Spatial Distribution of Cytoskeleton Intermediate FilamentsDuring Fetal Rat Hepatocyte DifferentiationJANY VASSY,1* THEANO IRINOPOULOU,1 MICHAEL BEIL,1,2 AND JEAN PAUL RIGAUT1

1Laboratoire d’Analyse d’Images en Pathologie Cellulaire, Universite Paris 7, Institut Universitaire d’Hematologie,Hopital Saint Louis, 75475 Paris Cedex 10, France2Department of Pathology, Humboldt University, 10098 Berlin, Germany

KEY WORDS cytokeratins; fetal rat hepatocyte; confocal microscopy; image analysis

ABSTRACT The construction of the liver parenchyma throughout fetal development depends onthe elaboration of intercellular contacts between epithelial cells and between epithelial andmesenchymal cells. During this time, the spatial distribution of cytokeratins in hepatocytes shows astriking evolution as demonstrated by confocal microscopy and image analysis.

In the early stages of fetal rat development, the liver is mainly a hematopoietic organ andhepatocytes represent fewer than 40% of all liver cells. At this time, cytokeratin filaments are scarceand are randomly distributed inside the cytoplasm. A coexpression of desmin and cytokeratin isfound in some cells. Intercellular contacts between epithelial and mesenchymal cells are morenumerous than between epithelial cells. Later in development, hepatocytes are arranged in a‘‘muralium duplex’’ architecture (two-cell-thick sheets). Contacts between hepatocytes become morenumerous and bile canaliculi become well developed. The density of cytokeratin filaments increasesand appears to be very high near the bile canaliculi.

In adult liver, hepatocytes are arranged in a ‘‘muralium simplex’’ architecture. Cytokeratinfilaments show a symmetrical distribution in relation to the nuclear region. The highest density offilaments is found near the cytoplasmic membrane.

Variations of the spatial distribution of intermediate filaments throughout hepatocyte differentia-tion were investigated in a pilot study using computerized image analysis. We found significantdifferences between the filament networks in fetal and adult hepatocytes. Microsc. Res. Tech.39:436–443, 1997. r 1997 Wiley-Liss, Inc.

INTRODUCTIONIn mammals, the liver is the first glandular structure

to appear in the fetal organism. It has a mixed origin:endoderm and mesoderm. Hepatocytes and bile ductsarise from endoderm cells, and the endothelia of sinu-soids, Kupffer cells, Pit cells, Ito cells, the connectivetissue of the hepatic lobules, and the capsule of Glissonare formed from mesoderm cells. In rodents (gestationperiod 5 21–22 days), the liver diverticulum appearson day 10 in the mouse and day 11 in the rat (Hous-saint, 1980). The hepatic diverticulum is a thickeningin the ventral floor of the foregut, near the yolk sac.Strands of parenchymal tissue penetrate the mesen-chyme of the septum transversum. Cell interactionswith mesenchymal cells induce the differentiation ofhepatoblasts (Casci and Zaret, 1991; Houssaint, 1980).On day 12 of gestation, hematopoietic cells invade thefetal liver (Housssaint, 1981). Hepatoblasts representonly 40% of the cells at this stage of gestation. Thispercentage will increase from day 14 to reach 66% byday 20 (Vassy et al., 1988; Vassy and Kraemer, 1993).

In the past, fetal liver development was studied toinvestigate whether crucial steps in hepatocyte differen-tiation exist (Cornelius, 1985; Kraemer et al., 1981).More recently, hepatocyte differentiation has come to beregarded as a more continuous phenomenon concerningthe morphology of hepatocytes and cytoplasmic organ-elles (Daimon et al., 1982, 1984; Houssaint et al., 1980,

1981; Luzzato, 1981; Medlock and Haar, 1983a,b; Vassyet al., 1988; Wong and Cavey 1992, 1993), the appear-ance of specific antigens of different membrane do-mains (Hubbard et al., 1994; Ihrke et al., 1993), plasmaprotein synthesis (Gray et al., 1985; Kraemer et al.,1981, 1986; Shioijiri, 1984; Yeoh et al., 1985), enzymeproduction (Shelly et al., 1989; Yeoh, 1986), and theexpression of specific intermediate filament proteins(Germain et al., 1988).

Moreover, fetal liver hematopoiesis has been studiedintensively for several years, with fetal hepatocytesdiscussed primarily from the perspective of nursingcells for hematopoietic components (Asano et al., 1987;Emura et al., 1984; Medlock and Haar, 1983b), andhematopoiesis is still one of the main topics studied inliver development (Fukumoto, 1992; Nagel and Nagel,1992; Ohneda et al., 1990; Sohn et al., 1993; Timens etal., 1990; Wong and Cavey, 1993; Yu et al., 1993).

Current research on fetal liver is focused on twoadditional topics. First, the interrelationship betweenepithelial and mesenchymal cell populations is animportant problem in embryogenesis (Casci and Zaret,

Contract grant sponsor: DAAD.*Correspondence to: Jany Vassy, Laboratoire d’Analyse d’Images en Pathologie

Cellulaire, Universite Paris 7, Institute Universitaire d’Hematologie, HopitalSaint Louis, 1 avenue Claude Vellefaux, 75475 Paris Cedex, France.

Received 15 February 1995; Accepted in revised form 17 October 1995

MICROSCOPY RESEARCH AND TECHNIQUE 39:436–443 (1997)

r 1997 WILEY-LISS, INC.

1991; Gall and Bhathal, 1990; Houssaint, 1980). Thisphenomenon is also interesting in the study of carci-noma differentiation. This important interaction be-tween stromal and epithelial cells is also an interestingtopic in various studies on carcinoma differentiation(Chung, 1993; Chung et al., 1992; Dinges et al., 1992;Hu et al., 1993; Sakakura, 1990). Second, the problemof epithelial cell lineage and the hypothetical existenceof a stem cell population in adult liver have become ofprime importance for a better understanding of hepato-cellular carcinoma genesis (Brill et al., 1993; Evarts etal., 1993; Sell, 1990, 1994; Sell and Pierce, 1994;Shiojiri et al., 1991; Shiojiri and Mizuno, 1993; Stein-berg et al., 1994; Thorgeirsson, 1993; Thorgeirsson etal., 1993).

Studying the expression of intermediate filamentproteins permits distinguishing different epithelial celllineages and epithelial and mesenchymal cells. In thenormal adult liver, parenchymal cells express only onepair of cytokeratins: C8 and C18 (Moll et al., 1982,

1993). Bile ducts cell express, in addition to this pair,C7 and C19 (Carthew et al., 1989; Franke et al., 1979;French, 1994; French et al., 1982; Germain et al., 1988;Ishii et al., 1985, 1991; Katsuma et al., 1988; Marceauet al., 1985, 1986, 1992; Van Eyken et al., 1987; VanEyken and Desmet, 1993). In the fetal rat development,rat hepatoblasts express C8 and C18 from day 12 ofgestation (Van Eyken et al., 1988a; Vassy et al., 1990,1993a,b; Vassy and Kraemer, 1993). Bile duct cellsdifferentiate from parenchymal cells on day 16 ofgestation. They appear as strings of pearllike struc-tures around large vascular branches and display anincreased reactivity for anti-C8 antibodies (Shiojiri,1994; Shiojiri et al., 1991; Van Eyken et al., 1988a,b).On day 18 of gestation, bile duct cells express C19,followed only later by C7 (Desmet et al., 1990; Germainet al., 1988; Shah and Gerber, 1989; Shiojiri, 1994;Shiojiri et al., 1991; Stosiek et al., 1990; Terada andNakanuma, 1993; Van Eyken et al., 1988a,b). However,it has recently been reported that some undifferenti-

Fig. 1. C8 distribution in rat liver shown by confocal microscopyat low magnification (pixel size 5 0.275 µm). a: Fetal liver, day 13 ofgestation. All hepatocytes contain C8. Few hepatocytes contact eachother. b: Fetal liver, day 20 of gestation. Hepatic cords constitute a‘‘muralium duplex.’’The strongest C8 labelling is found along canalicu-lar and lateral membrane domains. c: Adult liver. Hepatic cordsconstitute a ‘‘muralium simplex.’’ All the cytoplasm contain C8filaments, but the most intense labeling is found at the cell periphery.Bar, 25 µm.

437INTERMEDIATE FILAMENTS IN FETAL HEPATOCYTES

ated rat liver epithelial cell lines can express, togetherwith vimentin, C14, a cytokeratin normally found inthe basal cell layers of pseudostratified and stratifiedepithelia (Bisgaard et al., 1994a,b; Blouin et al., 1992;Butschat et al., 1992). This finding could be an argu-ment for the hypothetical existence of a cell populationthat is less differentiated than the stem cells usuallyrecognized (epithelial cells of the Hering canal). Theexistence of this population may be related to thetransitory presence of some cells (days 12–15 of gesta-tion) that coexpress cytokeratin and desmin in fetal ratliver (Vassy et al., 1993a,b). In adult liver, desmin isrestricted to Ito cells (Yokoi et al., 1984) and smoothmuscle cells of arteries. It has not yet been establishedwhether this cell population that coexpresses cytokera-tin and desmin belongs to a mesenchymal or an epithe-lial cell lineage. The more likely hypothesis is that thecells belong to a nonepithelial cell lineage; several cellsof mesenchymal origin, especially smooth muscle cells(Gown et al., 1988; Jahn et al., 1993; Jahn and Franke,

1989), have been reported to contain cytokeratins(Franke et al., 1980, Franke and Moll, 1987; Kuruc andFranke, 1988). In this case, as hepatocytes and Ito cellsare important in the adult liver, the special interrelation-ship between hepatoblasts and fetal Ito cells is ofcrucial importance. Moreover, it has been demon-strated that the extracellular matrix components, syn-thesized by both Ito cells and hepatocytes, play a crucialrole in hepatocyte differentiation in both normal liverand pathological conditions (Friedman et al., 1993;Mooney et al., 1992; Reid et al., 1992). In fetal liver, ithas been suggested that development of intrahepaticbile ducts is the result of hepatoblast transformationunder the influence of connective tissue (Gall andBhathal, 1990). In adult liver, the chemical compositionof the extracellular matrix in the space of Disse variesfrom the portal triads to the cental vein (Reid et al.,1992); for example, periportal hepatocytes are associ-ated with laminin and type IV collagen, but pericentralhepatocytes are surrounded by fibronectin and by type

Fig. 2. C8 distribution in rat liver shown by confocal microscopyat high magnification (pixel size 5 0.092 µm). a: Fetal liver, day 13 ofgestation. C8 filaments are scarce but uniformly distributed through-out the cytoplasm. b: Fetal liver, day 20 of gestation. C8 filamentsform a dense basketlike network throughout the cytoplasm. Thelabeling is more intense on the lateral domain (arrow) and on thecanalicular domain (arrowhead). c: Adult liver. C8 network displays aloose mesh. The network is more dense at the cell periphery (arrow-head) and around bile canaliculi (arrow). n, nuclei in a binucleatedhepatocyte. Bar, 10 µm.

438 J. VASSY ET AL.

I, II, and VI collagen. In early fetal liver, laminin is themost prevalent component of the extracellular matrix(Baloch et al., 1992; Rescan et al., 1989). Therefore,fetal hepatoblasts are associated with a matrix similarto the periportal one in adult liver.

Another question is whether the special expression ofintermediate filament proteins in fetal liver cells reflectthe particular microenvironment of the fetal liver interms of the extracellular matrix composition andintercellular contacts. In addition to a variable expres-sion of intermediate filament proteins, the intracellulardistribution of these filaments could be influenced bythe cellular environment.

SPATIAL DISTRIBUTION OF THECYTOKERATIN FILAMENT NETWORK

IN HEPATOCYTESSome relationship between the levels of expression of

C8 and C18 and the state of differentiation of maturecultured hepatocytes has previously been suggested(Baffet et al., 1991). The pattern of the cytokeratinfilament distribution in hepatocytes has been describedas a sensitive marker of liver damage (Steinmann et al.,1992). Moreover, the three-dimensional distribution ofintermediate filaments in hepatocytes has been wellshown by electron microscopy on cultured liver epithe-lial cells extracted by detergents (Katsuma et al., 1987,1988; Kawahara et al., 1989a,b; 1990). Connections ofintermediate filaments with nuclear lamina and thecell periphery have also been described with the sametechnique (Katsuma et al., 1987). Furthermore, it hasbeen shown that the differentiation of hepatocytes iscontrolled not only by the chemical composition of theextracellular matrix but also by its density (Friedmanet al., 1993; Mooney et al., 1992). For a given chemicalcomposition of the substratum, it has been demon-strated that hepatocytes are round and maintain highlevels of liver-specific protein secretion when they arecultured on low-density extracellular matrix but thatthey spread and proliferate when cultured on a higherdensity extracellular matrix. Thus, the concept oftensegrity and mecanotransduction introduced by Ing-ber (Ingber, 1993a,b; Wang et al., 1993; Wang andIngber, 1994) could be applied to hepatocyte differentia-tion. Connections among extracellular matrix, cytoskel-eton (especially intermediate filaments), and nuclearmatrix could direct, in some way, expression of genes(Bissell et al., 1982; Chung et al., 1992; Getzenberg etal., 1991; Roskelley et al., 1994; Traub and Schoeman,1994).

Hepatocyte differentiation throughout fetal rat devel-opment may be subdivided into three major stagesaccording to cell shape, enzyme differentiation, andglycogen synthesis.

Stage 1 (days 12–14) is characterized by potato-shaped hepatoblasts scattered throughout the livertissue (Asano et al., 1987; Vassy et al., 1988, 1990). Thehepatoblasts do not contain glycogen but they synthe-size and export plasma proteins (Kraemer et al., 1981,1986; Yeoh, 1986). At this stage, hematopoietic cells arepredominant and many cells express vimentin (Vassy etal., 1990; Vassy and Kraemer, 1993). Hepatocytes sel-dom contact one another (Fig. 1a), and bile canaliculiare not well differentiated. All hepatocytes express C18(Vassy et al., 1990) and C8 (Vassy et al., 1993a,b), but

the overall immunofluorescence labeling is weak. Cyto-keratin filaments are scarce but seem to be distributeduniformly throughout the cytoplasm (Figs. 1a, 2a), witha pattern that appears to be roughly symmetricalaround the nucleus (Vassy et al., 1990).

Stage 2 (days 15–17) is characterized by a morecompact liver, with hepatic cords of about five cellsthick and spherical hepatocytes. Days 16 and 17 areconsidered as a key period for enzyme differentiation(Gaasbeek et al., 1987; Kassner et al., 1991; Scott et al.,1988; Yeoh, 1986). Cytokeratin intermediate filamentsare more numerous than in the previous stage, and theoverall labeling is more intense, but the spatial distribu-tion is similar to that in stage 1.

During stages 1 and 2, hepatoblasts have an imma-ture organization and are not yet polarized (Stamato-glou and Hughes, 1994).

Stage 3 (days 18–21) is characterized by the emer-gence of glycogen deposits in cubic hepatocytes. Hepaticcords constitute a ‘‘muralium duplex’’ (Elias and Scher-rick, 1969), consisting of a set of two-cell-thick tissuesheets (Asano et al., 1987; Medlock and Harr, 1983a,b;Vassy et al., 1988, 1990). Prenatal hepatocytes formacinar structures (Stamatoglou and Hughes, 1994). Atthis stage, especially at day 20 of gestation, C18 and C8intermediate filaments are numerous, and the overalllabeling is very intense (Figs. 1b, 2b). Hepatocytes,decorated by anticytokeratin antibodies, have a charac-teristic appearance around the bile canaliculi (Fig. 2b,arrow). Cytokeratin filaments form a basketlike net-work, with a fine mesh, throughout the cytoplasm.However, the labeling is more intense at the peripheryof hepatocytes, where plasma membranes of neighbor-ing cells are connected on lateral domains (Fig. 2b,arrow) or on canalicular domains (Fig. 2b, arrowhead).

The typical structure of the adult liver, the ‘‘mu-ralium simplex’’ (Elias and Scherrick, 1969), consistingof one-cell-thick sheets, appears after birth. The charac-teristic distribution of cytokeratin filaments is shown inFigures 1c and 2c. The labeling is located mainly at thecell periphery and around bile canaliculi. In the cyto-plasm, the intermediate filament network has a ratherloose mesh, except in the areas close to the plasmamembrane (Fig. 3).

In summarizing these results, our hypothesis is that,in different steps of fetal liver differentiation, theinfluence of the cellular environment (cell–cell contactsand extracellular matrix contacts) is reflected by varia-tions of the spatial distribution of intermediate fila-ments, especially cytokeratins in hepatocytes. We arecurrently investigating these variations by automatedimage analysis of immunofluorescent images from C8localizations in fetal and adult rat hepatocytes, asvisualized by confocal microscopy, which allows sharpdefinition of images, even at higher magnifications(Vassy et al., 1993b, 1996).

QUANTITATION OF CYTOKERATINFILAMENT DISTRIBUTION

To validate our results on the variations of the spatialdistribution of cytokeratin filaments between fetal andadult stages, we performed a quantitation of the differ-ences in the filament network patterns.

In a pilot study, we considered adult and fetal (days13 and 20 of gestation) rat hepatocytes. Immunofluores-

439INTERMEDIATE FILAMENTS IN FETAL HEPATOCYTES

cent images of C8 localization were visualized by confo-cal microscopy and then transferred to an image analy-sis system.

The quantitation of the cytokeratin filaments re-quires appropriate image analysis models. In our study,the cytokeratin network is regarded as a line structure.To transform the original gray-scale image in a linestructure, a mathematical dealing, named skeletoniza-tion, was used. The concept of gray-scale skeletons,derived from the theory of mathematical morphology(Serra, 1982), provides an efficient tool for the analysisof line structures. We implemented two automatedmethods of line segmentation based on the skeletoniza-tion functions of the image analysis system AMBA(Roth et al., 1992). The first one detects only closed arcsof the skeleton, and the second one preserves the openbranches of the filaments (Beil et al., 1995; Vassy et al.,1996). The segmented line network was analyzed as agraphlike structure. Features such as the mean lengthof lines and the total number of lines in an image werecomputed to describe the network and the variations ofits pattern during development.

Another suitable concept to describe the filamentdistribution is a statistical model of texture. The Mark-ovian analysis of texture used is based on a transitionmatrix of gray levels within the gray-scale image(Haralick, 1979). The texture features derived from thismatrix (entropy, contrast, homogeneity) estimate thequantity of cytokeratin filaments and their spatialrelationship to each other. Entropy provides an indica-tion of the complexity within an image: the morecomplex the image, the higher the entropy value.Contrast measures local variation in the image: highcontrast values indicate high amounts of local varia-tion. Homogeneity is a measure of the distribution ofgray levels in the image: higher values indicate feweramplitude or intensity changes in the image.

The automated measurements of the filament net-work was performed at two microscope magnifications:3.6 pixels/µm (with 360 and electronic zoom 1) and 11pixels/µm (with 360 and zoom 3). Measurements at lowmagnification indicate global variations in the filamentdistribution. Measurements at high magnification indi-cate fine intracellular variations of the networks. Thefeatures were determined in an image frame of 512 3512 pixels.

In this pilot study, the number of lines and the meanlength of filament line segments, measured at lowmagnification in a frame of 512 3 512 pixels, increasedgradually from day 13 of gestation to the adult, butdifferences were statistically significant (P , .001) onlybetween adult liver and fetal liver at day 13 (Table 1).This result indicates that the global amount of fila-ments in hepatocytes increases gradually from day 13of gestation to adult.

For high magnification measurements, two features,entropy and contrast, increase from day 13 to day 20 ofgestation and then decrease by the adult stage (Table2). The differences between day 13 and day 20 andbetween day 20 and adult were statistically significant,but there were no significant differences between day13 and adult. Homogeneity decreases from day 13 today 20 of gestation and then increases by the adultstage. The differences between day 13 and day 20 andbetween day 20 and adult were significant, but therewas no significant difference between day 13 and adult.This result indicates that hepatocytes on day 20 ofgestation produce more complex, contrasted images,with higher amplitude of intensity changes, than hepa-tocytes on day 13 and in the adult.

Thus, the quantitation of the cytokeratin networks(by all implemented methods) shows, on the one hand,that the global amount of filaments increases through-out the development and, on the other hand, that thearchitecture of the network changes. These changescould be correlated with the increase of intercellularcontacts and the establishment of the hepatocyte polar-ization: the typical acinar structure of day 20 fetal liveris associated with significant differences detected withthe Markovian analysis of filement texture.

CONCLUSIONMechanical stress from the extracellular matrix has

been recently investigated and emphasized (Roskelley

Fig. 3. C8 distribution in adult rat hepatocyte shown by confocalmicroscopy at high magnification (pixel size 5 0.092 µm). A focalsection near the plasma membrane of an hepatocyte. The intermediatefilament network is denser than in the cytoplasm. Bar, 10 µm.

TABLE 1. Texture analysis at low magnification (3.6 pixels/µm)of immunofluorescence localization of C8 in adult and fetal

(days 13 and 20 of gestation) rat liver1

Features

Stages (mean 6 SD)

HS forP # 0.01*

Day 13(n 5 15)

Day 20(n 5 15)

Adult(n 5 15)

Mean length oflines (inpixels) 5.60 6 0.52 6.10 6 0.32 6.70 6 0.48

(A-13) 0.001,HS

(A-20) 0.041(13-20) 0.018

Number oflines in aframe 598 6 234 745 6 205 959 6 295

(A-13) 0.007,HS

(A-20) 0.075(512 3 512pixels) (13-20) 0.153

1HS, highly significant.*t test, A-13 5 comparison between adult and day 13.

440 J. VASSY ET AL.

et al., 1994; Wang and Ingber, 1994), but few data areavailable concerning mechanical influences from inter-cellular contacts. The development of fetal hepatocytescould be a useful model for these studies becausevariations in cytokeratin networks can be correlatedwith different steps in cell differentiation.

Both physical and biochemical signal transductionshave been used to explain the influence of the extracel-lular matrix on gene expression in mammary epithelialcells (Roskelley et al., 1994). Stress fibers and focaladhesion kinases are key processes in the mechanism.Authors who evoke a tissue matrix system, whichwould connect the extracellular matrix, the cytoskel-eton, and the nuclear matrix (Bissell et al., 1982;Ingber, 1993a; Wang and Ingber, 1994), do not alwaysspecify which cytoskeleton components would be in-volved, although microfilaments are often implied. How-ever, physiochemical properties of intermediate fila-ments suggest that they may play a role in determiningthe stiffness of epithelial cells (review in Nagle, 1994).Intermediate filaments are connected to the cell surface(desmosomes and hemidesmosomes) and to other cyto-skeleton components (Skalli and Goldman, 1991), andthey have affinities to nuclear lamins (Traub andSchoeman, 1994). Thus, they could be good candidatesfor a possible mechanical transduction of signals fromthe cell environment. However, further investigationsusing image analysis models are necessary to measurevariations in the stiffness of the cytoskeleton.

Computer-based image analysis provides the opportu-nity to distinguish between different intermediate fila-ment networks. Characteristics of the networks differaccording to the state of hepatocyte differentiation. Onequestion to be answered in the future is whether thesedifferences reflect variations in the cytoskeleton stiff-ness, which themselves could be the result of mechani-cal stress from the cellular environment.

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TABLE 2. Texture analysis at high magnification (11 pixels/µm)of immunofluorescence localization of C8 in adult and fetal (days 13

and 20 of gestation) rat liver1

Features(arbitraryunits)

States (mean 6 SD)

HS forP # 0.01*

Day 13(n 5 15)

Day 20(n 5 15)

Adult(n 5 15)

(A-13) 0.441

Entropy 74.00 6 8.16 80.30 6 6.17 72.30 6 9.70(A-20) 0.0002

HS(13-20) 0.0009

HS(A-13) 0.275

Contrast 33.00 6 9.55 44.30 6 9.29 35.80 6 10.70 (A-20) 0.0009HS

(13-20) 0.0001HS

(A-13) 0.647Homo-

geneity2,489 6 722 1,998 6 420 2,572 6 743 (A-20) 0.0003

HS(13-20) 0.0015

HS1HS, highly significant.*t test, A-13 5 comparison between adult and day 13.

441INTERMEDIATE FILAMENTS IN FETAL HEPATOCYTES

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