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4r.-ii.trA)* i !Q'4131 :q'-- "-r.0 Ar 293 Developmental chances in the expressionof crenesin,,olved in cholesterolbiosynthesisAnd lipidtransportin human and ratfetaland neonatal livers Marc S.Le,.in'A,lan J.A. pitt2,Alan L. SchwartZ 3.1,PeterA. Edwards and Jeffrey I.Gordon 1-2 DeparfrtieonfttWedicine:.Biuchemisiann-d.WoleculBairopkrsicJsP.ediatriacnsd ' Pharmacolog1v+.*ashinv1t-o'n#7tvIe-Srcs;iltool of.WedicinSt.Louis.WO and @Departmentosf.4,fediacnidnBeiologicCahlemisri@L,'.CL4Schooolf-Wediciple. Los.4ngel'es. CA (L.S.A.) (Receive2d3 December1988) Key words: Development;lipid transportE:xpression:Cholesterol Cloned cDNAs encodinga number of enzymes involvedin cholesterobliosynthesiass wellas extraceuularand intracellullairpidtransportwere used to compare thedevelopmentalmaturationof thesebiologicfunctionsin thefetal and neonatalratand human liverT.le restdtosf RNA blothybridizatioannalysesindicattehatsteady-statleevelsof mt HMG-COA synthase,I-LMC,-CoA reductaseand prenyltransferasmeIL14As are highestin latefetallifeand undergo precipitou(supto 80-foldc)o-ordinatreeductionismmediatelyafterparturitioTnh.ese changesreflecttheabilitoyf the fetalmt liverto produceI Pe quintaittoifecsholesteroals wellas the repressioonfthisfunctionduringthesucklim zriod in nse to exogenous dietary olesterol@.trfldngco-ordinatepafte-ms of IG@@syn@ r@tas@e and prenyl-transfemsMeRNA accumtdationwere alsoobservedin fourext-ahepatircattissues(brain.,Iunign,testine and kidney)duringtheperinatalperiod'.ne concentrationosf allthreemRNAs inthe8-week-oldhuman fetaliverare similartothoseobservedthroughoutsubsequentintrauterindeevelopmentwithlessthan2-foldchangesnoted between the 8ththrough25th weeks of gestationA.nalysisof the levelsof human apo AI, apo AII,apo B and liverfattyacid bindingproteinmRNAs duringthisperiodand in newborn liverspecimensalsoindicateldessthan 2-3-foldchanges. These observationsuggestthatthe 8-week human liverhas achieveda high degreeof biochemicaldifferentiatiwointh respecto functionsinvolvedin lipidmetabolism/transporwthich may be comparabletothatpresentin 19-21 day fetal ratliverF.urtheranalysisof human and ratfetalliverRNAs usingclonedcDNAs shotddpermitconstructioonf a developmentaltime scalecorrelatinhgepaticbiochemicaldifferentiatitoonbe constructebdetween thesetwo main- malianspecies. Introduction developmentalalterationisn the rateof incorporation of radiolabeledacetateintosterols[2].Bruengerand Marked changes in the requirementsfor products derivedfrom isoprene(e.g.cholesterolo)ccur during Rilling[3]documented chan esin the activitioefs two other ch,oiis-te-rox-er-,u@.-icn-etnhzeymdeesveloping rat development.The enzvme which catalyzesthe key,__ liver:sq Fenesyn@theta.-asned prenyl transferas(eor rate-liraitisnig@epin cH-oTe-sterboilosynthi-gis-raicro- farnesyl/pyrophosphatseynthetase)P.renyltransferase somal 3-hydroxy@3-methylglutaryl-CnA reductase isone of riveenzymes thatparticipatien theconversion (HMG-COA reductase)-undergoelsarge fluctualiqns 0 the preculIoL-oL-u=ls. duringriLliym-onLaMny(1.2]E._n_r,Oe activiti!yLbigh Bo squ ene syntetaseand prenyltransferasdeis2lay priorto birthd,eclinesto low leveldsurinizthesuckling similardevelopmentalactivitpyrofileisn ratliver:.Ibej p-e-rt,-Z@,nsat@tat@,we13a@@yvs@- )@and@demmooltn@nssascttriravaitte0v"s@aa fallfollowinbirth.risetoa peak duriniztherrddto]ac s-.cinite@nitncreaseatweaaruinngi.L@aaMnsg@eisninitsactivitpyarraall@lleel sucklin-te)eriod(postnatadlays 10-12).falLnnc&_More durinxthesuc ling-weanintirzansitiorneachinga nadir Currespondence: University School M.S. Levin. Deparu-nent of Medicine. WasW..,.-.. of Medicine. 660 South Euclid Ave- Box $124. SL by postnatalday 20.0 weaning.The mechanisms which resultin thesealterat% ionsin enzyme activitmyay be quitecomplex as Louis. MO 63110. U.S.)L illustratebdy the factthat the mevalonats.-mediated 0005-2760/89/SO3.50 0 1999ElwvictSciencePublisherDs.V.(BiomedicalDivision) decreasein H.%[G-CoA reductasleevelosbservedin adult animals reflecesdecreases in gene transcriptionas well as increasedratesof proteinde-radation(4.,@l. Littleis known about the de-ve'loo@mentalhist'orvof the activitieosf these enzymes in human fetaland neonatal liveror about the ontogeny of expressionof crenesinvolved in the transportand metabolic prociuing of lipidsin thistissue.The ratliverprovidesa convenient referencefor a comparative study of such developmental changes. For example in additionto the information about expression of cholesterogenicenzyme activitiesr.ecent analyses of apolipoproteingene expression in the developinc,rat liverindicatethata complex patternof activationoccurs during latefetaland culy neonatal lifeA.polipoprottin AI and E mP,,NAs begin to accumulate in thistissueplus itsembryonic horaoloaue (the fetalyolk sac endoderm) betweea days 15-21 of the 21-day gestation period (6-9]. Remarkable increasesin thelevelsof thesemRNAs occurduringthe sucklingperiodas the animalsadapt to the high fat (principaltlryiacylglycerdoile)tof mothersmilk(10,11]. By contrastr,atliverapo B MRNA levelrseacha peak by the 18th fetalday thatisnot exceededatany time durin- subsequentdevelopment (121.Followin-birth. hepaticapo B MRNA concentrationpsrogressivelfyaU duringthesucklingand weaaing periods(121T.his fall appears to be mediated by thyroxine(13].A third patternof activatioinsexhibitebdy theapo AIV gene which remainsdormant untilthesucklingweaninc.t,ransition(days13-14)when the-atliverbeginsto export largeamounts of triacylglyceroilc-hlipoprotein(s6]. We have begun a comparativeanalysiosf the accumulationof mR.NAs encodin-proteinsinvolvedin lipid metabolismin the fetaland neonatalhuman and rat liverA. panel of cloned cD','AsencodingapoupoproteinsAl. AII.and B. an ineracellulfaarttyacidbinding proteinas wellas H%IG-COA reductaseH,NfG-CoA synlhaseand prenyt transferasweere used to chafacterizethe stateof enzymaticdifferentiatioofnthe human fetalliverfrom weeks 8 to 25 of development and in the newbom. The resultisndicatethatthese mRNAs appear at an earlyphase of human fetallife (by week 3)and unde--zoonlyminimal(lestshan3-fold changes)in theircorc::nt,.t-oantintotalliveRrNA durin,t,herestof intrauterinaes wellascarlvpostnatallife. This earlyexpressionof lipidmetaboliccapabilitcyontrastswith the more marked changesinMRNA levels observedin theperinatalracliver. .viateriaalnsd intethods Preparation of R.V,4 front rat atid human liver Timed-pregnant.neonataland young adultSprague- Dawley ratswere obl-iinedfrom Sasco(St.Louis.MO). Weaned animals were maintainedon a standardchow dietad libitumand a fixed12 h (6:00a.m. to 6:00 p.m., tight cvcle. Animals (n - 10-40 for each time point@ were @illedbetween 1200 and 1400 h and theirlivers, tunas.brains.kidneys and small intestinesimmcdiatcl,, frozen in liquidnitrogen.Human fetalliversamp[C@ from rtrstand second trimesteraborted fetuseswere obtained by Schwartz et at. (141 and maintained a- 90 * C for 15-20 years. Fetal age was estimated frorr. crown-rump lengthsusing nomograms developed b.,, Tanimura et al.(15).Breitfeldand Schwartz (161and imichaelsonand Orkin (171have used thesehuman feta, liversamples previouslyto successfullyprepare RNA forin vitrotranslationA.dditional human liversample@ were obtained from a preterm newborn and a fullterrr newborn, both of whom died of acute respiratoryfailure These were storedat - 90 * C for about 15 years(141.A singleadult liverspecimen was procured from a male organ donor who died of trauma and had no historyor clinicaelvidenceof hepaticdysfunction(18]. Total cellulaRrNA was extractedfrom frozen pulverizedtissuesusing the guanidinethiocyanate, cesium chloridperocedure(19].RNA integritwyas as sessedby denaturingmettivimercuryagarosegelclec trophoresi(s201. R.,VAblothybridizatsitoundies Dot blotswere preparedby applyingfouramount of each tissueILNA sample (0.5.1. 2. and 3 ttg)t; nitrocellulofsielterass describedin a previouspublica tion(6].Yeast TRNA was added to each tissueRN.- sample priorto denaturationso thatthe totalRN. inputper dot was alwavs3 Blotscontainin-sample of ratliveri,ntestinkei,dney.brainand lungRNA wet probed with 3:P-labelcdd.ouble-strandedcDNAs e,- codin0e hamster HNIG-COA reductase(22].ratH-.viC CoA synthase[231.and ratprenyltransferas(e241.Dc blotsof human liverRNA sampleswere probed wi-. 32P-labeledcDLNAs specifyin-human preproapo @ [251,human preproapo All human preapoB (2- human liverfattyacid binding protein[281.hum;: a-fetoprotei(n291,human H'v[G-CoA reductase(301.r prenvltransferasaend rat H%,[G-CoA synehase.Conc tionsselectedforfiltehrybridizatioannd washing a listedin Ref. 12. These strin-enciewsere equivalent thoseused by othersto produce specifiicnteractio between thesecDtNAs and theirrespectivmeRNAs. T' relativaebundance of each mRi4A in the tissueRiN preparationswas determined by quantitativsecanni, laserdensitometryor filterautoradiographussing LKB XL UltroscandensitometerO.nly siaonalsin t linearran,,eof film sensitivity were utilizedfor caleu tions of re'lCa'tive MRLNA concentration. LNorthcrnblotsof totalcellulaRrNA were produc followianaclectrophoretifcractionatiotnhrough aaarosegetscontainingformaldehyde [31@ 295 Results and Discussion a remarkable simila 11the p,.tttt!orfnschange of each mPN4 'A durina-rat. atand neonataldevelopmenl .4cctirunlation of H.@IG-CoA ki-,Ifhase.H.%,IG-CO,4 re- H@iLhle@-cisar@c oted e@e,@.est-@1a1l@,1,0ioT-nh.e-se fall citiactipitrdiesntevrlanifmeRr.a'sVie.nt4hideevelopianbgrupwtiltyh2i4nh aftcearrturrietaicohcnionng- centratthiaotrnaessmucahs30-floolwdtehratnhose ralticer e@ncoun@teredp@r-iotro@irt@(t.g.. see the Cloned CDNAS encoding H,@w(G-COA synthase. H@N,IG-CoA reductase.and prenyl transferasewere used middle panel of Fi0g.I which shows chanZcPes in prenyl to probe dot blotsof totalcellularRNA preparedfrom transferaseMRN*A concentration)A. transien2t-4-fold ratliversharvestedduring fetaldays 16-21. the suck.- risein HNIG-COA synthastand prenyt transferasbeut lingperiod(derinedas the first13 postnataldays,Ref. not H,'v(G-CoA reductasemR4A accumulationoccurs 32).theweaning phase (days 14-28) and from animals duringthe niid-sucklinpgeriod(day 8) followedby yet proceedingthrough sexual maturation (postnataldays another riseduringearlyadulthood.Itism-portant to 35-70).),Iateriwaals collectedfrom 10 to 40 male and note teat-thispatternot change isnot a generalphe- female animalsrepresenting1-4 litterast each develop- nomenon in thedevelopingratliverF.or example,when mental stage seudied.The resultsof our dot blot the developmentalprofilesof apo AI, apo AIV and L-FABP mRSAs were studiedusingtheseR-N'As,quite hybridizatioannalysm are presentedin Fig.I and show differentontologicchanges were noted (see,forexam- Liver pie,Ref.6). The developmentalprofileof rat liverHLMO-C:OA HMG CoA Syntha30 reductaseand prenyl transferismeRNAs pararclthe changes in the activitieosf theseenzymes which have 3.0 been previouslydocumented by severalgroups [3,33-351. 2.0 - Birth to 3.0 2.0 E to irth I PronylTtansferase The correspondingactivitieosf Hi'viG-CoA synthase have not been reported.The rat fetusobtainsoilly about 105cof the sterolrequiredforgrowth and (levelopment from itsmother [36].Therefore.itisnot surpnsi@-fvt-h-arty-high levelsof thesemRNAs are observed in ratfetalliverdurin0athe periodsurveyed.DLe hi levelsof apo B mRiNA occur during theS2,m@has@of 1.E liverdeve ooment (i.e.ti.nn7a-te--Cc-staa-tiLco@@_-@t@-@@e followedbv an abrupt.ear y postnat drop 1121.This Or-o@@sionfor expressingthe principalapofipoprotein involvedin cholesterotlransportat a time of maximal endogenous productionemphasizes the co-ordinatenature of these developmentalchanges in the fetalrat 0.40.3 0.2 i Birth 0.1 MMG CoA Reductase 16IAMI2I4 3 ',1 24 35 70 1921 Days of Development FiS.1. Ck-elopmenLd changes in rat liverH,%(G-COA Synth=. H%10.CaA Reductisaend PrcnyTtr=ufcrawMRNA levelT&otal %:cliuRlNaAr w-.upreparefdrompooledratliver(sn- 10-AOanimals rw time;xlintC)l.onedmt cDNAs encodingthethreeenzymeswere u%cdt,.pirobelotbloucontainingfourcowmtmtions ofeachRNA. The tviati%ccun%:cntration *( each miLNA was calculated based on .wanninglawr%icnsiwnwtroyffiltearutoracrwgmphsand cipressedin 4rbitr4rJycnsitOrneturniiCtlNote thatthespedrtewtivitkosf the proti,w"eren,,iitdenticalnd thereforneo comparisonsmn be made abouttherelatilvevelosfeachmIL14Aata particulsatrarof liver 4ev.op L liver. The sic.,naflosrthe rapid postnatalfallin the capacityof the liversof sucklingrats to svntCS e c o esterolare not known. Itmay representan aiLa2ke response to ade uatesu liesof ics=ol Sefiv@erevdia*m thees milk [3,33,37)B.ased on the RNA blothy )ridizatiodnata.itappears thatthe mechanism involvesat leastin part a reductionin the steady-state levelsof mRN-As encoding these kev enzymes in cholesterolbiosynthesisT.he data do not allowus tosay to what extentsuch alterationrseilecea chlicnca Ingene transcriptionr mR','A stability. The changes observed in the levelsof prenyltrans- feraseMRINA durina- the latesucklinact,hrouaahwe.aning phases can be directlycorrelatedwith changes in the levelsof thisenzyme activit(y3).By the 14th-poo.s,tnat- al day, H.@v[G-CoA reduct,, Ii-nireart hasfallen t ry-un-det_t-"-@@ (361.However, duringthis second postnatalweek. risesIn nr,-n!itlr!insfer-aOsccur- @jirsi@L Lui*nLi@cs@with@@@@' ell- zy-me-inv-orvvteinet--htnd-hdce me valonate to squalent pathw@Y:- 296 Lung Intestine 1.2- 1.0. 0.8. O.S. 0.4. 0.2 Cl) Birth HMG CoA Synthass L 1.21.0O.S. o.e- 0.4 0.2 Birth HMG CoA Synthass 2.0. Pronyl Tran3fora38 0 1.0- E 0 O.S. I cn (D 1 0.2 Birth Birth HMG CoA Reductase 0.1. n I liti i i 21 Day of Development 2.0- Birth 1.0- E 0.5 cc illir Pranyl Tran3fora39 HMG CoA Rackietase 0.2- Birth 1 Rd IF mli '48 n 24 21 Day of Development Brain Birth 1.2- HMG CoA Syntha36 0.8- 0.4 Kidney 1.2. Birth O.S. HMG CoA Syntha3a 0.4- 2.0. CD 1.0- E O.S. Cl) c CD Birth 0.2Birth Pranyl Tran3fora39 HMG CoA Reducta3$ 2.0. Birth OD 1.0- E 0 O.S. co c 0 Birth 0.2- Pranyt Tran3forase HMG CoA Reductase gig n I'Ii4i 1 i la Day of Development InI Iiii i v 24 a Day of Development Fig2.. Coordinatepatternsof iccumulation of H16IG-CoA synthas-.p.renyl transterasea.nd HNIG-COA reductasemRNAs during lung.intestinal brainand kidney dcwlopMCnL ToW cellulaRrNA was isolatedfrom poolcd tissuesh:irvestefdrom 10-40 mLs at each day of fetaland posuutai lifenoted on the x-axis.Rel.2tivMeRNA concentrationwsere determined by scanning dot blot autoradiographsand expressedin arbitrary dwsiuxoetricunitlThe onlycomparison which ispermittedby thisform of data expressionisthatwhich involvesthesam rnRNA witwn a given dslue. 297 iquatene synthetase.These changes in prenyt trans(eraseand squalent synthetaseactivitydo not corre- A cloned human H,*v(G-CoA reductaseCDNA plus cDtNAs encoding rat HNIO-COA synthase-and rat pre. zipond to any known change in cholesteroslynthesis. Brut:n-,earnd Rillina(.31note,.t'hatsince the primary metabolic destinationof isoprenoid precursorsis cholesterolc.hanges in the.activitieosf theseenzymes nyt transterasewere used for thesestudies.Since the lasttwo representhetero[ogouscDiNAs. a preliminary experiment was performed.Northern blotsof RNA prepared from a human hepatoccitulacrarcinoma cell may reflectas yet unknown developmental alterations in the metabolictargetinogf mevalonate to othercompounds (e.g.d.olichotand itsderivativesu.biquinones. or isopeneenytERNAs (381and prenylatedproteins[391). Although informationabout the activitpyrofileof H',Iv[G-CoAsynthast during rat liverdevelopment has not been documented in the literatureb.ased on the MP,NA data presentedin Fig. 1. itsdevelopmental line(Hep G2) were probed withthe ratcDNAs employ. ing the same hybridizatiosntringencielsistedin Ref. 12 but the finalwash temperaturewas reduced from 55aC to 45 *C. The resultsindicatedthat each rat probe reactedwith a unique human MRNA species-3.1and 2.2 kb HN,(G-CoA synthaseMRNA and a 1.2kb prenyl transferasMeRNA (data not shown). These sizesare comparable to those previouslyreported for the corre- profilewould be.predictedto more closelyresemblethat of prenyt transferasethan H,%,(G-CoA 'reductaseM.oreover,the blothybridizatiosntudiesdemonstrate forthe firsttime that there are co-ordinatedevelopmental spondina ratmR,\[As (23,401D.ot blotsof totalceuular RNA is@latedfrom 8.5-25-week fetallivers(n - 1-3 individualpser time point)plustwo newborns and one adultwithout evidenceof Uver dysfunctionwere then changes in the levelsof thesethreeniRiNAs encoding enzymes involvedin cholesterobliosynthesis. prepared and probed with the threecDNAs using hybridizationand washing conditionsestabushed above. As in thecaseof theratliverRNA dot blots,multiple E.rpres,sioofn the H,@fG-CoA s.-nihaseH,.%,IG-CoAredliclasweid prenyltransferasge*nes in e.-ctrahepattiisc- concentrations(0.5-211-)of each human liverRNA sample were includedin thedot blots(seepanel A of sl,esdilriiriagtdeveloprnent The remarkablesimilaritiyn the accumulation'pro- fileso'f thesethreemRNAs observed durin0a the perinatalperiodof ratliverdiffer'entiatiwoans not unique to thisorgan.This isillustrateidn Fig.2 which sum- marizesresultsobtained from probin- dot blotsof lung, small intestinablr.ainand kidney Ri@As preparedfrom 10-40 animals ateach ofas manv as tendifferensttacres of development.Two obvious corclusionscan be made afterinspectionof the data.Firs-,t.hetiming and direc- tioriof developmental variationin r-eT-ra@tR--iivNeA con- rationwas vtrtualiivdenticalfo n e tissueS.econd. as in the liver,the highestcon- c trationof each MRNA durin- lun-.intestinanld kidney development was encountered in the lateand earlypostpartum periodwith subsequent declinesoc- currinain the sucklin-and/or weanin- phases.The notableexception was brain where a 12cogtessivr-)eost- nlicalrisein the concentrationof each MRNA occurred @.*1@-@d ciirkl0ina p Lod.Thus expressionof the rat H.N,[G-CoA synthase, H.@,[G-CoA reductastand pren,,-tlransferastcenes ap- pe:irsto be elaboratelyprogrammed to respond in a similartemporal fashionboth within and between dif- fe.rentissuesfrom latefetallifethrough the end of weaning. Fig.3). Inspectionof Ficl,3.B revealsa relativelmyonotonous developmentalprorileforallthreemRNAs. By 9 weeks of -estationthese mRNAs have achieved steady-state levelswhich do not change more than 2-foldduring the remainin- 17 weeks of fetalifethat were surv4@,veadnd lessthano3-5-foldwhen compared to the two newborn and singleadult RNA preparations.When therewas an opportunityto compare more than one sample at a given staze of human fetaldevelopment, remarkably littliendividualvariatiownas noted in the relativleevel of a givenMRNA. This less than 2-foldchance in cholesterocenic MRNA levelsduring fetalifeis not a cencralphenomena. Itcontrastswith resultsobtained when thesesame RNA preparationswere probed with other cDNAs encoding proteinsnot involvedin Lipidmetabolism.For example. a controlexperimentwhich examined thelevels of a-feeoproteiMnRNA disclosedthe approx. 10-fold reductionexpected between firsttrimesterliverand liverharvestedat thebe-inninaof the lasttrimeste(rsee panels A and B of Fig.3) [411.In addition.a recent study revealedmarked changes in the levelsof epsilon. gamma and theta globinmRNAs as well as cerutoplasmin MRNA in the 10-25-week human fetalliver RNA samples (421.L*vtorcovetrh.e patterns of change of these fivedifferentmRN.@k!iwere quite ctistincftrom .4(.-citipiLoiflnatiRt.uYn.4sencodingcholesterobliosynthetic ett:.I,Pa)lIiedslipidtratispoprrtoteiiiisnfetali,teonataalnd ildi,hiutnittilriver Fig.3 providesthe resultosf our analysisof develop- mental changes in the concentrationof the three cholesterolbiasynthetiecnzyme mRNAs in human liver. ' one another. There is littlienformationabout the activitieosf thesecholesterolbiosyntheticenzymes durina human fetaldevelopment. However. the relativelmyonotonous HMG-COA reductase.H,*viG-CoA synthaseand pranyt transferasMeRNA level-aire compatible with observa- 298 A PrenylTransftrcseCONA GestationalAge (weeks) 8.5 10 11 IZ5 13 14 15 16.517 21 25 a -fetoorateinCONA GestationaAlge (weeks) 8.5 10.11 12.513 14 15 165 17 16.521 25 10 0.5 lk Z .0 40m.b0L---skitAb A CL5 z 05 newborn 6y ad '7r 0.5 n"lrn Slyo@ FBI 6 4 a 6 4 E2 0 CD G4L L KW COA SynftSS HmG C*A Ro"tase Tal PrWIl TraMISfa3@ u voom :io 11 :ia *13 914 017 xii 4 2 c 4- E2 (D a4 2 Apo At Apo An Apo 5 L-FAW A 4 2 U 10-12 13-14 16-18 21 - 25 NowI som Aduft FetalAgo (weeks) 2 1 8.5 10-12 1:@14 16-18 21 2D FetalAge (weeks). New- Adult Born Fig.3. Dc%-clopmentalchanges in the concentrationof mRNAs encoding cholesterogenicenzymes in samples of human fetalliver.cDNAs encoding human H@@,10-CoA reducms@-plus r2t H,%IG-CoA synthase,rat prenyl u-2nsfer3se(plus human a-fetoproteinw)ere used to probe dot blols conwain$ sever2lconcerLtmtioeL(s0.5-2;Lg)of eachhuman Uvcr totalcellulaIrL'4A sample. Fetalage isbased on crown-rump lengths(151.Fetal age isbased on crown-rump lengths(151.'4otethatthetwo 14-we-eksamples came from scp&ratefetuses(seethe key definingthe symbol ascribed to each R4A preparation)P.anel A displaysreprescntalidvoet blou probed with 3P-labeledprenyltransferasacnd a-fetoprotciCnDNAS. Panel B presentsdevelopmental profilesof mIL4As encoding the threecholesterobliosynthetiecnzymes and a-fetoproteinA.s in Ft& I arbitrary densitomeuicunitswere used to expresstniLNA levellNo conclusionscan be made about therelativceoncentrationsof each of thefour mRiNAs in a tiverRNA sample prepared at a particuladr4evelopmenW stage- Fi& 4. De%-ctopmentatprofileof human liverrnRNAs specifyingproteinsinvolvedin lipidtransport.StoLsprepared with the same- fetaln.eonaw and 2dulthuman liverRN*A samples u those used to generatethe data sh&3wn in Fig.3.were probed with @:P-lab@elecdloned CD-NA specifying hum= apolip@oproteianpso A[. apo All.apo B.and liverfattyacidbindingproteiniL-FASP). tionsmade by Carr and Simpson (431 thatonly slight alterationosccur in the rateor cholesteroslynthesiisn the developinghuman feW liver(.Thisratewas estimated to be appro.-9,.mg/day duringtheIgthweek of gestation.) When the levelsOf other MR@NAS encodingproteins involved in Upid transport were exartuned in huma n fetalliver.sinlilar'flat'developmental profileswere observed (Fi& 4).mRNA3 specifyingthe two principal human HDL apolipoprot6ns, Al and All, plus the principalproteincomponent of LDL, apo B achieve steady-statcco:ncentrationbsy 8 weeks of acestationthat are similarto thosedocumented during.weeks 10-25 and in term infantsV.ariationswerc lessthan 2-foldin allcases.'L@viorcotvheerM.RNA encodinglivqE_!?_tly-Aci ein bindineprotein(L-FABPI. a s be ieve to artici atdj 3 : - . c -,nd/oc -ohnilc p.r_ocemtnL_01_-Ce.-x--Oof-antnXtousaci4- (reviewed in ReL 44).displaysa similarlack of change in itsconcentm- tion during this period (Fi& 4).Together these data 299 .iugaesatn earlybiochemicalmaturationOf Lhehuman fe"ta.iliiverwith respectto itscapacityfor cholesterol ,@vniliesain.dilipidtransport. populationsare obviouslyimportantwhen considc:ring regulatioonf gene expressionin thistissueF.or example.therelativelcyonstantlevelosf mRNAs encoding proteinsinvolvedin lipidmetabolismthatwere docu- Dil.)rc!rentoifcitthiehounr?taannd ratfetaliver There are very few studieswhich have provided informationabout the biochemicalmaturityof human liverfrom midgestationtoterm.so itisverydifficutlot put ourdataconcerningexpressionof genes involvedin lipidmetabolismin any sortof comparativemetabolic contextat the presenttime.Greengard has conducted an extensiveanalysisof theliteraturdeealingwithen- mented intotalhuman fetaUlverRNA duringgestation may *mask'relativeldyramaticchanges in theirconcentrationwsithina givencelltypeand/or changes in theirexpressionin differenctelltypes.Therefore,itwUl be veryimportantto extendtheseanalysestlsingin situ hybridizatioannd immunocytochen-dcaltechniquesto examine changesin thecellspecifiecxpressionof thm genes in thefetalhuman and ratliver. zymatic differentiatioofnhuman and rat liver[451. Although the data for human are both limitedin the Acknowledgements number of.enzymes examined and the scope of the analysesthrough fetallifeb.ased on publishedreports We thank David Sweetserforhisassistancien pre- concerningapprox. 30 enzymes itappears thatmost paringtotalcellulaRrNA from human fetaliversam- liverenzymes exhibitsignificanqtuantitativdeifferences plm Aldons J. Lusis (UCLA) for supplyinga cioned in theiractivitiebsetween the second trimesterand apo B CDNA, and RobertSimoni(Stanford)forprovid- adulthood.For example. enzymes involvedin synthesis inga clonedhamsterHMO-COA reductaseCDNA. Sup- of DTNA. pentoses.nonessentialan-Linaocids.as wellas portedby Grants DK 37960and HL 30568 from the glycolysihsave activitieisn the activelygrowing fetal NationalInstituteosf Health.A.L.S.and J.I.G.are liverwhich are differenftrom thosein theadulttissue. F-stablisheIdnvestigatoorfs theAmerican Heart Associ- In the few examples where developmentalprofileasre atlon. availablien fetaland newbom human and r,,laitveri.t- a!nea@rshat enzvv@ee@* inn tnhce-s5.i, ti;_@i@th@mam@arta@@@ithough thetim- References ing of such changes has not been wellenough characterizetdoprovidea comparativetimescaleforhuman and ratliverdifferentiatiCoonm.parison of our ob:;er-vaticoonnscerninghuman apo Al. apo B and LF,-\BPMRN*A levelsin totalhuman fetalliverRNA \,viephr,:viousptuvblishedstudiesin ratliver(6.12.461 susaoesesthatthe8-12-week human fetaliverisatleast as %vei'ldifferentiateads*the 19-21-day fetalratliver \,vitrhespectto expressionof thesemRiNAs. Further studiesb.asedon RINA hybridizationtechniquess.hould prove valuableinestablish0int-hiscomparativedevelopmental timescalefora varietyof differentiatfeudnc- tions. 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