D 19/ D 19 ) would have an abnormally timed patternof  Thpo expressionthatmightimpactonmegakaryopoiesis.Totest this, bone marrow and liver samples were obtained from12–20-week-old Clock D 19/ D 19 mice at two time points, corre-sponding to the times of nadir and peak Thpo expression(Fig. 3A). The diurnal fluctuation of  Thpo wasevident in wild-type and heterozygous mice, but disrupted in Clock D 19/ D 19 mutants. Although a diurnal pattern of  Thpo expression wasobserved in Clock D 19/ D 19 mutants, it appeared to be reversedrelative to wild-type animals. As well as the Clock D 19/ D 19 mutation disrupting the timing of  Thpo expression, there wasalso increased plasma TPO in Clock D 19/ D 19 mutant mice(Fig. 3B). Clock also regulates a diurnal rhythm of  Mpl expression TPO acts on its cognate receptor, MPL, expressed onmegakaryocytes and megakaryocyte progenitors to promoteand maintain megakaryopoiesis. It has recently been demon-strated that Mpl  expression is regulated by Period 2 [17], atranscriptional target of CLOCK. We therefore examinedwhether loss of temporal control of  Thpo expression in Clock D 19/ D 19 mutant mice was associated with abnormal Mpl  expression.Inwild-typemice, Mpl  expressionwasdiurnal,withpeak expression at ZT20 ( P < 0.01; Fig. 4A). This was notassociated with a change in the number of megakaryocytesisolated from the marrow at this time ( P > 0.05; Fig. 4B),suggesting an intrinsic change in expression levels. In AC D EB 0.80.61.01.20.40.000 4 8 12162024283236404448510151 6 11Circadian time (h)Circadian time (h)16 21siControlsi CLOCK  CLOCKControlsiRNACLOCKControlsiRNA26 1 6 11Circadian time (h)16 21 260.20.80.61.00.40.00.2     N   r    1    D    1    /    T    B    P    T    H    P    O   m   R   N   A   (   f  o   l   d  c   h  a  n  g  e   ) 0.80.61.01.2mRNA Protein**0.40.00.2    C   L   O   C   K   /   G   A   P   D   H 0.80.61.01.20.40.00.2    C   L   O   C   K   /   G   A   P   D   H     N   r    1    D    1    /    T    B    P Fig. 2. CLOCK regulates thrombopoietin gene ( THPO ) expression in vitro . The expression of  THPO was measured in two human cell lines (HepG2 andHuh7).Cellsweresynchronizedbybriefexposuretodexamethasone,andsamplesofcellswerethencollectedevery4–5 hfor26–48 h(circadiantimeisthetime in hours after cells were synchronized). (A) Rhythmic expression of the circadian gene NR1D1 verified that HepG2 cells were synchronized andexpressing a circadian clock. (B) Expression of  THPO was also rhythmic ( P < 0.01 by one-way ANOVA for the effect of time). (C) THPO expression wasrhythmicinHuh7cells,andtransfectingcellswithsmallinterferingRNA(siRNA)to CLOCK  abolishedthisrhythm. N  = 6independentculturespertimepoint. (D) CLOCK  mRNA levels in Huh7 cells treated with control siRNA or siRNA to CLOCK  , N  = 4, * P < 0.01. (E) CLOCK levels relative toglyceradehyde-3-phosphate dehydrogenase (GAPDH) levels determined from two independent western blots of lysates from Huh7 cells treated withcontrol siRNA or siRNA to CLOCK. * P < 0.01. Clock regulates thrombopoietin 665 Ó 2012 International Society on Thrombosis and Haemostasis  Clock D 19/ D 19 mutant mice, the diurnal fluctuation in Mpl  expression was confirmed in wild-type littermates ( P < 0.01)butwasnotpresentinmutantmice(Fig. 4C).Thus,both Thpo and Mpl  expression rely on CLOCK  for their normal diurnalpattern. The Clock D 19/ D 19 mutation leads to an increased megakaryocyte yield from the bone marrow We reasoned that megakaryocyte numbers and/or modalploidy levels may also be affected by the CLOCK mutation.Inspection of histologic femur sections revealed that marrowmorphology was similar between the genotypes and thatmegakaryocytes were localized normally throughout the mar-row(Fig. 5A).Despitethis,thenumberofmegakaryocytesperfield of view (with · 400 magnification) was slightly increasedin Clock D 19/ D 19 mutant mice as compared with controls(Fig. 5B; P < 0.05). In addition, the number of megakaryo-cytesisolatedfromthefemursandtibiaewasincreasedby25%in Clock D 19/ D 19 mutant mice as compared with either wild-typeor heterozygous mice (Fig. 5C; P < 0.01). However, analysisof the DNA content of megakaryocytes by flow cytometryindicated that there was no change in the relative frequency of CD41-positive cells ( P = 0.94 by Student Õ s t -test, data notshown) or the different ploidy classes (data for the modal 16Nploidy class are shown in Fig. 5D; P > 0.05 by one-way ANOVA for the effect of genotype). In vitro megakaryocytecolony-forming unit (MEG-CFU) assays did not reveal anyeffect of the Clock mutation on the number of MEG-CFUs(Fig. S3), and nor was there evidence of any gross defects inproplatelet formation by megakaryocytes cultured in vitro (data not shown). The Clock D 19/ D 19 mutation affects platelet counts In humans and wild-type mice, platelet counts rise and fallaccording to the time of day (for a review, see [23]). Wefound that whereas wild-type mice exhibited a diurnalfluctuation in the platelet count between ZT8 and ZT20,this was reversed in Clock D 19/ D 19 mutant mice, indicating thatthe biological timing mechanism controlling circulatingplatelet abundance is abnormal in Clock D 19/ D 19 mice(Fig. 6A). When platelet counts at ZT8 and ZT20 werepooled to obtain the daily average for each genotype, therewas evidence that the Clock D 19/ D 19 mutant mice had increasedplatelet numbers (Fig. 6B; P < 0.05). However, the propor-tion of reticulated platelets in the circulation was similarbetween genotypes (Fig. 6C). In addition, a diurnal fluctu-ation in the percentage of thiazole orange-positive platelets(a measure of young Ô reticulated Õ platelets [24]) in the plateletpopulation was unaffected by the Clock D 19/ D 19 mutation,being higher at ZT8 than at ZT20 in both genotypes (both P < 0.01), suggesting that diurnal changes to the proportion 1.41.81.51.20.90.60.30.01.01.64035302520151050NSNS* CBA 1.20.80.60.40.20.0 Δ 19/  Δ 19ZT20 ZT20ZT8ZT888 12 16 20 24420ZTZTWT     M   p    l    /    T    b   p    M   p    l    /    T    b   p    T  o   t  a   l   M   K  s   i  s  o   l  a   t  e   d   (  ×   1   0   ) Fig. 4. The Clock D 19/ D 19 mutation affects Mpl  expression. Mice were entrained as described in Fig. 1. (A) Mpl  expression was measured in bone marrowsamplescollectedfromwild-type(WT)miceovera24-hperiod.Theblackbardenotesthetimesoflights-off(fromzeitgebertime[ZT]12toZT24).(B)Theyield of megakaryocytes (MKs) from bone marrow aspirates did not change between ZT8 and ZT20 (NS, not significantly different). (C) The diurnalfluctuation in Mpl  expression in WT mice was disrupted in Clock D 19/ D 19 mutants. (* P < 0.01, n = 4–6 mice per time point for all data). 1.41.01.6 A B 140120100806040200**#1.20.80.60.40.20.0ZT 8WTWT Δ 19/  Δ 19 Δ 19/  Δ 19ZT 20 ZT 20ZT 8     T    h   p   o    /    T    b   p    P   l  a  s  m  a   T   P   O   (  p  g  m   L   –   1    ) Fig. 3. Effect of CLOCK mutation on thrombopoietin gene ( Thpo )expressionMicewereentrainedasdescribedinFig. 1.(A) Thpo expressionwas measured in liver samples collected from wild-type (WT) and Clock D 19/ D 19 mice ( D 19/ D 19) at ZT8 and ZT20. There was a diurnal fluc-tuationin Thpo expressioninbothgenotypes(* P < 0.01forthedifferencebetween expression at ZT8 and ZT20); however, the pattern was reversedin Clock mutants. (B) The plasma TPO concentration was increased in Clock D 19/ D 19 mutants). (* P < 0.01, P < 0.05, n = 4–6 mice per timepoint). 666 C. J. Tracey et al Ó 2012 International Society on Thrombosis and Haemostasis
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