cell lineages and oval cell progenitors in rat liver ... · preneoplastic growth of adult rat...

[CANCER RESEARCH 51, 2611-2620, May 15. 1991]

Cell Lineages and Oval Cell Progenitors in Rat Liver Development1

Nobuyoshi Shiojiri, Joan M. Lemire, and Nelson Fausto2

Department of Biology, Faculty of Science, Shizuoka University, Shhuoka, Japan [Â¡V.S.f, and Department of Pathology and Laboratory Medicine, Brown University,Providence, Rhode Island 02912 Â¡J.M. L, N. F.J

ABSTRACT

We determined whether the formation of the hepatic primordium inthe rat is associated with the expression of liver-specific markers. Further, we examined the origin of intra- and extrahepatic bile ducts andtried to establish whether there are cell types in the developing liver thatmight correspond to "stem-like" cells ("oval cells'1) that proliferate during

carcinogenesis and toxic injury in adult livers. Using in situ hybridizationand immunohistochemical methods, we show that a-fetoprotein (AFP)mRNA is detected in cells of the ventral foregut at 10.5 days of development and that the protein is first detected 1 day later. Thus, AFPtranscription occurs before liver morphogenesis, and translation of theprotein is first detected when liver cords are being formed, indicating thatAFP expression in endoderma! cells signals their commitment towardthe liver lineage. Although albumin is considered a trait of differentiatedhepatocytes, its mRNA was first detected just 1 day later than the AFPmessage. An analysis of the expression of lineage-specific cytokeratins(cytokeratins 7, 9, 18, and 19), surface markers, and histochemicaldetermination of f-glutamyl transferase activity and glycogen revealedthat (a) hepatoblasts undergo gradual maturation throughout liver development, (b) AFP- and albumin-containing hepatoblasts gave rise to intra-and extrahepatic bile ducts, and (c) hepatoblasts forming primitive intra-hepatic bile ducts during liver development have markers similar to thoseexpressed by stem-like cells that proliferate during liver carcinogenesis.

INTRODUCTION

The morphological development of the liver from the ventralforegut endoderm has been described in detail in vertebrateembryos (1, 2). The liver primordium has both endoderma! andmesodermal components, the first giving rise to hepatoblasts3to form the hepatic parenchyma while sinusoidal-lining cellsand connective tissue components originate in the mesenchymaltissue invaded by the liver cords (3). Various aspects of liverdevelopment have received considerable emphasis including theinductive interactions between mesenchyme and epithelium (3,4), the emergence of various molecular markers of liver cellmaturation (5-9), the sequential appearance of enzymes involved in various hepatic functions (10), and the origin anddevelopment of intra- and extrahepatic biliary structures (11-13). The analysis of the expression of AFP4 during liver devel

opment in rodent and human embryos is of special interestbecause this protein is present only in very small amounts inthe adult liver but is expressed in relatively large quantitiesduring hepatocarcinogenesis and in most liver neoplasms (re-

Received 11/12/90; accepted 3/4/91.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1Supported by Grant CA3S249 from the National Cancer Institute.2To whom requests for reprints should be addressed.3The term "hepatoblast" refers to n-fetoprotein-expressing cells that are first

recognized in the ventral foregut endoderm at 10 to 11.5 days of rat liverdevelopment. Through a series of differentiation steps and depending on theirlocation in the parenchyma, hepatoblasts give rise to the common bile duct (atapproximately 11.5 days of development), hepatic duct (at approximately 15days), intrahepatic bile ducts (after 15 to 16 days of development), and maturehepatocytes (maturation to hepatocytes is gradual: parenchymal cells that expressthe surface marker HES after 16 days of development and arbitrarily referred toas "immature hepatocytes"), see Fig. 9.

4 The abbreviations used are: AFP, a-fetoprotein; ALB, albumin; GGT, y-glutamyl transferase; PAP, peroxidase-antiperoxidase; CK, cytokeratin; PBS,phosphate-buffered saline; FITC, fluorescein isothiocyanate: DAB, 3,3'-diami-

nobenzidine: PNA, peanut agglutinin.

viewed in Ref. 14). Moreover, it has been established in thepast few years that in most models of experimental hepatocarcinogenesis, the cells that express AFP and its mRNA at theearly stages of the process are not typical hepatocytes but areinstead less mature cells that can function as progenitors forthe hepatocytic and ductal lineages (15-18). These findings,together with the observation that cells of presumptive biliaryorigin but capable of AFP expression proliferate in liver injuryproduced by diverse agents (19-21), give particular importanceto studies of the derivation and maturation of biliary structuresduring liver development (13, 22, 23).

Given this background, we conducted studies to examine celllineages during rat liver development. The basic premise forthis study is that the identification and characterization ofprecursors for mature hepatocytes and bile duct cells duringembryonic development are essential for the understanding ofboth liver morphogenesis and the cellular aspects of toxic injuryand neoplastic growth in the adult liver. We have focused onthe following goals: (a) determining whether the formation ofthe liver primordium from endoderma! cells is associated withthe expression of tissue specific markers; (b) identifying matu-rational changes in hepatoblasts from 10 days of gestation tobirth; (c) establishing the origin of intrahepatic bile ducts andsearching for progenitor cells that express both hepatocyte andbile duct markers; and (d) identifying the origin and steps inthe development of extrahepatic bile ducts. For these studies,we used in situ hybridization and immunohistochemical techniques to detect AFP and ALB mRNAs and the encodedproteins. We also analyzed by immunohistochemical methodsthe expression of cytokeratins and cell surface markers that areexpressed in different cell types in adult rat liver and usedhistochemical methods to demonstrate GGT activity and glycogen. We found that AFP mRNA is made by endoderma! cellsbefore the start of liver morphogenesis and that ALB mRNAappears 1 day later when cord formation starts. We show thathepatoblasts at 15 to 18 days of development give rise tointrahepatic bile ducts and that cells in these structures continueto express AFP, albumin, and other hepatocyte markers as theybegin to acquire phenotypic traits typical of ductal cells. These"dual phenotype" cells are similar to cells that proliferate in

preneoplastic growth of adult rat liver.

MATERIALS AND METHODS

Animals. Embryos from Fischer 344/CrlBR rats (Charles RiverBreeding Laboratories) were used. Pregnancy was determined by thepresence of sperm in the vaginal smear. The noon of the first day atwhich sperm was detected was considered as 0.5 days of gestation.

Demonstration of AFP and ALB by Immunohistochemistry. AFP andALB localization in fetal liver was done as previously described (12,24). Livers (after 15.5 days of gestation) or whole embryos (10.5 to14.5 days of gestation) were fixed overnight in a mixture of cold 95%ethanol and glacial acetic acid (99:1, v/v) and embedded in paraffin.Incubation with the primary1 antibodies, sheep anti-rat AFP antiserum(Nordic Laboratories; 1/100 dilution) and rabbit anti-ALB antiserum(Organotechnic; 1/100 dilution), was done for l h at room temperature.The avidin-biotin complex method was used for AFP staining (anti-sheep immunoglobulin G biotinylated antibodies; Nordic; 1/100 dilution). For ALB staining the PAP procedure was used (goat anti-rabbit

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DIFFERENTIATION OF RAT LIVER CELLS

immunoglobulin G antibodies and rabbit PAP complex). Controls forunspecific staining were done using the same procedures but addingexcess purified AFP or ALB to the primary antibody solution.

Detection of AFP and ALB mRNAs by in Situ Hybridization. Tissueswere frozen in n-hexane cooled in a dry ice-ethanol bath or fixed inparaformaldehyde and embedded in paraffin. Hybridization with 35S-

labeled probes was as described by Evarts et al. (25) except that thehybridization reaction was carried out at 43Â°C.Slides were coated with

NTB-2 emulsion, exposed for 1 to 14 days, developed, and stained withhematoxylin and eosin.

The probes used for AFP mRNA detection were riboprobes (positiveprobe and negative control) derived from plasmid RAÃ•(a gift of Dr. S.Nishi) that contained a nearly full-length fetal rat AFP complementaryDNA clone. Two fragments were excised from this plasmid (700-basepair Pstl/BamHl and 800-base pair BamHl/Pstl fragments) encodingthe 5' end and the central portion of fetal AFP mRNA, respectively.

These fragments were ligated into the corresponding sites of the transcription vector pBSM13+ (Stratagene Cloning Systems) in oppositeorientations in relation to the T7 polymerase promoter for the construction of antisense (700 base pairs) and sense (800 base pairs, negativecontrol) probes. The plasmids (pBAF700 and pBAFSOO) were linearized and transcribed with T7 RNA polymerase as described by thesupplier (Stratagene). using [Â«-/A/o-35S]UTP(5 MMfinal UTP concentration). After digestion with DNase I, the "S-labeled RNA probes

were subjected to limited alkaline hydrolysis (26) to obtain fragmentsof 75 to 200 base pairs.

Riboprobes (sense and antisense) for ALB mRNA were derived fromthe mouse ALB complementary DNA sequences of the pmalb2 plasmid(27). The 700-base pair ///mllll fragment was removed from pmalb2and subcloned in both orientations into the //indi 11 site of pGEM-3(Promega Corp.). Plasmids pGmAlb2-l (positive probe) and pGmAlb2-4 (negative control) were linearized with BamHl. In vitro transcriptionand alkaline hydrolysis were as described for the construction of AFPriboprobes, except that Promega buffer and SP6 polymerase were usedin the transcription reaction. Hybridization reactions with antisenseand sense (control) probes were run in parallel in each experiment.Further controls for hybridization with antisense probes were done bytreating the sections with 100 Mg/ml of RNase A (Sigma) for 30 minat 37Â°Cbefore hybridization.

Immunohistochemical Methods. Whole embryos at 10.5 to 14.5 daysof gestation and livers from 15.5 days of gestation to the postnatalperiod were frozen in n-hexane cooled in a dry ice-ethanol bath,sectioned at 8 ^m in a cryostat, and fixed in cold acetone for 10 min atâ€”20'C for staining of cytokeratins and cell surface markers. The

following antibodies were used: (a) mouse monoclonal antibodiesagainst human cytokeratins 7, 8, 18, and 19 (designated CK7, CK8,CK18, and CK19; Amersham Corp.; 1/10 dilution); (Â¿>)mouse monoclonal antibodies against rat M, 39,000 and 55,000 cytokeratins (a giftfrom Dr. N. Marceau; 1/500 dilution) (28, 29); (c) guinea pig polyclonalantibody against cow hoof prekeratin that recognizes a M, 52,000 ratkeratin (a gift from Dr. N. Marceau; 1/50 dilution) (30); (d) rabbitpolyclonal antibody against calf muzzle keratin (Dako Corp.; 1/300dilution); and (e) mouse monoclonal antibodies HES6 and BDS7 thatrecognize surface antigens of, respectively, hepatocytes and ductal cellsin adult rat liver (a gift of Dr. N. Marceau; 1/200 dilution for HES6and undiluted for BDS7) (29). Incubation with the primary antibodywas for l h at room temperature and, after thorough washing with 0.01M PBS, the sections were incubated with secondary antibodies also forI h at room temperature. The secondary antibodies used were (a) formouse primary antibodies, FITC- or peroxidase-labeled goat antibodies(Cappel and Dako Corp.; 1/50 dilution); (b) for guinea pig primaryantibodies, FITC-labeled rabbit antibodies (Dako; 1/50 dilution); and

(c) for calf muzzle keratin as the primary antibody, goat anti-rabbitantibodies (Jackson ImmunoResearch; 1/50 dilution, 1-h incubationfollowed by rabbit PAP complex at 1/100 dilution, incubated for 30min at room temperature). Every staining reaction was carried out inparallel with control sections in which the primary antibody was replaced by PBS or normal rabbit serum (Jackson). Sections stained withfluorescent dyes were examined in a Zeiss fluorescent microscope;peroxidase-treated sections were stained with DAB. Some sections were

stained with both HES6 and calf muzzle keratin polyclonal antibody.For this procedure, sections were incubated with a solution containingthe two antibodies, followed by incubation with a mixture containingFITC-labeled anti-mouse immunoglobulins and goat anti-rabbit anti

body. After examination of HES6 staining by immunofluorescence,sections were incubated with rabbit PAP and stained by the DABreaction.

Lectin Binding. Sections were incubated with biotinylated PNA (50Mg/ml; Vector Laboratories) for l h at room temperature and then withavidin-biotin complex (Vector) according to the manufacturer's instruc

tions. Control sections were incubated with PNA solution containing0.1 M lactose (Sigma Chemical Co.).

GGT Activity and Glycogen. GGT activity was demonstrated withthe method of Rutenberg et al. (31). Glycogen was visualized with theperiodic acid-Schiff reaction.

Transplantation of Fetal Liver Fragments. Fragments from livers atDay 13.5 of gestation were transplanted into the testes of Fischer 344rats (140 to 160 g) as previously described (32). The testes of theseanimals were removed after 2 mo, and the areas containing the transplants (identified with a dissecting microscope) were fixed in Bouin's

fluid and embedded in paraffin.

RESULTS

Developmental Commitment: AFP and ALB Expression. Theliver originates from the ventral foregut endoderm in a thickened area that appears at the anterior intestinal portal regionat 10.5 days of gestation (1, 2). We wanted to determine howsoon during the process of formation of the liver primordiumwould endoderma! cells express genes that indicate developmental commitment to liver morphogenesis. We analyzed theexpression of AFP and ALB by in situ hybridization for thedemonstration of mRNAs and by immunohistochemistry forthe localization of the protein. At 9.5 to 10 days of development(0 to 4 somites) neither protein nor their mRNAs could bedetected in the endoderm, although AFP mRNA was abundantin the yolk sac (data not shown). At 10.5 days of development,when thickening of the endoderm in the ventral foregut regionstarts, AFP mRNA was clearly detected in areas of the ventralendoderm that were in contact with the septum transversummesenchyme (Fig. 1). By 11.5 days, both AFP mRNA and theprotein were present in hepatoblasts. These cells formed cordsthat extended from the foregut endoderm into the septumtransversum (Fig. 1). Both the immunohistochemical stainingof AFP and AFP mRNA detection by in situ hybridization werespecific as determined with appropriate controls that includedremoval of or competing out the primary antibody for theimmunohistochemical method and the use of the oppositeorientation AFP riboprobe and RNase treatment for the in situhybridization procedure.

ALB mRNA was detected approximately 1 day later thanwas AFP mRNA, at 11.5 days of gestation, when hepatic cordsare formed (Fig. 2). Expression of ALB and its message wasclearly detectable by 12.5 days, at which time almost all hepatoblasts contained AFP and ALB mRNA and stained for theproteins. Some ALB staining was detected in endodermal cellsof the region of the liver primordium at 10.5 days of development, but we could not completely exclude the possibility thatthe staining was nonspecific.

Stages of Maturation of Hepatoblasts. We studied the expression of various cytokeratins and surface markers by immunohistochemistry and determined GGT activity and glycogen byhistochemical methods in the liver of rats during fetal development and the perinatal period. We chose markers that distinguish hepatocytes from bile duct cells in adult rat liver. Thedistribution of cytokeratins in these cell types is particularly

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Fig. 1. AFP localization during rat liver morphogenesis. A. general features of 10.5-day rat embryo (H & E, x 100); neural tube (/), foregut (2), liver primordium(3), septum transversum mesenchyme (4), and visceral yolk sac (5). B, AFP immunostaining in 11.5-day liver (x 250); arrows indicate AFP-positive hepatic cords. Cand D, AFP mRNA at 10.5 days detected by in situ hybridization (C, bright field; D, dark field; X 250; paraffin-embedded sections; 2-wk film exposure). Large arrow,liver primordium: small arrow, septum transversum mesenchyme. fand F, AFP mRNA at 11.5 days detected by in situ hybridization (E, bright field; F, dark field; x250; 1-day exposure). Large arrows, hepatic cords; small arrow, extrahepatic bile duct.

helpful for this analysis as adult hepatocytes contain only CK8and CK18, while duct cells have CK7 and CK19 in addition toCK8 and CK18 (33, 34). The other markers (22) selected forstudy are also specific for either hepatocytes (cell surface component HES6 and glycogen) or bile duct cells (cell surfacecomponent BDS7 and GGT activity).

We found that during fetal development hepatoblasts show agradual change in the expression of some of these markers. At11.5 and 12.5 days of development, CK8 was the only cytoker-atin uniformly expressed by hepatoblasts (Table 1; Fig. 3). Themouse monoclonal antibody prepared against M, 55,000 rat

cytokeratin gave an identical staining pattern as that obtainedwith human CK8 antibodies. Neither HES6 staining nor glycogen was detected at this stage of development, but the vastmajority of the cells stained for CK18 and had GGT activity(Table 1), although the expression of these two markers variedfrom cell to cell (Fig. 3). As development progressed, immaturehepatocytes began to express HES6 at 15 to 16 days of development and glycogen at 19.5 days. Expression of CK18 andGGT activity remained patchy until birth, but during the postnatal period GGT activity was lost and CK18 staining becameuniform (Table 1).

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Fig. 2. ALB mRNA localization during ratliver morphogenesis. Detection of ALBmRNA by in siiu hybridization at 11.5 days (Iand B, x 250; 2-day film exposure) and 13.5days (C and D, x 125; 2-day exposure) ofgestation. // and /' are dark-field photographsto demonstrate silver grains in the same sections shown in A and C, respectively. Largearrow, liver parenchyma; small arrow, extra-hepatic bile duct (determined by serial sectionsthat showed the connection from liver to gut).

Table 1 Markers ofhepatocyte development as detected by immunoHistochemicalstaining or enzyme histochemistry

The markers listed stained 75 to 100% of cells.

Days of development

10-11.5AFPCK8*12-17AFPALBCK8CK18rGGT<17.5-19AFPALBCK8CK18GGTHES619.5-NB"AFPALBCK8CKI8GGTHES6GlvAdultALBCK8CK18HES6Gly

" NB, newborn; Gly, glycogen.* CK8 staining was identical to that of a monoclonal antibody against rat M,

55,000 cytokeralin.'CK18 staining and GGT activity were present in variable amounts in all

parenchyma! cells from Day 12 until birth.

The Origin of Intrahepatic Bile Ducts. Intrahepatic bile ductsdevelop from hepatoblasts surrounding the large vascularspaces (1, 2, 13, 23, 32, 35, 36). We wanted to determinewhether intermediate cells containing markers for both thehepatocyte and ductal lineages would be present during liverdevelopment and be the progenitors of intrahepatic bile ducts.The major interest in searching for these cells is that they mightcorrespond to the immature, progenitor-type cells that proliferate during liver carcinogenesis in adult rats. At 15.5 days ofdevelopment, hepatoblasts around the large portal vein close tothe hilus began to form pearl-like structures, as described byVan Eyken et al. ( 13). These primitive bile ducts were heavilystained by both CK18 and CK8 antibodies and had high GGTactivity (Table 2; Fig. 4). CK18 was a particularly good markerfor these cells because of the strong staining of primitive biliarystructures in comparison with parenchyma! cells. In addition,some cells in these structures expressed CK19 (a bile ductmarker), and 50 to 75% of cells contained AFP and ALB. AFP

and ALB mRNAs were detected by in situ hybridization in 25to 50% and 10% of primitive bile duct cells, respectively (Figs.5 and 6). As development progressed, maturing bile ductsgradually expressed CK7 and CK19 and the cell surface markerBDS7 (Table 2; Fig. 4).

Primitive bile ducts continued to be formed throughout fetaldevelopment and the first 1 to 2 wk after birth. Cells of maturebile ducts did not contain AFP and ALB mRNAs and did notstain for these proteins. Double staining in the same sectionwith the hepatocyte surface marker HES6 and the bile ductmarker poly-CK, or staining with HES6 and CK19 antibodiesin consecutive sections of 17.5-day and neonatal livers, showedthat both the hepatocyte and the ductal cell markers werepresent in the same cells in primitive ducts (Fig. 6). Two wkafter birth, intrahepatic bile duct cells no longer stained withHES6 (Table 2). These results show that bile ducts originatefrom intermediate cells expressing hepatocyte and ductal markers present in the liver parenchyma near portal spaces startingat 15.5 days of development. Cells of primitive bile ductsundergo maturational changes during development but continue to express hepatocyte traits for 1 to 2 wk after birth.

Differentiation of Hepatoblasts into Large Hepatocytes andIntrahepatic Bile Ducts in Transplanted Fetal Liver. We wantedto determine whether hepatoblasts at a developmental stagethat precedes the emergence of intrahepatic bile ducts couldgive rise to both hepatocytes and duct cells. For this purpose,we transplanted fragments of livers at 13.5 days of development(about 2 to 3 days before the appearance of intrahepatic bileducts) into the testes of four syngeneic rats. In all animals, thetransplanted cells gave rise to large hepatocytes and to typicalbile ducts (Fig. 7).

The Origin of Extrahepatic Bile Ducts. It has been reportedthat extrahepatic bile ducts develop independently from intrahepatic duct formation (13, 37, 38). We have confirmed theseobservations and further analyzed the formation of extrahepatic

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Fig. 3. Stages in hepatocyte maturation. A,CK8 staining of cells in hepatic cords (arrow)at 11.5 days of development. B, GGT activityin parenchymal (large arrow) and perivascular(small arrows) cells at 17.5 days. C and D,CK18 staining in 19.5-day and neonatal liver,respectively, demonstrating heavily positiveduct structures around a portal vein; parenchymal cells in the background are lightly stained.Thick arrow, intrahepatic bile ducts; thin arrow, portal vein. E and F, HES6 staining in14.5- and 19.5-day liver, respectively. Noteabsence of staining in Â£(14.5 days) and staining at 19.5 days in both parenchyma! andperivascular cells. Arrow, portal vein. Allpanels, x 250; A and E, immunoperoxidasemethod, counterstained with hematoxylin; Cto F, immunofluorescence.

Table 2 Markers of intrahepatic bile duct development as detected byimmunohistochemical staining or enzyme histochemistry

Unless otherwise indicated, markers listed were detected in 75 to 100% ofcells.

Days ofdevelopment15.5Â»AFP

(50-75%)ALB(50-75%)CK8CCK18Poly-CKGGT17.5*AFP

(50-75%)ALB(50-75%)CK8CK18CK7(10%)CK19

(25-50%)Poly-CKGGTHES6

(10-25%)Newborn"ImmatureductsAFP

(10-25%)ALB(<5%)CK8CK18CK7CK19Poly-CKGGTHES6

(<5%)BDS7MatureductsCK8CK18CK7CK19Poly-CKGGTBDS7

Â°Newborn liver contains both immature and mature ducts. Mature ducts are

the only structures found in adult livers.* Intrahepatic bile ducts develop from hepatoblasts located near the vascular

spaces at 15.5 days of development. The markers listed are for hepatoblasts thatform primitive bile ducts.' ("KS staining was identical to that observed using monoclonal antibodies

against rat M, 55,000 Bunkeratili.

ducts. These ducts were first detected at about Day 11 ofdevelopment; the cells forming the common bile duct containedAFP and AFP mRNA (Fig. 8). The hepatic duct originated at15 to 16 days of development from hepatoblasts that containedAFP mRNA, AFP, and ALB. At 19.5 days of development,cells of extrahepatic ducts no longer expressed AFP or ALB.Starting at 11.5 days of development, extrahepatic bile ductsstained for CK8, poly-CK, and CK19 and, at later stages (19.5days, and newborn animals), they stained for CK7, CK8, CK18,CK19, and poly-CK, as well as the lectin PNA (Table 3; Fig.8). The results indicate that portions of the extrahepatic bileducts originate from AFP-producing hepatoblasts, while otherstructures (hepatic duct) emerge a few days later from AFP-and ALB-containing cells.

DISCUSSION

An important issue in the analysis of development is themechanism by which cells from the various layers becomedetermined or committed to form a specific tissue. In particular,

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Fig. 4. Development of intrahepatic bileducts. Demonstration of cytokeratins in peri-portal cells forming primitive bile ducts (arrows). Staining of 17.5-day liver for CK8 (A)and CKI9 (B). Staining of neonatal liver forCK8 (C hepatocytes lightly stained), CK18i/'). CK7 i /â€¢.hepatocytes counterstained withhematoxylin are CK7 negative), and CK19 (F).All panels, x 250.

it is important to find out the time at which commitment takesplace and whether or not the expression of tissue-specific markers precedes morphogenesis of that tissue. Specifically, wewanted to determine whether the start of transcription andtranslation of the AFP message would occur in endoderma!cells of the ventral foregut before the liver was formed. Furthermore, we wanted to find out when ALB expression wasfirst detected during liver development.

During rodent development, AFP is expressed in the visceralcells of the yolk sac by 6 to 8 days of gestation and several dayslater in the fetal liver (5, 39). Most studies have involvedmeasurements of liver AFP mRNA by nucleic acid hybridization, starting at 14 to 15 days of development in rats or mice(6-8). Muglia and Locker (6) could not detect AFP message bydot-blot hybridization with RNA preparations obtained fromthe foregut region of 10-day-old rat embryos, while at 12 days,both AFP and ALB mRNAs were apparently present. Thesingle in situ hybridization study of AFP and ALB expressionduring rat liver development, an analysis of the expression ofboth genes in late gestation and the perinatal period, showedthat all hepatocytes of 17- to 19-day-old rat fetuses contain

AFP and ALB messages (40). We show that the AFP messageis detectable by in situ hybridization in cells of the ventralforegut in the anterior intestinal portal region at 10.5 days ofdevelopment (9 to 14 somite stage) and that the protein is found1 day later. This indicates that AFP transcription occurs beforeliver morphogenesis and that translation of the mRNA into theprotein is first detected when liver cords are being formed andextended into the mesenchyme. In contrast, ALB mRNA wasfirst demonstrated at 11.5 days of development, coincident withliver cord formation, while the protein was detected 1 day later,when cord formation is completed. Endoderm of the foregutregion becomes determined for liver formation as early as the5-somite stage in chick embryos, and the determination requiresinductive interaction between the ventral foregut endoderm andthe hepatocardiac mesenchyme (4). Based on the time of appearance and localization of the AFP message revealed by ourexperiments, AFP transcription seems to be one of the earliestmarkers of liver-specific gene expression in determined endo-dermal cells. It is likely that the start of AFP and ALB genetranscription during liver morphogenesis requires tissue-specific transcription activators (reviewed in Ref. 41). If this is the

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Fig. S. Localization of AFP and albuminmRNAs in hepatoblasts forming primitive bileducts. AFP (. I and B; arrows indicate the alignment of the sections) and ALB mRNAs (fandF) detected by in situ hybridization in peripor-tal cells forming duct structures at 16.5 daysof development. C and D (controls), hybridization with AFP probe in 16.5-day liver sections pretreated with RNase. A, C, and E,bright field; II. D, and /'. dark field photo

graphs. All panels, x 500; 2- and 3-day filmexposure for AFP and ALB message, respectively.

Fig. 6. Origin of intrahepatic bile ducts fromALB- and HES6-positive cells at 17.5 days ofdevelopment. A and B, biliary ducts formed byALB mRNA-positive hepatocytes (arrows);biliary cells at the opposite side of the ductsare ALB mRNA negative (arrowhead); (A,bright Held; B, dark field; x 500; 3-day filmexposure). C and D, double staining with poly-CK (C) and HES6 (D) in the same section (x250). Large arrows indicate periportal cellsforming ducts around the portal vein (smallarrows).

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â€¢Ã•"

MÂ«/

Fig. 7. Fragment from 13.5-day liver transplanted into the testes of a syngeneicrat. The testes were examined 2 mo after transplantation and show large hepa-tocytes (arrowheads) and differentiated intrahepatic bile ducts (arrow). H & E, x250.

case, the necessary factors must be present before or at the timewhen AFP mRNA is first detected in foregut endodermal cellsat the 9- to 14-somite stage of rat and mouse development.

Previous work (12) demonstrated by immunohistochemical

methods in the developing mouse that AFP is present in someendodermal cells of the anterior intestinal portal region, particularly in the cranial diverticulum. It was also observed that inmice, ALB was detectable at 10.5 to 11.5 days of gestation,approximately 1 day later than AFP. Although the previousstudies did not include the analysis of AFP and ALB transcriptsby in situ hybridization, our work in rats entirely agrees withthe published results.

AFP-producing hepatoblasts acquired hepatocyte-specificcharacteristics in a sequential pattern that included productionof ALB, expression of HES6, and finally glycogen accumulation. Albumin, which is considered a marker for differentiatedhepatocytes, is already produced by hepatoblasts at 12 days ofdevelopment; in contrast, HES6 staining appeared several dayslater, at 15.5 days of development. The changes in the patternof cytokeratin expression in hepatoblasts were relatively minorthroughout development, with the exception of cells locatednear the vascular spaces (see below). Other investigators haveshown that both in vivo and in culture, enzymes associated withvarious liver functions also appear in a stepwise fashion duringliver development (42, 43).

Van Eyken et al. (13) and Shiojiri (12, 24, 32) demonstratedthat intrahepatic bile ducts develop from immature cells located

Fig. 8. Development of exlrahepatic bileducts from AFP-containing cells. Localizationof AFP mRNA in cells forming extrahepaticbile ducts at 16.5 days of development (A.bright field; B. dark field: 2-day film exposure).C and D are controls for AFP hybridizationusing a section from 16.5-day liver pretreatedwith RNase. Â¿"andF. staining of extrahepatic

bile ducts with CK7 (17.5 days) and PNA(neonatal liver), respectively. All panels, x250.




Table 3 Markers of extrahepalic bile duel development as detected byimmunohistochemical staining or enzyme histochemistry

Unless otherwise indicated, the markers listed stained 75 to 100% of cells.

Days of development

Days orDevelopment

11.5"AFP

(50-75%)'

CK8CK 18 (25-50%)Poly-CK15-16*AFP

(50-75%)ALB (25-50%)CK8CK18CK19Poly-CKPNA1

9.5/newbornCK7

CK8CK18CK19Poly-CKPNABDS7

" Common bile duct.* Hepatic duct.' AFP mRNA was present in 50 to 75% of cells at this stage.

near the large vascular spaces. We confirmed and extendedthese observations with the use of a large number of markersand in situ hybridization techniques. One of our goals was tosearch for cells that would have a pattern of expression of AFP,ALB, cytokeratin, and cell surface markers similar to that ofepithelial cell populations that proliferate during liver injuryand carcinogenesis. Such cells have markers of both hepatocyteand ductal lineages (30, 44, 45) and apparently constitute areserve stem cell compartment in adult rat liver (46-49). Wefound that cells of immature bile ducts expressed hepatocytemarkers (AFP, ALB, HES6) during development and for 1 to2 wk after birth as they gradually acquired bile duct markers(CK7, CK19, poly-CK, and BDS7). Based on the simultaneousexpression of hepatocyte and duct markers (AFP, ALB, CK7,CK19), we suggest that "oval cells" that proliferate at the earlystages of hepatocarcinogenesis as well as AFP-containing "ductal cells" detected after galactosamine injury correspond to cells

of primitive bile ducts that appear at Day 15.5 to Day 17.5 ofdevelopment and express hepatocyte and bile duct markers.5

The types of markers expressed by primitive bile duct cellschange during development. For instance, at 17.5 days ofgestation, AFP predominates over CK7 but, at birth, CK7expression is much higher. Similarly, single cells in oval cellpopulations of preneoplastic or injured liver express hepatocyteand duct markers in variable proportions, presumably indicating different stages of maturation and lineage commitment (16,30).

Germain et al. (22) showed that, in culture, liver cells from12-day fetal rats express markers corresponding to the hepatocyte or ductal lineages, depending on growth conditions. Herewe show that fragments of 13.5-day fetal liver transplanted tothe testes of young adult rats give rise to large, mature hepato-cytes as well as bile duct structures. The transplanted fragmentswere obtained several days before the emergence of intrahepaticbile ducts and contained only immature parenchymal cells. Invivo, only those hepatoblasts surrounding the vascular spacesdevelop into duct cells and express markers for both lineages.However, some hepatocytes scattered through the parenchymadistant from the vascular spaces also contain some of thesemarkers. Although both the perivascular and parenchymal hepatoblasts might be dual lineage progenitors, duct formation inliver development may require the inductive interaction betweenhepatoblasts and the matrix components of the venous spaces(32). These results are in general agreement with the studies ofShah and Gerber (50, 51), who found that laminin might berequired for bile duct differentiation in humans. They alsosuggested that some immature duct structures persisting in

10 II 12 I] 14 IS 16 17 18 19 20

f oregut â€”Â»"hepatoblastsendoderm

commonbile duct

5J. M. Lemire et al., submitted for publication.

potential oval cell origini

Fig. 9. Cell lineages in rat liver development. The diagram represents hepato-blast differentiation and the developmental stages at which these cells give rise tointra- and extrahepatic bile ducts. Potential origins for oval cells that proliferatein adult liver and stages of hepatocyte maturation are also indicated.

adult human liver may constitute a stem cell compartment forboth ducts and hepatocytes.

Van Eyken et al. (13) could not demonstrate staining of cellswith CK7, CK17, CK18, and CK19 in fetal rat liver sectionsembedded in paraffin. We show here that these cytokeratinscan be detected in acetone-fixed frozen sections. Using this typeof preparation we confirmed the observation of Germain et al.(22) that staining with BDS7 antibody shows a patchy distribution in the liver parenchyma during fetal development. However, we found that this antibody also stained some hemopoi-etic, endothelial, and connective tissue cells in addition to bileduct precursors. Although BDS7 staining in the developingliver was not completely specific, the antibody stained only bileduct cells in livers of adult rats. In rodents, extrahepatic bileducts form independently from the intrahepatic ducts (13, 37,38). We confirmed these observations and found that the hepatoblasts that give rise to the common bile duct expressedAFP and its message. At a later stage of development, hepatoblasts containing both AFP and ALB formed the hepatic duct.

In conclusion, this work and previous studies (12) establishthat AFP expression in endodermal cells signals their commitment toward liver morphogenesis and that all endodermalepithelial cells constituting the adult liver (hepatocytes, intra-and extrahepatic bile ducts) originate from AFP-producing

hepatoblasts (Fig. 9). Furthermore, the hepatoblasts locatednear vascular spaces that give rise to intrahepatic bile ducts andtransiently express both hepatocyte and duct markers maycorrespond to facultative stem-cell populations that proliferateduring carcinogenesis.

ACKNOWLEDGMENTS

We thank Dr. N. Marceau for his generous gift of BDS7, HES6, andvarious cytokeratin antibodies; Dr. S. Nishi for giving us the RAÃ•AFPplasmili: M. Panzica for the construction of AFP transcription vectors;and C. White and A. Baxter for their help in preparing the manuscript.

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1991;51:2611-2620. Cancer Res Nobuyoshi Shiojiri, Joan M. Lemire and Nelson Fausto DevelopmentCell Lineages and Oval Cell Progenitors in Rat Liver

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