Document zb3mkm06OMLkpLDBMEypELjpR

r )93 Developmental changes in the expressionof genes in,,,olved in cholesterolbiosynthesisand lipidtransportin human and ratfetalan'd neonatal livers iviarcS. Le--@i1n,Alan J.A.Pitt2 ,Alan L. Schwar tZ 3.,'P.eter A. Ed,%-ards and JeffreyI.Gordon'-:: Deparrnientosf 1 Wedicine.: Bt(jchemisiarntd-.VoleculaBriopkrjicsJ.Pediatricasnd 'Pharmacologv.14,ashinjrr(o-n'Rivcr1s-Stcrhool of.'4edicinSet..Louis..%40and @ Deparrmentiof.4fedicianned BiologicaClhemisfty.L'CL4 Scbootof.%fedicinLeo.s.4ng'eles. t CA (L*.S.A.) (Received23 December 1988) Key words: Development;Upid transportE;xpression;Cholesterol Cloned cDNAs encodinga number of enzymes involvedin cholesterobliosynthesiass well as extraceuulaarnd intracellullairpidtransporwtere usedto compare thedevelopmentalmaturationof thesebiologicfunctionsin thefetal and neonatalmt and human Uver.'ne resultosf RNA blothybridizatioannalvsesindicattehatsteady-statleevelsof mt HMG-COA synthase,KMCCOA reductaseand prenyltransferasme%NAs arehighestin latefetallifeand undergo precipitou(supto Wfold) co-ordinatreeductionismmediatelyafterparturitioTnh.ese chmilesreflecttheabilitoyf the fetalratliverto produceINOM titi f cholesteroals wellas the repressionof thisfunctionduringthesuckling x2riodinrmgnse to ex dietary trikingco-ordinatpeatternsofEM CZ:@o synthase,reductase and prenyl-transferaMsReNA accumtdationwere alsoobservedin four extrabepatimct tissues(bmin,.Iungi,ntestine f and kidney)duringthepednaw periodI.Me concentrationosf allthreemRNAs inthe8-week-oldhuman fetalliverare similarto thoseobservedthroughoutsubsequentintrauterindeevelopmentwithlessdm 2-foldchangesnotedbetween theSththrough25thweeks of gestationA.nalysisof the levelsof human apo AI,apo AII,apo B and liverfattyacid bindingproteinmRNAs duringthisperiodand in newborn liverspecimensalsoindicatedlessthan 2-3-foldchanges. These observationsuggestthatthe 8-week human liverhas achieveda high degreeof biochemicaldifferentiatiwointh respectto functionsinvolvedin lipimdetabolism/transportwhich may be comparabletothatpresentin 19-21 day fetal ratliverF.urtheranalysiosf human and ratfetaliverRNAs usingclonedcDNAs shotddpermitconstructioonf a developmentaltime scalecorrelatinhgepaticbiochemicaldifferentiatitoonbe constructebdetween thesetwo main- malian species. Introduction developmentalalterationisn the rateof incorporation Marked changes in the requirementsfor products of radiolabeledacetateintosterols[2].Bruenger and Rilling[3]documented chanizesin theactivitioefs two derivedfrom isoprene(e.g.cholesterolo)ccur during development. The enz.-me which catalyzes the key, other cholesterogetuc enzymes in the developing rat ---------liver:squaiene syntnetaseand prenyl transferase(or rate-iimitinsgtep in c oesterol biosyn-tb=is-rr@cro- farnesyl/pyrophosph ate synthetase).Prenyl transferase somal 3-h-vdrc@x-y-3-methylglutaryl-CoAreductase isone of fiveenzymes thatparticipate'inthe conversion (HMG-COA reductase)-underg ,s lar&c fluctuatnis of-TpEg@_na@e to@squalene, the precursnr nf tterpls. durinstrat livernnt e;ny [1.2].Em Ene activtiy.!ilbigh Both squalene e and orenyl transferasedivjay priorto birth.declinesto low levelsdli@n the suckling similaidevelopmental activityprofilesin rat liver:they @@er,,@@natal days-T--13)-2neidemonstrates a tran- fallfollowin-b&irth,riseto a peak durinstthe mid tojate sientiii-creaasttwearung. t-nanges in itsactivityparallel sucklinyt)eriod(postnataldays 10-12). fallnnr&More during the sucklina-weaningtransitionreaching a nadir I Currcspondence: M.S. Levin. Department o( Medicine. Wastii..,.-.. University School of Medicine. 660 South Euclid Am. Box 8124. SL LA-misM.O 63110.U.S-k by -postnatalday 20. oD ly tn ri-cnin, 0- 9 weaning. ne mechanisms which resultin these alter- N ations in enzyme activitymay be quite complex as illustratebdy the fact that the mevalonati.-mediated 0005-2760/19/$030@510989ElscvicSrcicwcPubUdmn B.V.(BiomedieDWivision) decrease in H-N,[G-COA rcduccase levelsobserved in adultanimals retlectdsecreasesin gene transcriptioans well as increasedratesof proteindeeradation(4.-il. Littleiskno-n about the developmental historvof the activitieosf these enzymes in human fetaland neonatal liveror about the ontogeny of expressionof c,entsinvolved in the transporcand metabolic proce-@sing of lipidsin thistissueT.he ratliverprovidesa convenient referencefor a comparative study of such developmental changes.For example in additionto the information about expression of cholesterogenicenzyme activitiesr.ecentanalyses of apolipoproteingene expression in the developing rat liverindicatethata complex patternof activationoccurs during latefetaland early neonatal life.Apolipoprote'LnAI and E mRNAs b4n to accumulate in this tissueplus itsembryonic homologue (thefetalyolk sac endoderm) between days 15-21 of the 21-day gestation period (6-91. Remarkable increasesin the levelsof thesemIL4As occur during the suckling period as the animals adapt to the high fat (principalltyriacylglycerodli)etof mothersmilk[10,11). By contrast,ratliverapo B MR,.NA levelsreacha peak by the 13th fetalday that isnot exceeded at any time durin- subsequent development (121.Following birth, hepaticapo B MRNA concentrationsprogressivelyfaU during the sucklingax@d weaning periods [121.This fall appears to be mediated by thyroxine[13).A third patternof activatioins exhibitedby the apo AIV gene which remains dormant untilthe sucklingweanina transition(days 13-14) when the-atliverbeginsto export largeamounts of triac.vlglyc.-rrioclh-lipoproteins(6]. We have begun a comparative analysisof the accumutation of mRSAs encoding proteinsinvolvedin lipid metabolism in the fetaland neonatal human and rat liver.A panel of cloned cDNAs encodina apoupoproLeinsAI. All, and B. an intracellulafrattyacidbinding protein as well as H'.IvIG-CoA reductase,HLNIG-COA synthase and prenyl transferasewere used to characterizethe stateof enzymatic differentiationf the human fetalliverfrom weeks 8 to 25 of development and in the newborn. The resultsindicatethat these mRNAs appear at an earivphase of human fetallife (by week 3) and unde-,zoonl. y rninimat(lessthan 3-fold cl,iangesi)n theirconc,-nt.-atEon in totalliverRNA durin- the restof intrauterineas wellas earlypostnatallife. Th'tsearlyexpression of lipidmetaboliccapabilityconcrastswith the.more markell changes in MRNA levels observed in the perinatalratliver. .N,(uteriaalnsd ,'viethods Preparationof R@V,4froni ratatidhuman liver Timed-pregnant, neonatal and young adultSprague- Dawley catswere obtained from Sasco (St.Louis,MO). Weaned animals were maintained on a standard chow dietad libitumand a fixed12 h (6:00a.m. to 6:00 p.m.) lightcycle.Animals (n - 10-40 for each time point) were killedbetween 1200 and 14CO h and theirlivers. tuncs.brains.kidnevs and small intestinesimmediate(,,. frozen in liquidnicroat:n.Human fetalliversampi from firstand secony trimesteraborted fetuseswecs obtained by Schwartz et al.[14] and maintained at -90*C for 15-20 years.Fetalage was estimatedfrorr. crown-r-ump lengths using nomograms developed by Tanimura et al.(151.Breitfeldand Schwartz (161 and LMichaelsonand Orkin (171 have used these human fe(a: liversamples previouslyto successfullyprepare RLNA for in vitrotranslationA.dditional human liversa.mplcz 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 (14).A singleadult liverspecimen was procured from a male organ donor who died of trauma and had no historyot clinicalevidence of hepatic dysfunction 118). Total ceuular RINA was extracted from frozen pulverizedtissuesusing the guanidine thiocyanate,, cesium chlorideprocedure [191.RNA integritwyas as sessed by denaturine,methvlmcrcury agarose gel clec trophoresis(201. R.,VAblothybridizatiosntudies Dot blotswere prepared by applyin- four amount of each tissueR.LNA sample (0.5.1. 2. and 3 Ac, t nitrocellulosfeilterass described in a previous public:! tion (6].Yeast TR.NA was added to each tissueRN.sample prior to denaturation so that the totalRN.input per dot was always 3 iLa.Blotscontainingsample of ratliver,intestinek,idnev. brain and lung RNA weprobed with 3@P-labeled.double-stranded cDNAs e7 codina hamster Hi)viG-CoA reductase(221,rat HNIC CoA synthase (231.and rat prenyt transferase(241.D( blotsof human liverRNA samples were probed wi: "P-labeled cDNAs specifying human preproapo i 125].human preproapo All [26].human preapo B (2human liverfattyacid bindin- protein [281.humia-fetoprotein(291.human H*vfG-CoA reductase(301,r prenyt transfeme and rat H.'v(G-CoA synthase.Conc tionsselectedfor filterhvbridizationand washing a listedin Rc:f.12.These scrin-,enciewsere equivalent those used by others to produce specificinteractio between these cDtNAs and theirrespectivemRLNAs. T' relativeabundance of each MRNA in the tissueRiN preparationswas determined by quantitativescanniWer densitometry of filterauturadiographs usinco, LKB XL Ultroscandensitometer.Only sianalsin t linearranue of rilmsensitivitwyere utilizefdorcalcu tionsof relativMeRNA concentration. Northern blotsof totalcellularRi'4A were produc following clectrophoreticfractionationthrough a-,arosegetscontaining formaldehyde [31@ 295 Retuitsand Discussion .4ccitmulutionf*H.VIG-Co,4 st-itthasHe.,VfG-CoA retilictcazisredp,-entt-,I-ansferamsRe.V.4s in thedeveloping raihve,- Cloned cD'4As encodina,HNIG-COA synthase, H*v[G-CoA reductasea.nd prenvl transferasweereused to probedot blotsof totalcelluiarRINA preparedfrom ratliversharvestedduring fetaldays 16-21. thesucklingperiod(definedas the first13 posmatal days.Ref. 32).theweaning phase(days14-28) and from animals proceedingthroughsexualmaturation(postnatadlays 35-70).Nlateriawlas collectedfrom 10 to 40 maleand femaleanimalsrepresentin1g-4 litterast eachdevelopmental stagestudied.The resultsof our dot blot hybridizatiaonnalyseasrepresentedinFig.I and show 3.0 2.0 - Birth to Liver HW CoA Synthase PronytTransfora36 2-.0 ,ui to Birth 0.40.31 0.2i- Birth 0.1 HMG CoA Reducta36 16 2 4 5 -4 24 35 7,,0 1921 Days of Development i. C@-vtiopmenLcihluges in mt liverHN,(G-CoA Synth=. Rc:du%:taawnd Pmn)rtTr=sfawe mR.'4ALevelTso.tal celluiRaNrA --.ipsreparedfrompooledr3tliver(sm - 10-4 aninub pertime;xvicn).Clonedmt cDNAs tra*dingthethreenzymeswere u,.,CtdiprobeJutblotcsonWainSfourconmntmtionsofeachRNA. The mlati-:.ocn-.;cntmotfieoanchmiLSA was calcuL2tbeadud on .urinninltuer%knsiwcnetroyf filtearutoradiographasnd exprcoodin arbitr.Iictnysitt)mcturnictlNote thatthespmiric,2ctivoifctihees prc)b*cvsfvfklitdenticuaWl therefonroecomparlwncsanbe made abojuttherelatilveeveolfseachmL*4Aata particusluaprofliver dtvt[OPMML a remarkablesimilaritiyn the p@ictcrnosf change of each MRN*A duringratfetaland neonataldevelopmenl Hi h levelsare noted durin--Li-tge-stationT.hese fall abruptlywithin 24 h afterparturitionrt:a n con- cenEratLotnhsatareasmuch as30-folldowerthanthose pe-arTev,!ie-nscounteredpriorto -6t-r-(t;h.g.s.ee the middle panelof Fig.I which shows chancesin prenyl transfecasMeRNA concentrationA).transien2t-4-fold risein HIvtG-CoA synthastand prenyttransferasbeut not HNIG-COA reductasemRtNA accumulationoccurs duringthemid-sucklinpgeriod(day8) followedby yet anotherriseduringearlyadulthood.Itistmportancto n-ot-c-trat--Egs-p-atE-er-n-o-its-ncogtana.-g-ecneralphe- nomenoa in thedevelopingratliverF.or example,when the developmentalprofileosf apo AI, apo AIV and L-FABP mR.NAs were studiedusingtheseP-'4*Asq,uite differentontologicchangeswere noted (see,forexam- ple,Ref. 6). The developmentalprofileof rat liverHMG-COA reductaseand prenyltransferasmeRNAs paralletlhe changes in the activitiesof these enzymes which have been previouslydocumented by severalgroups(3,33-351. The correspondingactivitieosf Hk'v(G-CoA synthase have not been reportedT.he rat fetusobtains0 about 1OFoof thesterolrequiredforgrowth and devel- opm!nt from itsmother(361T.hereforei.tisnotsurpris- in-at,hatvery highlevelsof thesemRNAs are observed inratfetaliverdurinagtheperiodsurveyed.The hi&hkst levelsof apo B mRiNA occurduringthesame phasc-of ratliverdevelopment(i.e.i.nlategestationonl to followedby an abrupt.earlypostnat top 121.This proVISLonfor expressin-the principalapoupoprotein involvedincholesterotlransportat a time of maximal endogenous productionemphasizesthe co-ordinatena- lureof thesedevelopmentalchanves in the fetalrat liver. Thet-precise5:i-.naflosrthe rapidpostnatalf,allin the capacttvof the liversof sucklin&_Et2@,o svi ie CKLoraers-te;rot not known. Itmay representan ad c rlspo_nseto ad igrholesterot deliveredviainother'msilk[3,33,37B1a.sed on theRNA blothy)ridizatiodnata.itappearsthatthemechanism involvesat leastin parta reductionin thesteady-state levelsof mRNAs encodin-,theseke.venzymes in cholesterol biosynthesis. The data do not allow us to say to what extent such alterations reflect a change in Sent Eranscriptionr MR.N'A stability. The chan-es observed in the levelsof prenyl trans- ferasemR.N'.Z durin,-t,he latesucklin- throuah weaning phasescan be directlcyorrelatewdith changesin the levelsof thisenzyme activie[y31.B the 14 ostnatal day,H'.Iv[G-CoAreductaseactivitiyn ralliverhas fallen Fynu- 4eecUblg@ (3,61Ho.we ver,duringthis second postnatalweek.risesirL12renyflrnnsferasotc:currisecoincidewsitihAthhaaUl_d&hLbuc4i-b*-.,nLeLnb-a, zymfin-voiv atent:paLhwaYr 296 Lung 1.2- Birth 1.0. 1 O.S. O.G. 0.4. 0.2- HMG CoA Synthase Intestine 1.2- 1.0- o-a0.0- 0.4- 02- Birth HMG CoA Syntha36 2.0. 1.0- Birth E 0 O.S. I (D . 0.2- Pranyt Tran3f@rass p 9 9v HMG CoA Redwtass 2.0. Birth 1.0- E 0 O.S. co ll&q ip Pronyl Tran3fora39 0.2Birth HMG CoA Rockictass li24 1 14 t4 21 x 74 Day of Development 0-1- 1v 24 3 21 Day of Development am 1.2- Brain HMG CoA Syntha36 0.4- Kidney 1.2. Birth HMG CoA Syntha36 0.8- 0.4. co 2.0. 1.5- CD 1.0- Birth E 0 0.5 I Pranyl Tran3fSra39 2.0. Birth CD 1.0- r= 0.5 0 Pronyl Tran3forass 0.2- HMG CoA Reducta39 . Birth 0.1- n @41, 4 1 Ia Day of Development CD Birth 0.2. HMG CoA Reductase 0.1. n Day of Development Ft& L Coordinatepatternsof accumulationof HNfG-CoA syntham.prenyltrawferue.,2ndHNIG-COA reductasemRNAs duringlung.inteStia2l. brainand kidneydevelopmenl TotalcellulaRrNA was isolatefdrom pooledtissuehsarvestedfrom 10-40 catsat each day of fetaalnd postnatal fifenoted on the x-axis.RelativemR,*4A concentrationwsere detern-Anebdy scanningdo( blot autoradiagraphsand expressedin arbitrary dc"teowuk unitsM.w onlycompafimn whichispermittebdy thisform of dataexpressioinsthatwhichinvolvesthesanw fnRNA withina Siven 297 squatene synthetase.These changes in prenyt transferaseand squalene synthetasca:ctivitydo not correspond to any known change in cholesterolsynthesis. Bru,:n,,erand PIlling(31 note"' that since the primary mctabolic destination of isoprenoid precursors is cholesterol.changes in the acti@.ieicosf these enzymes may reflectas yet unknown developmental alterations in the metabolic targetingof mevalonate to other compounds (e.g..dolichot and its derivatives,ubiquinones, or isopencenyltRNAs (381and prenviated proteins(39]). Although information about the activityprofileof H,N,[G-CoA synthase during rat liverdevelopment has not been documented in the literature,based on the mR'4'A data presented in Fig. 1. its developmental profilewould be predicted to more closelyresemble that of prenyt transferasethan HN,(G-CoA reductase.Moreover,the blot hybridizationstudies demonstrate forthe firsttime that there are co-ordinate developmental changes in the levelsof these three mRNAs encoding enzymes involved in cholesterolbiosynthesis. A cloned human fi4lvtG-CoAreductast CD%N*A plus cDiNAs encoding ratH4@10-CoA s.vnthaseand rat pre. nyt transferasewere used for these studies.Since the last two represent heterologous cDtNAs. a preliminary experiment was performed. inorthern blots of RNA prepared from a human hepatoccitularcarcinoma cell line(Hep G2) were probed with the rat cDLNAs employ- ing the same hybridizationstringencieslistedin Ref. 12 but the rinalwash temperature was reduced from 55*C to 45 * C. The resultsindicated that each rat probe reacted with a unique human mRiNA species-3.1 and 2.2 kb HN,[G-CoA transferastMRNA synthase MRNA and a 1.2 kb prenyt (data not shown). These sizes are comparable to those previously reported for the corre- spondin- rat mRNAs (23,401.Dot blots of totalceuular RNA i's@tated from 8.5-25-week fetallivers(n-1-3 individualsper time point)plus two newbonls and one adult without evidence of liverdysfunction were then prepared and probed with the three cDNAs using hybridizationand washing conditions established above. As in the case of the ratliverRNA dot blots,multiple E.rpressionof the HMG-COA svnihase, H.VfG-CoA redticiasewid prenyl transferasegenes tn exfrahepaticrissiiesdilringrat development The remarkable similarityin the accumulation 'profiles*or these three mRLNAs observed during the perinatal period of rat liverdiffer@ntiationwas not unique to thisor-an. This is illustratedin Fig. 2 which summarizes resultsobtained from probina dot blotsof lung. small intestinalb.rain and kidnev RNAs prepared from 10-40 animals at each of as many as ten differentstages of development. Two obvious corc!usions can be made afterinspection of the data. First.the timing and direction of developmental variation in r-erat-iMvR-NeA conc@nrat@ionwa@@ virtu@av ei@ntic@al foLtub-s@-@n exen-ti-ssS-euceo-.nd. as in the Liver,the highest concentraeion of each mR,14'A during lung. intestinaland kidney development was encountered in the late and early postpartum period with subsequent declinesoccurrin- in the suckling and/or weaning phases. The notable exception was brain 'A,-herae t)roa v C)OSTnatal risein the.concentration of each,m.R"-4,-o\ccurred Eti-ac@enerally@reac@aheodcak in the mid-to lategnekling ps-r@tedT.hus -@.xpressionof the rat H.'v[G-CoA synthase, H.%(G-CoA reductase and pren,..ltransferasegenes apPeLirsto bc claboratclv programmed to respond in a similar temporal fashion both within and between different tissuesfrom late fetallife through the end of weaning. concentrations (0.5-2 ;Lg)of each human liverRNA sample were included in the dot blots (see panel A of Fi-. 3). "inspectionof Fig. 3B revealsa relativelymonotonous developmental profilefor allthree mRNAs. By 8 weeks of gestation these mRNAs have achi@ved steady-state levelswhich do not chan-C more than 2-fold during the remaining 17 weeks of fetallifethat were surve@ved and less than 3-5-fold when compared to the two newbom and single adult RNA preparations.When there was an opportunity to compare more than one sample at a given stase of human fetaldevelopment. remarkably littleindividualvariationwas noted in the relativelevel of a given MRNA. This less than 2-fold chanae in cholesterogenic MRNA levelsduring fetallifeis not a general phenomena. Itcontrasts with resultsobtained when these same RNA preparations were probed with other cDN.-^%sencoding proteins not involved in lipid metabolism. For example. a control experiment which examined the levels of a-fetoproteinMRNA disclosed the approx. 10-fold reduction expected between first trimester liver and liverharVdSEed at the beginnin- of the lasttrimester(see panels A and B of Fia.3) (411.In addition.Li recent study revealed marked chanoes in the levelsof epsilon. gamma and theta globin mRNAs as well as ecruloplasmin MRNA in the 10-25-week human fetalliver R@NA samples (421. 'Lviorcovert.he patterns of change of these rive differentmRNks were quite (tistineftrom .4ccci)@itilaiotju*mniR.V.4sencoding cholestereb)ilosynthetic ett:.%-Papilledslipidtruitsporptroteiiuinfetal,treonataalnd tidtilhtuntit.l-iiver Fig.3 provides the resultsof our analysisof develop- mental changes in the concentration of the three cholesterol bicsynthetic enzyme mRNAs in human liver. ' one another. There is littleinformation about the activitieosf these cholesterolbiosyntheticenzymes during human fetaldevelopment. However. the relativelymonotonous HL*vtG-CoA reductase, HLMG-COA synthase and prenyl transferaseMRNA level.a-rie compatible with observa- 298 A Pr"l Transte, CONA GestationalAgo (week3) 8.5 10 11 12.5 13 14 15 16.517 21 25 2 0 0 0 41 0 a - fetaproteinCONA GestationaAlge (wteks) 8.5 10 11 12-513 14 15 16.5 17 -18.521 25 .. 2 0.5 iz r CL 5 Z x.tt o.s e- -9, reiftrn6y ad a5 newborn6y ad 4 2 F- 2 a6 41 a2 < M4 CoA Synthue KIWOC4A Reductase CL Pr"i TnLnstwa3a 13 ;14 a0lIeTs X21 %25 1 Cb r a-F*tWatW 42- 4. 1 E2 2 fA a 42 Apo At Apo All c:l Apo 5 # L-FABP 2 IL 21 -411 r4l U 10-1213-14 16-18 21 25 NB-ar-" Adult Fetal Age (weeks) 1 8@5 10:12 13:14 16'-18 21 25 NOW- Adult awn Fetal Ag* (weeks). Fig.3.DLN-clopmentalchanges in theconcentratioonf mR&*4As encodingcholesteroget%einczymes insamplesor human fetal Uver. cDNAs encoding human H&@,10-CoA reductase plus mt HV[G-COA synthase. rat pranyt tr=sferase (plus hum= a-fecoprocein) were used to probe dot biols containing several concentmcioas (0.3-2 ;tg) of each human Uver totalcellularILNA sample. Fetal age isbased on crown-rump lengths (15@ Fetal age is based on crown-rump lengths (151.-Note that the two 14-week samples came from separate fetuses (see the key dertaing the symbol ascribed to each RNA preparation). Panel A displays represetuativedot blou probed with 3P-labeted prenvi transferaseand a-fetoprotein cDNAs. Panel 8 prewnlsdevelopmental prordes of mRNAs encoding the three cholesterol bicsynthetic enzymes and a-fetoprotein. As in Fig. I arbitrary densicometric units were used to express MLNA lcvcL4tN.o conclusions can be made about the relativeconcentrations of each of the four mRNAs in a liverRNA sample prepared at a particulardevelopmental stage. Fig. 4. De%-elopmenLal profile of human liverfnR,4As spc:cifyingproteins involved in lipid transporl BloLs prepared with the same femi. neonaw ind .adult human liver RNA samples as those used to generate the data shown in Fig. 3. were probed with @@P-I;ibeted4:loncd CDNA spccifyiny, hum= apulipoproceins apo At. apo All. apo B. and liverfatty teid bindin$ protein iL-FxBP). tiorumade by Carr and Simpson 1431 thatonly slight alteraliooncscurinthe rate of cholesterolsyntheisnis the developing human fetal liver.(This rate was esti- mated to be approx. 9 mg/day during the 18twheek of gestation.) When the levels of oeher mR.4As encoding proteins involved in lipid transport were examined in human fetal liver, similar 'flac developmental profiles were obsernd (Fig. 4). MPNAs specifying the two principal human HDL apoupoproteism Al an'd All. plus the principalprotein component of LDL, apo B,'achieve steady-stateconcentrationsby 8 weeks ofgestationthat are similar to those documented during.weeks 10-25 and in term infantsV.ariationswere lessthan 2-foldin allcases.Lv(oreover. the MRNA encoding liver.[altygcid binding protein -FABP). a s Livillcxtnal"qmk-pmtein octieve to particiico)atien, 'hel,tptn-k%eAA,IL%ggg$CtabaiC procEFintof-evotenous fatt3f aCi4. (reviewed in Re[- 44). displays a similar lack of change in its concentm- tion during this period (Fig. 4). Together ihae dat2 2" @,ugcresatn early biochemicalmaturation of the human (;:t@liilverwith respectto itscapacity for cholesterol ,;vntl-iic.ia.nid lipidtraniport. Dti.fe,rentiatoifonthe hunian and ratfetalliver Ther,:are very few studieswhich have provided information about the biochemical maturity of human liverfrom midgestationto term.so itisvery difficultto put our data concerning expression of genes involvedin lipidmetabolism in any sort of comparative metabolic context at the present time.Greengard has conducted an extensiveanalysisof the literaturedealing with enzymatic differentiatioonf human and rat liver(45). Although the data for human are both limitedin the number of.enzymes examined and the scope of the analyses through fetallife.based on pubushed reports concerning appro-, 30 enzymes it appears that most liverenzymes exhibitsignificanqtuantitativedifferences in theiractivitiebsetween the second trimesterand adulthood.For example. enzymes involvedin synthesis or DLNA. pentoses.nonessentialamino acids.as well as glycolysishave activitieisn the activelygrowing fetal liverwhich are differentfrom those in the adulttissue. In the few examples where developmental profilesare,, availablein fetaland newbom human and ratliveri.t- a c.ars th@at er7tYz@_m@e@,e lcih@a-n-vee inn tnhee@ssaa -@th@mam@alia@@@lthough thetim- inaa of . such changes has not been well enough char- actertzedto provide a comparative time scaleforhuman and rat liverdifferentiatioCno.mparison of our ob- servationsconcernin- human apo Al. apo B and LF,-\BP MRNA levels in total human fetal liver RNA with previouslv published studies in rat liver (6.12.461 su-zo,:ststhat the 8-12-week human fetalliver isat least 0 as well *differentiated*as the 19-21-day fetal rat liver %vith respect to expression of these mRNAs. Further I studies. based on RNA hybridization techniques. should prove valuable in establishing this comparative developmental time scale for a variety of differentiated func- tions. One important caveat needs to be mentioned con- cernino,interpretation of these data. Total cellular RNA pr,:PLlr,:fdrom fetal raL and human liver bv definition in,:Iudes mPNAs from all cellular constituents in the liver. These cellular populations undergo remarkable chan,,es = durin,a, development. A limited number of quanti'tative morphometric studies of human fetal liver indicate that hepatic parenchymal cells account for Ltppro.-.5.0-70"o of its cellular population between the ,Sth to 23th weeks of gestation with ernthroid cells rcprescntina- 40-50'c of fetal liver nuclei @nd granulocytic precursors less than 1OFe (471. In the fetalrat liver, approx. 50"c or liver cells are involved in ema:opoile4@s1i-5, just prior to parturition with this number raptl_ cre:Lrln-gto less than 5?o' bv the end of the suckling equences of these shifts in ccfi populationsare obviouslyimportant when considering regulationof gene expressionin thistissue.For exampie.the relativelcyonstantlevelsof mRNAs encoding proteinsinvolvedin lipidmetabolism thatwere documented in totalhuman fetalliverRNA during gestation may 'mask' relativetdvramatic changes in theirconcentrationswithin a givencelltype and/or changes in theirexpressionin differentceU types.Therefore,itwill be very important to extend theseanalyses using in situ hybridizationand immunocytochemical techniques to examine changes in the cellspecificexpressionof tb=c genes in the fetalhuman and ratliver. Acknowledgements We thank David Sweetser for his assistancein preparing totalcellularRNA from human fetalHversampIM Aldon3 J. 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