an in vivo look at vertebrate liver

An In Vivo Look at Vertebrate LiverArchitecture: Three-DimensionalReconstructions from Medaka

(Oryzias latipes)RON C. HARDMAN,* DAVE C. VOLZ, SETH W. KULLMAN, AND DAVID E. HINTON

Duke University, Nicholas School of the Environment and Earth Sciences,Durham, North Carolina

ABSTRACTUnderstanding three-dimensional (3D) hepatobiliary architecture is

fundamental to elucidating structure/function relationships relevant tohepatobiliary metabolism, transport, and toxicity. To date, factual infor-mation on vertebrate liver architecture in 3 dimensions has remainedlimited. Applying noninvasive in vivo imaging to a living small fish ani-mal model we elucidated, and present here, the 3D architecture of thislower vertebrate liver. Our investigations show that hepatobiliary archi-tecture in medaka is based on a polyhedral (hexagonal) structural motif,that the intrahepatic biliary system is an interconnected network ofcanaliculi and bile-preductules, and that parenchymal architecture inthis lower vertebrate is more related to that of the mammalian liver thanpreviously believed. The in vivo findings presented advance our compara-tive 3D understanding of vertebrate liver structure/function, help clarifyprevious discrepancies among vertebrate liver conceptual models, andpose interesting questions regarding the ‘‘functional unit’’ of the verte-brate liver. Anat Rec, 290:770–782, 2007. � 2007 Wiley-Liss, Inc.

Key words: liver; hepatobiliary; biliary; liver architecture;comparative hepatology; fish; 3-dimensional struc-ture; liver structure and function

Several conceptual models emerged in the 19th and20th centuries to describe structure/function relation-ships of the vertebrate liver; lobular mammalian livermodels, and a tubular liver model to describe the lowervertebrate livers of birds, fish, reptiles, and amphibians.The mammalian ‘‘classic,’’ ‘‘modified,’’ and ‘‘portal’’ lobulemodels describe morphological features encountered intwo-dimensional (2D) single sectional views of the liver(e.g. histological preparations, electron micrographs) andattempt to characterize the relationship between vascu-lature, biliary passageways, and the hepatocellular com-partment (Kiernan, 1833; Mall, 1906; Elias and Bengels-dorf, 1952; Rappaport, 1958; Fig. 1). While these modelsshare similarities, discrepancies exist in describing hep-atobiliary structure/function. For instance, Kiernan’sclassic lobule is a hexagonal structure with portal tractsat the hexagon corners and a central hepatic venule,whereas Mall’s portal lobule places the portal tract asthe central axis of the model. Rappaport’s acinar model

of the liver has a physiological rather than morphologi-cal basis (emphasizing afferent and efferent sinusoidalflow within the parenchyma), and attempts to describemetabolic variance along a periportal to centrolobulargradient (Jungermann, 1988). In the past 20 years ‘‘pri-mary’’ and ‘‘secondary’’ lobule concepts have alsoemerged (Matsumoto et al., 1979; Saxena et al., 1999;

*Correspondence to: Ron Hardman, Duke University, NicholasSchool of the Environment and Earth Sciences, Durham, NC,27708. Fax: 919-684-8741, Phone: 919-741-0621.

Grant sponsor: NIH (NCRR); Grant number: 1 RO1 RR018583-02; Grant sponsor: National Institute of Health (NIH/NCI); Grantnumber: R21CA106084-01A1.

Received 11 July 2006; Accepted 20 February 2007

DOI 10.1002/ar.20524Published online 21 May 2007 in Wiley InterScience (www.interscience.wiley.com).

� 2007 WILEY-LISS, INC.

Reviews THE ANATOMICAL RECORD 290:770–782 (2007)

Fig. 1. A: The ‘‘classic’’ and ‘‘portal’’ lobule, and liver ‘‘acinus’’characterize mammalian hepatobiliary structure/function relationships.A ‘‘tubular’’ concept has been used to describe the lower vertebratelivers of birds, fish, reptiles, and amphibians. a: The tubular concept intransverse view; two rows of hepatocytes form a tubule lumen (TL) attheir apical membranes, while basal membranes border sinusoidal orintercellular/intertubular space. b–d: Findings from three-dimensional(3D) reconstructions reveal ‘‘tubular’’ architecture is more complexthan previously considered. The tubule lumen is actually an intercon-nected network of branching canalicular and bile preductule passage-ways. Findings from 3D reconstructions also suggest a common/shared structural/functional unit (indicated by brown oval in both con-ceptual models) among lower and higher vertebrate livers. This func-tional unit is comprised of (1) a ‘‘portal tract/hilus,’’ a single conduitcontaining two afferent blood supplies (hepatic portal and arterial ves-sels), and efferent bile duct(s); (2) a ‘‘primary efferent vascular con-duit,’’ central vein/hepatic vein/terminal hepatic vein; and (3) an anas-tomosing hepatic muralium (predominantly monolayered in mammals,bilayered in lower vertebrates), perfused by a canalicular network andsinusoidal bed, that bridges these two. B: Our investigations, in con-junction with extant mammalian liver information, suggest the verte-brate hepatobiliary system to be constructed on two fundamental ar-

chitectural motifs: fractal branching in portal tracts (larger bile ductulesand ducts) and a polyhedral (hexagonal) motif at the canalicular levelof organization. The polyhedral architectural motif (1) appears to orderthe arrangement of the interconnected canalicular network and indi-vidual cells, and (2) may provide maximal structural/functional relation-ships. The latter is suggested given hexagonal organization is the leastperimeter way for space filling, meaning hepatocellular (metabolic)space is maximized, while minimal material is used for constructionand maintenance of the hepatocellular membranes, and consequentlyin the creation of canaliculi. Because blood to bile distance is mini-mized (Figs. 3, 4), transport is maximized. Lastly, the presence of ahexagonal motif in both the ordered arrangement of the canalicularnetwork, and in the organization of the hepatic lobule, suggest a hex-agonal structural motif may provide structural integrity to the organsystem in total through the cellular and tissue level of organization.Similarly, fractal branching of the blood and bile conduits in the portaltracts confers maximal afferent and efferent transport efficiencythrough these fluid compartments. Numbered segments in the hilusand bile duct regions indicate the branching order (fourth order)observed in medaka. These underlying structural principles may serveto order the system toward maximal structural integrity and metabolicand transport efficiency.

Teutsch et al., 1999). These concepts, based on 3D recon-structions of serial sections of human (Matsumoto et al.,1979) and rodent liver (Teutsch et al., 1999) describecone-shaped units observed at the tissue level of organiza-tion (branching of portal tracts). For instance; secondarylobules (synonymous with the ‘‘classic’’ lobule), comprisedof six to eight primary lobules, have a central terminal he-patic vein and six portal tracts at the periphery. Althougheach of these models remain valuable for communicatingnormalcy and disease, they are incomplete in conceptu-ally describing vertebrate hepatobiliary structure andfunction, particularly in 3 dimensions, and, understand-ably, disparities remain in applying these models acrossvertebrate species. As such, lobular and acinar models ofthe liver have never been completely accepted (Rappa-port, 1958; Lamers et al., 1989). Moreover, none of theseconceptual models have seen successful application tolower vertebrate hepatobiliary structure/function. Suchdiscrepancies likely belie inadequacies in the conceptualmodels themselves, or rather, our lack of comparativeunderstanding of vertebrate liver architecture.Much less is known about the nonmammalian verte-

brate liver. The prevailing model for these livers is thehepatic tubule, which emerged from the predominanceof observations of a two hepatocyte thick parenchymaand the fact that the vast majority of studies in lowervertebrate liver over the past century have shown noclear lobular formation (Elias and Bengelsdorf, 1952;McCuskey et al., 1986; Hampton et al., 1988; Rochaet al., 1994; Hinton and Couch, 1998). The current tubu-lar concept describes of two rows of hepatocytes (in lon-gitudinal section), the adjacent apical membranes ofwhich form a tubule lumen (bile passageway), intowhich bile is actively secreted (Figs. 1, 2). Basal mem-branes of hepatocytes face sinusoidal or intertubularspace. Of interest; the ‘‘tubular’’ formation is widely heldas a common hepatobiliary architecture found among allvertebrate species; it is the shared predominant pheno-type in all embryonic vertebrates, to include humans,and has been used to characterize the adult phenotypefor amphibians, birds, reptiles, and fishes (Elias, 1949;Arias, 1988). While the hepatic ‘‘tubule’’ formation pre-dominates among lower vertebrates throughout their lifespan, mammalian liver undergoes transition from atubular to laminar (muralium) architecture that is com-plete in man by age 5 (Arias, 1988). ‘‘Tubule’’ formations(denoting more than one row of hepatocytes) are alsoobserved in liver regeneration in mammalian species af-ter severe injury, such as after hepatectomy and frommarked acute exposure to toxins/toxicants (Van Eykenet al., 1989; Vandersteenhoven et al., 1990).From the brief review above it can be understood that

our comparative understanding of the vertebrate liver isincreasingly important. Not only is the tubular liverstructure the most ubiquitous hepatic phenotype in theworld, a better comparative understanding of the verte-brate liver enhances our ability to interpret and commu-nicate normalcy and disease across the variety of animalmodels employed in research. By example, small fish ani-mal models such as medaka and zebrafish are provingincreasingly invaluable to the study of vertebrate devel-opment, carcinogenesis, and for investigation of molecularmechanisms of disease and toxicity (Wittbrodt et al.,2002; Shima and Mitani, 2004; Berghmans et al., 2005;Alestrom et al., 2006).

The 3D in vivo findings we present here not only elu-cidate hepatobiliary architecture of the lower vertebrateliver phenotype in medaka, but may help better ourcomparative understanding of various conceptual modelsapplied to vertebrate liver structure/function.

MATERIALS AND METHODS

For decades, various color mutant strains of medaka(Oryzias latipes), acquired from natural and commer-cially available populations, have been maintained inthe Laboratory of Freshwater Fish Stocks at NagoyaUniversity, Japan. Cross-breeding from these stocks wasused to produce a stable ‘‘transparent’’ strain of medaka(STII), homozygous recessive for all four pigments (iri-diophores, leucophores, xanthophores, melanophores;Wakamatsu et al., 2001). STII medaka allow high reso-lution (<1 mm) noninvasive in vivo imaging of internalorgans and tissues at the subcellular level. Using laserscanning confocal microscopy (LSCM) and fluorescentprobes to elucidate the hepatobiliary system, we imagedliver structure and function at various stages of develop-ment in over a hundred individual living medaka. Ofthese, 15 medaka were used for 3D reconstructions pre-sented here (3 medaka at 8 days postfertilization [dpf], 4medaka at 12 dpf, 3 medaka at 30 dpf, 3 at 40 dpf, 2medaka at 60 dpf). With widefield and confocal fluores-cence microscopy, salient features of the organ systemsuch as canaliculi, space of Disse, endothelial cells, bili-ary epithelial cells, red blood cells, and hepatocytes andtheir nuclei, were clearly resolved in vivo (Fig. 2). Confo-cal stacks from in vivo imaging of the hepatobiliary sys-tem were used for 3D reconstructions, and from these,architectural, morphometric, and volumetric analyseswere made.In vivo investigations included medaka embryos, lar-

vae, and juveniles, from organogenesis (�50 hours post-fertilization) through 60 days. Medaka were exposed bymeans of aqueous bath (238C) to the fluorescent probesBodipy C5 Ceramide, Bodipy C5 HPC and fluoresceinisothiocyanate to facilitate in vivo investigation of hepa-tobiliary structure/function. Each of the fluorophoressaw hepatobiliary uptake and transport and excretioninto the gut lumen. Exposed cohorts were typically com-prised of 10 individual medaka. At various time points,individual medaka were removed from exposure cohorts,sedated with 10 mM tricaine-methane sulfonate (MS-222), mounted in aqueous medium (Embryo Rearing Me-dium for dechorionated embryos and hatchlings and de-ionized water for 20 dpf larvae and older) on depressionwell glass slides with cover slip, and imaged live usingbrightfield, and widefield and confocal fluorescence mi-croscopy. Time points of study varied from 10 min to 2hr depending on the fluorophore used and the aspect ofthe hepatobiliary system being studied. For instance,Bodipy C5 HPC and fluorescein isothiocyanate accumu-lation in the hepatic parenchyma is first observed at 10min after exposure, with maximal saturation of the fluo-rophore in the hepatic parenchyma occurring at �30min. Time points between 15 and 45 min post fluoro-phore exposure were commonly used for in vivo studies.Imaging systems: Confocal fluorescence microscopy

was performed with a Zeiss 510 Meta system and ZeissLSM 5 Axiovision image acquisition software, an Argon

772 HARDMAN ET AL.

and HeNe laser, Carl Zeiss C-apochromatic 40x/1.2, andC-apochromatic 10x/0.45. For wide-field fluorescence mi-croscopy, a Zeiss Axioskopp with DAPI/TRITC/FITC fil-ter cubes was used. Excitation/emission parameters forfilter cube sets were: DAPI/UV (Ex 360–380 nm/Em AllVis >400 nm); FITC (Ex 450–490 nm/Em 515–565 nm);TRITC (Ex 528–552 nm/Em 578–632 nm). Gross ana-

tomical imaging was performed using a Nikon SZM1500 dissecting microscope with a Nikon DXM 1200 digi-tal capture system (brightfield). Regarding software,image analysis and compilation was performed withEclipseNet (Nikon, USA), Adobe Photoshop (Adobe,Inc.), Amira 3D (Mercury Computer Systems, Berlin),ImageJ (V1.32j), IP Lab software (Scanalytics, Inc., ver-

Fig. 2. Noninvasive in vivo imaging of hepatobiliary structure/func-tion in living STII medaka. A: STII medaka embryo 6 days postfertiliza-tion (dpf), left lateral view. The fluorescent cytochrome P4503A metab-olite resorufin (red) in transport through the intrahepatic biliary net-works (IHBPs) of the embryonic liver (L), and concentration in the gallbladder (GB). Otic vesicle (Ov), yolk sac (Y). B: Tubule-like formations,seen in transverse section, in the developing parenchyma, observed invivo at 6 dpf. C: Concentrative transport of resorufin from hepatocyteinto the tubule lumen (TL/IHBP). D1: STII medaka, 30 dpf, left lateralview, brightfield microscopy. Green algae can be seen transiting thegut lumen. D2: An in vivo confocal image of liver from the region of in-terest (grey square) in frame (D1), showing fluorophore transport(FITC) through IHBPs. E1–E4: In vivo confocal imaging of blood to biletransport. E1: Two rows of hepatocytes and their nuclei (HN) are seenin longitudinal section. Red blood cells (elongated ovate structures)

were observed circulating through sinusoids (S/r) abutting the basalmembranes of hepatocytes. E2: Single section from in vivo confocalimage stack showing conservative b-Bodipy C5 Phosphocholine(green fluorescence) in transport from sinusoid (S) to canalicular space(IHBP), imaged in vivo 30 min. Post administration of the fluorophoreto medaka (aqueous bath). E3: Composite of E1 and E2, localizing flu-orophore concentrate to IHBPs. E4: Surface map of region of interest(ROI) of inset (white square) in frame E2; illustrating concentrativetransport of the fluorophore from sinusoidal space (S) to bile space(IHBP). The ROI, showing a 17-mm span from blood to bile (across he-patocyte), was quantitatively assessed, and revealed b-Bodipy C5Phosphocholine concentration to be �66 times greater in the biliarypassageway than in sinusoidal space. These studies demonstrated invivo evaluation of concentrative blood to bile transport of fluorescentprobes was possible.

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sion 3.55), and Zeiss Image Browser (Carl Zeiss). Fluo-rescent probes used were 7-benzyloxyresorufin (10–50mM); b-Bodipy C5-HPC [BODIPY1 581/591 C5-HPC (2-(4,4-difluoro-5- (4-phenyl-1,3- butadienyl)-4-bora-3a,4a-diaza-s- indacene- 3-pentanoyl)-1-hexadecanoyl- sn-glyc-ero-3-phosphocholine), (30 nM–10 mM)]; Bodipy FL C5-ceramide [N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3- pentanoyl) sphingosine, (500 nM–5 mM)];and fluorescein isothiocyanate (1 nM–50 mM). Fluoro-phores were acquired through Invitrogen/MolecularProbes (Carlsbad, CA). All fluorescent probes were admin-istered to STII medaka by means of aqueous bath in con-centration ranges given, at room temperature, under darkconditions.All transmission electron microscopy (TEM) was per-

formed at the Laboratory for Advanced Electron andLight Optical Methods (LAELOM), College of VeterinaryMedicine, North Carolina State University. Individualmedaka were anesthetized and fixed in 4F:1G fixative(4% formaldehyde and 1% glutaraldehyde in a monoba-sic phosphate buffer with a final pH of 7.2–7.4 and afinal osmolality of 176 mosmol). Thin sections (Spurrresin embedded) were then examined using a FEI/Phi-lips EM 208S Transmission Electron Microscope.

FINDINGSPolyhedral (Hexagonal) Architecture of theIntrahepatic Biliary System in Medaka

The 3D reconstructions from in vivo imaging revealedintrahepatic biliary passageways (IHBP) in medaka tobe predominantly an interconnected network of canali-culi and bile preductules, the organization of whichappeared to be based on a polyhedral (hexagonal) struc-tural motif (Figs. 3–6). The polyhedral motif was mostclearly observed in the arrangement of IHBPs, and wasnot evident in sinusoidal architecture. Empirical obser-vations found IHBP branching to be distinctly angular,and statistical analysis of IHBP branching angles (in3D) revealed clustering of angles into three main groups:120 degrees (74.9%), 90 degrees (14.8%), and 58 degrees(10.3%). The majority of angles measured averaged120 degrees, the interior angle of the hexagon (Fig. 4).The 3D angle measurements were made by randomlyselecting intrahepatic biliary passageway bifurcationand trifurcation points in 3D reconstructions, achievedby rotating the 3D model and randomly selectingbranching points. All angles of a bile segment branchingpoint were measured, inclusive of all 3D planes (Fig. 3).Further 3D analyses revealed IHBP architecture to bebased, conceptually, on a tessellating (3D) hexagonalmesh (Carle et al., 2001). This hexagonal motif was alsoapparent in the ratio between bile segment length andblood to bile distance (Figs. 3, 4). When bile segment(canaliculi) length was considered to correspond to oneside of a hexagon, the distance (radii) from hexagon side(canaliculus) to hexagon center (sinusoid) appeared tocorrelate to the hexagonal ratio given in Figure 3. Ahexagonal architecture has also been described, fromempirical observations, in the fine structure of the bili-ary system in rat (Mochizuki et al., 1988; Murakamiet al., 2001) and human (Yamamoto et al., 1990).Although a discussion of this topic is beyond the scope ofthis study, the presence of a polyhedral structural motif

likely imparts structural integrity, and metabolic andtransport efficiency, features provided by higher-orderorganizing principles such as hexagonal architecture,not uncommon in biological systems (Mainzer, 1996).

Biliary System and Hepatocytes

Linear segments of IHBPs (canaliculi) averaged 11 mmin length and 1.3 mm in diameter, and exhibited unary,binary, ternary, and quaternary branching. This findingshould not be confused with fractal branching patterns(Turcotte et al., 1998); the biliary system in medakadoes not appear (except for the localized area at the liverhilus) to follow a fractal branching tree motif. Rather,there is ubiquitous feedback/interconnectedness withinthe 3D biliary network (Fig. 5). Four to six hepatocyteswere associated with each linear segment of IHBP(Fig. 6) and arranged such that a single hepatocytecould contribute to two or three bile segments (canali-culi/bile-preductules). The same hepatocellular/canalicu-lar relationship has also been described in rat (Mottaand Fumagalli, 1975), where the same side of a hepato-cyte was observed to bound two or more bile canaliculi.While hepatocytes had a mean diameter of 11.3 mm, theyshowed varying morphology in vivo, apparently adaptiveto the space they were filling (e.g., at bifurcating or tri-furcating junctions). Hence, while the intrahepatic biliarynetwork showed a more ordered structure, hepatocellularorganization and morphology appeared much more adapt-ive to space filling and parenchymal organization.The IHBP network, composed of canaliculi and bile

preductules, occupied �95% of the liver corpus uni-formly, with each area of livers examined (n ¼ 15) con-taining approximately equal volumes of IHBPs relativeto hepatocellular and vasculature volumes, regardless ofhow distal/proximal an observed volume of liver wasfrom the liver hilus. Canaliculi were observed to mergewith bile preductules at unique morphological sitesformed by junctional complexes between bile preductularepithelial cells (BPDECs, discussed following) and hepa-tocytes (Fig. 6). Together, canaliculi and bile preductulescomprised the IHBP network. This interconnected net-work of IHBPs was observed to feed three primary intra-hepatic bile ducts (IHD), lined by cuboidal biliary epithe-lia. IHDs merged at the liver hilus into a common he-patic duct that was associated with the cystic andcommon bile ducts (Fig. 5). Cuboidal and large squa-mous biliary epithelial cells (non-BPDECs) wereobserved primarily localized to the hilar and peri-hilarregions of the liver.

Parenchymal Architecture

Of interest, while ‘‘tubule’’ like formations wereobserved (in vivo and in single sectional views) in em-bryonic livers (3 dpf to 7 dpf), tubule formations werenot readily apparent in 3D reconstructions (n ¼ 15) oflarval and older livers. Rather, parenchymal architec-ture in larval and later developmental stages of medakaappeared to be more consistent with an anastomosing,predominantly dual layered, muralium. First, 3D recon-structions revealed the ‘‘tubular’’ lumen to be composed,not of an extended linear biliary passageway that wouldcreate a classic ‘‘lumen,’’ but of a hexagonal network ofbranching, interconnected canaliculi and preductules,

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Fig. 3. Examples from three-dimensional (3D) reconstructions ofmedaka hepatobiliary system, illustrating the types of analyses thatled to findings on polyhedral (hexagonal) architecture. Grayscale back-ground images are sections from in vivo confocal image stacks, fromwhich 3D reconstructions were generated. In (A–D,F–H) the emptyspace surrounding biliary passageways (green/gold), and between si-nusoids (red), is hepatocellular space (hepatocytes not rendered forvisual clarity). Colored dots in A–C are given for anatomical reference.A–D: An isolated section of the intrahepatic biliary network, illustratingrelationship of canaliculi (green) to sinusoids (red). A: Branching canal-iculi (intrahepatic biliary networks [IHBP]) are shown between tworows of hepatocytes (confocal grayscale image). B: Same as A with si-nusoids present, and C is A&B viewed directly on the ‘‘z’’ axis (fromtop). C: Example of analyses of polyhedral structural motif, which wasevident in the relationship between blood to bile distance and canalic-ular length (see Fig. 4). Bile segment length (canaliculus, S) correlatedto calculated and measured values of blood to bile distance (r), whenassessed with the hexagonal ratio [r ¼ 0.5 (S) 1.44]. D: Empiricalobservations (3D) of angular canalicular and bile preductule branchingled to statistical analysis of branching angles, which suggested thepresence of a polyhedral structural motif (see Fig. 4). E: Schematicshowing modified ‘‘tubular’’ concept of lower vertebrate liver (noteIHBPs, and the hexagonal arrangement in relation to hepatocytes/tu-

bular profile). In medaka, we found this structural arrangement ofbranching canaliculi that comprises the ‘‘tubule lumen,’’ revealing ‘‘tu-bular’’ architecture more complex than previously considered (see Fig.1). F: 3D reconstruction of the relationship between IHBPs and sinu-soids. Between 2 sinusoids (red) can be seen a branching canalicularnetwork (green/gold). G–I: Examples of 3D morphometric analyses,which suggest medaka parenchymal architecture to be more consist-ent with an anastomosing muralium. 3D measurements of hepatocellu-lar space (between sinusoids) revealed a muralium-like architecture.G: Example dimensions given are 196.3 mm in length, 98.2 mm inheight, and 24.7 mm in width. H: Same as G viewed in a horizontallongitudinal section (from the top). Sample dimensions are shown. I:Single slice from in vivo confocal stack of hepatic parenchyma. Grayarrowheads indicate sinusoids, black arrowheads hepatocyte nuclei.Hepatocellular compartment width (analyzed in vivo and in 3D) wasfound to be predominantly two cells thick, although occasionally up toeight cells in some areas. Average parenchymal width was 24.58 mm.Parenchymal depth varied from 21.2 mm to > 200 mm. Note that typi-cal dimensions of 3D reconstructions were 100 mm in depth; hence,depth dimension is limited. Parenchymal metrics were derived from127 measurements (3D) in medaka at 8, 12, 30, and 40 dpf. Space ofDisse (SD), Sinusoidal Endothelial Cell (SE), Red Blood Cell (R), Sinu-soid (Sn, S1–S4).


which formed the inner parenchymal framework(Figs. 3–6). In other words, tubule lumens were found tobe canalicular segments with an average length between10 and 13 mm. Second, hepatocellular muralium-likestructures were apparent, which varied in height from�20 mm to >100 mm, and width from �23 mm to 83 mm,observations consistent with an anastomosing muralium

(because confocal stacks were �100 mm in depth, themaximum measurement for plate ‘‘height’’ was limitedto 100 mm). Third, the longest non-branching biliary seg-ments were observed to be �30 mm in length, and thesewere few in number (�2–3% of bile segments measured).For a tubular architecture to be present, tubules would,from these 3D investigations, be less than 30 mm in linear

Fig. 4. Morphometric and volumetric analyses (upper left graph) ofrandom measures (341 observations) of three-dimensional (3D)branching angles of bile segments in four individual livers (8–40 dayspostfertilization [dpf]) found branching angles to cluster into threeprimary groups: 120 degrees (�75%), 93 degrees (�15%), and64 degrees (�10%). Upper right graph: Using the hexagonal ratio inthe lower portion of the plot, a polyhedral structural motif was alsosuggested in the relationship between blood to bile distance and can-alicular length. Measured values of blood to bile distance (r) correlated

to calculated values, falling between the longest (R) and shortest (r)radii in the hexagon [r ¼ 0.5 (S) 1.44]. Where bile segment length (e.g.,canaliculus) ¼ S, blood to bile distance ¼ r. Pearson’s correlationcoefficient (0.049, P ¼ 0.6). The difference in the mean between calcu-lated and measured r values was 0.67 mm, half the width of the canali-culus. In vivo volumetric investigations in three individual medaka at8 dpf, 12 dpf, and 30 dpf revealed consistent ratios between intrahe-patic biliary passageway volume, blood (sinusoid) volume, hepatocel-lular volume, and parenchymal volume.

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length and would be highly branched and interconnected(following biliary network architecture). Hence, 3D in vivoobservations found medaka parenchymal structure tomore resemble a predominantly two- to three-cell-thick he-patic muralium, with an average width of 26 mm andheight that varied from 20 mm to > 100 mm. It follows thatmedaka parenchymal architecture appeared highly analo-gous to mammalian architecture; although predominantlytwo to three hepatocytes thick (and in rare instances up toseven or eight cells thick), as opposed to the predominantlyone- to two-cell-thick mammalian muralium (Elias andBengelsdorf, 1952).

Biliary Epithelia

3D investigations found BPDECs (Hinton and Pool,1976; Hampton et al., 1988; Okihiro and Hinton, 1999)to be located throughout the parenchyma of medaka liv-ers studied (from 8 dpf to 60 dpf). BPDECs are the puta-

tive correlary of mammalian progenitor/stem cells, giventhey share morphological characteristics ascribed tomammalian oval cells (bipotential progenitor cells).BPDECs, like oval cells, are phenotypically distinct fromhepatocytes, characterized by a high nuclear to cyto-plasm ratio with no basement membrane, and are inti-mately associated with the biliary system (Goldinget al., 1996; Fausto and Campbell, 2003). In vivo,BPDECs were observed to form unique junctional com-plexes with hepatocytes, at which were created IHBPstermed bile preductules. These junctional complexeswere morphologically distinct (Fig. 6). In single sectionview these unique morphological formations appeared asBPDECs surrounded by bile passageways on all sides(Fig. 6). 3D reconstructions from in vivo imagingrevealed BPDECs to occupy the ‘‘center’’ of these com-plexes, where one or more BPDECs formed multiplejunctional complexes with surrounding hepatocytes (Fig.6). Biliary passageways (bile preductules) formed at

Fig. 5. Three-dimensional (3D) architecture of liver hilus, bile duc-tules/ducts, and canalicular network. A: In vivo image capture of theliver hilus at 30 days postfertilization (dpf). Three primary intrahepaticducts (IHDs) conjoined a common hepatic duct (HD), which fed thecystic duct (CD), gall bladder (GB), and common bile duct (CBD). Theliver hilus of medaka contained two afferent blood supplies (the he-patic portal vein [HPV] and hepatic artery [not shown]), and hepaticduct. Mucosal folds (MF) of the gut (lower left) are seen caudal to theliver. B,C,F: The 3D reconstructions aided in elucidating the relation-ship between the canaliculo-preductule network, bile ductules andducts, and liver hilus. Images C (horizontal longitudinal section) and F(sagittal view) show an example of 3D reconstruction of the canali-culo-preductular network draining to a common IHD (one of threeIHDs shown in B). E: A hand-traced schematic of an isolated sectionof the canaliculo-preductular network (diamond-shaped region of inter-est [ROI] indicated in C and F). One individual IHD (green arrowhead)

was followed and the branching of the IHBPs mapped to a 2D sche-matic. Each colored line corresponds to one bile segment (�11 mm inlength). Colors are for illustrative purposes only. The 90-degree anglescorrelate to bile segment branching (not to scale or degree, for illus-trative purposes only). The schematic is used to illustrate the intercon-nectedness of the canaliculo-bile preductular network. For clarity, partof the network was abbreviated by using dashed lines to illustratefeedback and interconnectedness within the IHB network. Dashedlines represent a branching network of canaliculi (as illustrated in col-ored lines). The canaliculo-bile preductule network occupied �95% ofthe liver body (by area). D: Schematic showing overall biliary architec-ture of medaka liver. Bile ductules and ducts exhibited fourth orderfractal branching, and were largely found in hilar/peri-hilar region ofthe liver. Ductules terminated in a canaliculo-bile preductular network(illustrated in E), that followed a polyhedral (hexagonal) architecturalmotif.


these BPDEC/hepatocyte junctional complexes com-monly showed ternary or quaternary branching. WhileBPDECs varied in morphology and size, showing ovate,triangular and ellipsoidal nuclei, the majority ofBPDECs were commonly found to be �6 mm in diameter.Our in vivo findings on BPDECs in medaka are, inter-estingly, consistent with previous oval cell studies inrodents and humans (Farber, 1956; Golding et al., 1996;

Theise et al., 1999; Fausto and Campbell, 2003; Knightet al., 2005). While we have localized BPDECs to specificlocations within hepatic parenchyma, and these cellsshare morphological characteristics consistent withmammalian hepatic progenitor cells, BPDECs in medakahave only been partially characterized (Okihiro and Hin-ton, 2000). Cuboidal and squamous biliary epithelia(non-BDPEC), lining larger diameter intrahepatic biliary

Fig. 6. A–A2: Canaliculus/bile preductule and hepatocellular rela-tionships. A1,A2: Three-dimensional (3D) reconstructions from confo-cal image in A. Four to six hepatocytes (hepatocyte nuclei shown inpurple, numbered) were observed in relation to a given canaliculus(green). Circled red A indicates a bifurcating canalicular segment. Asingle hepatocyte was observed to contribute to one to three canali-culi. B1–B2: The 3D reconstructions of confocal image in (B), illustrat-ing the structure of a bile preductule (BPD) junction. Red arrowheadsin (B) indicate bile preductular epithelial cells (BPDECs) within the he-patic parenchyma. In 2D confocal sections BPDECs appeared tooccupy the ‘‘center’’ of these canaliculo-bile preductule structures.BPDECs formed unique junctional complexes with surrounding hepa-tocytes, which created biliary passageways, termed bile preductules

(BPD). The 3D reconstructions (B1,B2) elucidated the actual architec-ture of these canaliculo-bile preductule complexes, revealing bile pre-ductular epithelia form multiple junctional complexes with surroundinghepatocytes, at which bile preductules are formed. In B1 and B2, theempty space surrounding BPDEC is hepatocellular space (left‘‘empty,’’ or not rendered, for illustrative purposes). In vivo findingswere corroborated with ultrastructural studies (C1,C2), which aided inelucidating/validating in vivo observations. Frame C is a still imagefrom a 3D reconstruction illustrating the distribution of BPDECs(purple, indicated by red arrowhead) throughout the hepatic paren-chyma. Junctional Complex/Bile Preductule (Jc/BPD), Intrahepatic Bili-ary Passageway (IHBP), Hepatic Nuclei (HN), Sinusoid/red blood cell(S/r), Bile Preductular Epithelial Cell (BPDE/C).

778 HARDMAN ET AL.

passageways, were observed almost exclusively nearhilar and perihilar regions of the liver (Fig. 5).

Morphometric and Volumetric Information

3D reconstructions enabled highly accurate volumetricand ratiometric analyses of biliary, parenchyma, vascu-lature, and liver volumes (Fig. 4). We found prior exvivo volumetric studies (Hess et al., 1973; Blouin et al.,1977; Rocha et al., 1997; Hinton et al., 2001) in verte-brate livers to correspond well with our in vivo findings.

DISCUSSION

Surprisingly, we did not observe a well-developedarborizing biliary ‘‘tree,’’ well characterized in mamma-lian studies (Ludwig et al., 1998; Masyuk et al., 2001).This ‘‘tree’’ describes bile ducts of the liver hilus arboriz-ing into more highly branched, and numerous, bile ductsand ductules of diminishing diameter, as the biliary sys-tem infiltrates more distal regions of liver (relative tothe liver hilus). We attribute the overall lack of an arbo-rizing biliary tree in medaka liver to two factors: (1)mammalian studies are describing portal tract arboriza-tion, or interlobular stromal areas of the liver (meaningbiliary tree arborization describes the tissue level of or-ganization, lobule formation); (2) medaka liver is the ar-chitectural analogue of the intralobular mammalian pa-renchyma. The two points are discussed in detail below.These findings raise important questions regarding

current conceptual models of vertebrate liver architec-ture. In mammalian liver, hepatocytes have been de-scribed as irregularly shaped polygonal cells that form,predominantly, a one- to two-cell-thick wall/plate-likestructure (muralium), which anastomoses throughoutthe liver (Elias and Bengelsdorf, 1952). Observations ofdual layered hepatocytes in fish and other lower verte-brate livers, and the lack of observed lobular formationsin the majority of lower vertebrate livers studied(McCuskey et al., 1986; Hampton et al., 1989; Rochaet al., 1997; Hinton et al., 2001; Akiyoshi and Inoue,2004) led to the reasoning that lower vertebrate liversmay be composed primarily of anastomosing ‘‘tubules.’’In single section view (e.g. histological preparations withlongitudinal orientation), hepatic tubules appear as tworows of hepatocytes, the apical membranes of whichform a tubule lumen (bile passageway), and basal mem-branes of which border sinusoidal, intracellular, or inter-tubular space. As our 3D in vivo findings have shown,the true structure of the ‘‘tubular’’ liver is more complexthan previously understood. Because one hepatocytemay contribute to one to three canaliculi, ‘‘tubulelumens’’ are actually composed of an interconnected hex-agonally branching network of canaliculi and bile pre-ductules. Where ‘‘tubule-like’’ formations were observedin 2D transverse sectional views in the embryonic liver(in vivo), ‘‘tubule’’ formations were very rarely encoun-tered in larval and older fish, and were not readily appa-rent in 3D reconstructions. For a tubular architecture tobe considered, tubules in medaka would, from our 3D invivo investigations, average � 29 mm in linear length (�three hepatocytes), and would be quite rare (2–3% ofbile segments measured were found to span up to 29mm). The prevailing 3D structure of the biliary systemin medaka was a highly branched and interconnected

network of canaliculi and bile preductules (bile seg-ments) that averaged 11–13 mm in length (Fig. 5).Because 3D investigations reveal medaka hepatobili-

ary architecture, representative of the lower vertebratetubular phenotype, to more closely resemble a dual-lay-ered anastomosing muralium, akin to the single-cell-thick muralium described in mammals, important ques-tions are raised. If hepatic tubules comprise a two-hepa-tocyte-thick muralium, how are they integrated into amuralium-like structure? Although tubule formationsmay theoretically comprise a dual-layered muralium,this would necessitate an intertubular space, whichraises another question; if tubules are present, aretubules joined at the intertubular space? If so, does thisnot describe a muralium structure? These importantquestions remain to be answered, given our findingsfrom in vivo 3D reconstructions suggest a dual-layeredplate-like muralium predominates in larval and laterlife stages of medaka, whereas tubule-like formationswere observed in the embryonic liver.The tubular concept of the lower vertebrate liver was

derived largely from 2D observations of histological andelectron micrograph (EM) preparations. In histologicalsections, the fine structure of the biliary system is diffi-cult to discern (canaliculi average 1–2 mm in diameter),and when resolved, we now know, due to a hexagonal ar-chitectural formation, would show a random order ofappearance in a 2D section. For instance, using histolog-ical sections Rocha et al. (1994) described biliary passa-geways in trout as appearing randomly dispersedthroughout the liver; this would be an accurate 2D ob-servation, or how the 3D hexagonal architecture of thecanalicular network we have described would appear ina 2D histological section. Consequently, from a 2D per-spective, a dual-layered muralium may appear, or beinterpreted as, a tubule-like formation. Hence, given theinterconnected 3D biliary architecture we have eluci-dated in this study, it may be that a ‘‘tubular’’ formationand dual layered plate-like muralium are perhaps, oneand the same, and that discrepancies in understandingmuralium architecture in lower vertebrates have arisenfrom varied 2D viewpoints (and thereby varied interpre-tation), and a lack of 3D studies in vertebrate liverstructure at the canalicular level of organization.If fish and mammals share similar muralium architec-

ture, what can explain the absence of ‘‘portal tracts’’ andlobule formation in lower vertebrate livers? Addressingthese questions involves consideration of organ systemontogeny and the nature of the ‘‘functional unit’’ of thevertebrate liver. Although this is beyond the scope ofthis article, a brief discussion is warranted. First, whileportal tracts/triads are well described in mammalianliver and integral to current mammalian liver concep-tual models (lobular, acinar), they are not often observedin lower vertebrates. In lower vertebrates, anatomicalstructures that may be perhaps the evolutionary, oreven functional, precursors to portal tracts have beendescribed as venous biliary arteriolar tracts (VBAT), ve-nous arteriolar tracts (VAT), venous biliary tracts (VBT),biliary tracts (BT), and arteriolar tracts (AT) (Rochaet al., 1995; Hampton, 1988). These morphological fea-tures bear some anatomical resemblance to the portaltract, although no semblance of lobule formation inlower vertebrates has been found. Second, based on our3D reconstructions, it can be hypothesized that the hep-


atobiliary systems of both medaka and mammals sharea common fundamental functional unit: (1) a ‘‘portaltract/hilus,’’ a single conduit containing two afferentblood supplies (hepatic portal and arterial vessels), andefferent bile duct(s); (2) a ‘‘primary efferent vascularconduit,’’ central vein/hepatic vein/terminal hepatic vein;and (3) anastomosing hepatic plates/cords, perfused by acanalicular network and sinusoidal bed, that bridgesthese two. A similar conceptual functional unit, the he-patic microcirculatory subunit (HMS), was proposed byEkataksin et al. (1997). This wedge-shaped unit is com-posed of ‘‘base’’ (the portal tract/hilus), ‘‘apex’’ (the cen-tral vein/terminal hepatic vein/hepatic vein), and a con-tinuous system (muralium) of anastomosing hepaticplates (laminae hepatic) connecting the base and apex,perfused by sinusoidal bed (labyrinthus hepatic). Hence,it can be considered that, in the lower vertebrate liver ofmedaka; the liver hilus is the correlary of the portaltract, and hepatic vein the corollary of the central vein/terminal hepatic vein. In between these is the laminaehepatic, the fine structure of which, up to this study, hasremained a mystery in medaka and other lower verte-brates.One of the outstanding questions in human biliary

architecture has been; does each duct of Hering corre-spond to one canaliculus? Or do canaliculi form a conflu-ence before entering the canals of Hering (Saxena et al.,1999)? Our 3D studies in medaka show the canalicularnetwork to form a confluence before entering the hilarintrahepatic ducts, the anatomical corollary of the canalsof Hering. Given the findings presented here and that:bile canalicular diameter is conserved in vertebrate liv-ers (1–2 mm); early observations by Elias and Bengels-dorf (1952) suggest a polygonal hepatocellular formation;that a ‘‘chicken-wire’’ pattern of bile canaliculi has beendescribed in human liver (Ekataksin et al., 1995); hexag-onal architecture has also been described in the finestructure of the biliary system in rat (Murakami et al.,2001) and human (Yamamoto et al., 1990); the tubularstructure (dual layered parenchyma) appears to be con-served among all embryonic vertebrates; erosion caststudies by Murakami et al. (2001) show a hexagonalcanalicular network feeding intrahepatic bile ducts (rat);bipotential progenitor-like cells in both medaka andmammals are closely associated with the biliary system,it is not unlikely that all vertebrate livers share thesame fundamental functional unit. Hence, it is interest-ing to consider the medaka hepatobiliary system, repre-sentative of lower vertebrate architecture, as a singlefunctional unit (akin to the HMS), while mammalianhepatobiliary systems can be considered to be composedof multiple functional units organized into hepaticlobules (typically 1–2 mm; Fig. 1). In mammals, wehypothesize that portal tracts arborize within the liverto form the classic intrahepatic biliary/vascular trees,giving rise to lobulation (multiple functional units).Hence, as conceptual models go, the primary architec-tural differences between medaka and mammalian hepa-tobiliary systems appears to arise at the tissue/organlevel of organization (multiple lobules in mammals vs.single lobule in medaka). These differences can be at-tributable to the metabolic and structural demands/needs between lower and higher vertebrate hepatobili-ary systems (in the context of organ system ontogenyand functional capacity), where mammals see a compa-

ratively higher metabolic demand, typically higher bodytemperature, higher load bearing on the liver resultingfrom gravity, and relatively larger mammalian livermass. It follows that higher vertebrates likely show‘‘lobulation’’ of the liver in support of greater organ massand metabolic demand; the iteration of a single hepaticfunctional unit (e.g. HMS). In more massive mamma-lian livers the formation of hexagonal lobules, organizedby stromal tissue (portal tracts/triads), would impartstructural integrity to the organ (due to the physicalproperties of hexagonal packing; Weaire and Phelan,1994; Hales, 2001), much needed in an organ of suchmass (�3–5% of body weight in mammals), and onehighly perfused with a liquid medium (the liver of mam-mals can store up 30% of total blood volume). It follows,from an ontological viewpoint, that appearance ofVBATs, VATs, VBTs, BTs, and ATs in larger piscine spe-cies may be evidence of the emergence of ‘‘lobulation’’among lower vertebrates such as teleosts. Such a consid-eration begs more detailed investigation of larger fishspecies, and reptilian and amphibian livers. Lastly, thehexagonal structural motif appears to be conservedamong all vertebrates; found not only in the organiza-tion of the canalicular network (intralobular paren-chyma), but also in the formation of the classic lobule(arborization of portal tracts).Reaching a conceptual model of the ‘‘functional unit’’

of the liver has long been sought, and the spatial archi-tecture of the lower vertebrate liver, particularly the bil-iary system, has long been in question. Differences inextant conceptual models have arisen from how lowerand higher vertebrate livers have been viewed at vari-ous levels of biological organization (cellular, tissue,organ), in which anatomical plane, and whether in twoor three dimensions. Given various ways of viewing theliver and the prior technological constraints (lack of 3Dand in vivo tools), understandably, discrepancies haveappeared to exist between lower vertebrate and mamma-lian hepatobiliary conceptual models. Such discrepanciescan be illustrated in a study by Akiyoshi and Inoue(2004), who investigated two hundred different teleostspecies (histological preparations) and described varyingliver architecture among them (muralium as cord-like[one cell], tubular [two cells], and solid [>two cells]).Indeed, we observed all three structural forms in our invivo and 3D investigations, each comprising the hepaticparenchyma, although the muralium of medaka wasfound to be predominantly two cells thick. Similar obser-vations have been made in developing mammalian liverby McCuskey et al. (2003). Hence, it appears the limitsof spatial observation/understanding permitted by singlesectional views of the liver have led to these, while accu-rate descriptions from a 2D viewpoint, discrepancies ininterpretation, and thereby discrepancies in a compara-tive understanding of the 3D architecture of vertebrateliver. Such discrepancies can be understood consideringthe difficult task of extrapolating 2D information to 3Darchitectural models. Particularly when 2D observationsin single sectional views of the liver have accuratelycharacterized the 3D structure we have elucidated here.It can be said that in one sense, prior observations oflower vertebrate liver architecture that described a tu-bular conceptual model were correct, in so far as theywere communicating the 2D morphology encountered insingle sectional views of the liver.

780 HARDMAN ET AL.

SUMMARY

Collectively, these findings elucidate the 3D architec-ture of the medaka hepatobiliary system and improveour comparative understanding of vertebrate liver struc-ture and function. These findings also present interest-ing questions regarding the ‘‘functional unit’’ of the ver-tebrate liver; where the hepatobiliary system in medakacan be, as a conceptual model, considered a single func-tional unit, akin to the individual lobule. We have alsoaddressed prior discrepancies among conceptual modelsof vertebrate hepatobiliary system architecture, and thefindings presented should help with integration of priorobservations of vertebrate liver structure/function into amore cohesive conceptual framework. In summary,in vivo and 3D findings in medaka show: (1) parenchy-mal architecture is predominantly a two-cell-thick mura-lium, although tubule-like formations were observed inembryonic livers; (2) the hepatic muralium is organizedthrough a polyhedral (hexagonal) structural motif,revealed in biliary architecture; (3) the intrahepatic bili-ary system is an interconnected network of canaliculiand bile preductules; (4) the canaliculo-preductular net-work occupies the majority of the liver corpus (�95%)uniformly, with equidiameter IHBPs (1–2 mm) observedthroughout the liver; (5) larger bile ductules and ductswere observed predominantly at the liver hilus, conse-quently an arborizing biliary tree was largely absent,seen only in the rudimentary branching of intrahepaticducts from the hepatic duct; and (6) the livers of thesesmall fish are replete with BPDECs, the putative mam-malian correlative of bipotential progenitor/stem cells.

ACKNOWLEDGMENTS

Thanks to Dr. David Miller, Laboratory of Pharmacol-ogy and Chemistry, National Institute of EnvironmentalHealth Sciences, Research Triangle Park, for providingaccess to their laser scanning confocal microscopy facil-ity, and special thanks to the Duke University Inte-grated Toxicology Program. This publication was madepossible by a grant from the National Center forResearch Resources, a component of the National Insti-tutes of Health. Its contents are solely the responsibilityof the authors and do not necessarily represent the offi-cial views of NCRR or NIH.

LITERATURE CITED

Akiyoshi H, Inoue A. 2004. Comparative histological study of teleostlivers in relation to phylogeny. Zoolog Sci 21:841–850.

Alestrom P, Holter JL, Nourizadeh-Lillabadi R. 2006. Zebrafish infunctional genomics and aquatic biomedicine. Trends Biotechnol24:15–21.

Arias IM. 1988. The Liver: Biology and Pathobiology. Irwin M.Arias, William B. Jakoby, Hans Popper, David Schachter, DavidA. Shafritz, eds. Raven Press, Baltimore, Maryland.

Berghmans S, Jette C, Langenau D, Hsu K, Stewart R, Look T,Kanki JP. 2005. Making waves in cancer research: new models inthe zebrafish. Biotechniques 39:227–237.

Blouin A, Bolender RP, Weibel ER. 1977. Distribution of organellesand membranes between hepatocytes and nonhepatocytes in therat liver parenchyma. A stereological study. J Cell Biol 72:441–455.

Carle J, Myoupo J, Stojmenovic I. 2001. Higher dimensional honey-comb networks. J Intercon Netw 2:391–420.

Ekataksin W, Zou Z, Wake K, Chunhabundit P, Somana R, NishidaJ, McDonnell D. 1995. HMS, Hepatic microcirculatory subunits inmammalian species. Intralobular grouping of liver tissue withdefinition enhanced by dropout sinusoids. In: Cells of the HepaticSinusoid. Leiden: Wisse E, Knook DL, Wake K, eds. The KupfferCell Foundation, The Netherlands.

Elias H. 1949. A re-examination of the structure of the mammalianliver. II. The hepatic lobule and its relation to the vascular andbiliary systems. Am J Anat 85:379–456.

Elias H, Bengelsdorf H. 1952. The structure of the liver of verte-brates. Acta Anat 14:297–337.

Farber E. 1956. Similarities in the sequence of early histologicalchanges induced in the liver of the rat by ethionine, 2-acetyla-mino-fluorene, and 30-methyl-4-dimethylaminoazobenzene. CancerRes 16:142–148.

Fausto N, Campbell JS. 2003. The role of hepatocytes and ovalcells in liver regeneration and repopulation. Mech Dev 120:117–130.

Golding M, Sarraf C, Lalani EN, Alison MR. 1996. Reactive biliaryepithelium: the product of a pluripotential stem cell compart-ment? Hum Pathol 27:872–884.

Hales T. 2001. The honeycomb conjecture. Discrete Comput Geom25:1–22.

Hampton J, Lantz R, Goldblatt P, Lauren D, Hinton D. 1988. Func-tional units in rainbow trout (Salmo gairdneri, Richardson) liver:II. The biliary system. Anat Rec 221:619–634.

Hampton JA, Lantz RC, Hinton DE. 1989. Functional units in rain-bow trout (Salmo gairdneri, Richardson) liver: III. Morphometricanalysis of parenchyma, stroma, and component cell types. Am JAnat 185:58–73.

Hess FA, Gnagi HR, Weibel ER, Preisig R. 1973. Morphometry ofdog liver: comparison of wedge and needle biopsies. Eur J ClinInvest 3:451–458.

Hinton DE, Couch JA. 1998. Architectural pattern, tissue and cellu-lar morphology in livers of fishes: Relationship to experimentally-induced neoplastic responses. In: Fish Ecotoxicology. Braunbeck,T., D.E. Hinton, and B. Streit, eds., Birkhauser Verlag. Basel,Switzerland.

Hinton D, Pool C. 1976. Ultrastructure of the liver in channel cat-fish Ictalurus punctatus (Rafinesque). J Fish Biol 8:209–219.

Hinton D, Segner H, Braunbeck T. 2001. Toxic Responses of theLiver. In: New Perspectives: Toxicology and the Environment,Target Organ Toxicity in Marine and Freshwater Teleosts.D. Schlenk and Benson, W. eds. Taylor & Francis, New York.

Jungermann K. 1988. Metabolic zonation of liver parenchyma.Semin Liver Dis 8:329–341.

Kiernan F. 1833. The anatomy and physiology of the liver. PhilosTrans R Soc Lond 123:711–770.

Knight B, Matthews VB, Olynyk JK, Yeoh GC. 2005. Jekyll andHyde: evolving perspectives on the function and potential of theadult liver progenitor (oval) cell. Bioessays 27:1192–1202.

Lamers WH, Hilberts A, Furt E, Smith J, Jonges GN, van NoordenCJ, Janzen JW, Charles R, Moorman AF. 1989. Hepatic enzymiczonation: a reevaluation of the concept of the liver acinus. Hepa-tology 10:72–76.

Ludwig J, Ritman EL, LaRusso NF, Sheedy PF, Zumpe G. 1998.Anatomy of the human biliary system studied by quantitativecomputer-aided three-dimensional imaging techniques. Hepato-logy 27:893–899.

Mainzer K. 1996. Symmetries of nature: a handbook for philosophyof nature and science. Walter De Gruyter Inc., Berlin, Germany

Mall F. 1906. A study of the structural unit of the liver. J Anat5:227–308.

Masyuk TV, Ritman EL, LaRusso NF. 2001. Quantitative assess-ment of the rat intrahepatic biliary system by three-dimensionalreconstruction. Am J Pathol 158:2079–2088.

Matsumoto T, Komori R, Magara T, Ui T, Kawakami M, Tokuda T,Takasaki S. 1979. A study on the normal structure of the humanliver, with special reference to its angioarchitecture. Jikeikai MedJ 26:1–40.

McCuskey P, McCuskey RS, Hinton D. 1986. Electron microscopy ofthe hepatic sinusoids in rainbow trout. In: Cells of the Hepatic


Sinusoid 1. Kirn A, Knook DL, Wisse E., Eds. The Kupffer CellFoundation, The Netherlands

McCuskey RS, Ekataksin W, LeBouton AV, Nishida J, McCuskeyMK, McDonnell D, Williams C, Bethea NW, Dvorak B, KoldovskyO. 2003. Hepatic microvascular development in relation to themorphogenesis of hepatocellular plates in neonatal rats. Anat RecA Discov Mol Cell Evol Biol 275:1019–1030.

Mochizuki Y, Furukawa K, Mitaka T, Yokoi T, Kodama T. 1988. Po-lygonal networks, ‘‘geodomes’’, of adult rat hepatocytes in primaryculture. Cell Biol Int Rep 12:1–7.

Motta P, Fumagalli G. 1975. Structure of rat bile canaliculi asrevealed by scanning electron microscopy. Anat Rec 182:499–513.

Murakami T, Sato H, Nakatani S, Taguchi T, Ohtsuka A. 2001. Bili-ary tract of the rat as observed by scanning electron microscopyof cast samples. Arch Histol Cytol 64:439–447.

Okihiro M, Hinton D. 1999. Progression of hepatic neoplasia inmedaka (Oryzias latipes) exposed to diethylnitrosamine. Carcino-genesis 206:933–940.

Okihiro MS, Hinton DE. 2000. Partial hepatectomy and bile duct liga-tion in rainbow trout (Oncorhynchus mykiss): histologic, immuno-histochemical and enzyme histochemical characterization of hepaticregeneration and biliary hyperplasia. Toxicol Pathol 28:342–356.

Rappaport AM. 1958. The structural and functional unit in thehuman liver (liver acinus). Anat Rec 130:673–689.

Rocha E, Monteiro RA, Pereira CA. 1994. The liver of the browntrout, Salmo trutta fario: a light and electron microscope study.J Anat 185(Pt 2): 241–249.

Rocha E, Monoteiro R, Pereira C. 1995. Microanatomical organiza-tion of hepatic stroma of Brown Trout, Salmo trutta fario (Tele-ostei, Salmonidae): a qualitative and quantitative approach.J Morphol 223:1–11.

Rocha E, Monteiro RA, Pereira CA. 1997. Liver of the brown trout,Salmo trutta (Teleostei, Salmonidae): a stereological study at lightand electron microscopic levels. Anat Rec 247:317–328.

Saxena R, Theise ND, Crawford JM. 1999. Microanatomy of the humanliver-exploring the hidden interfaces. Hepatology 30:1339–1346.

Shima A, Mitani H. 2004. Medaka as a research organism: past,present and future. Mech Dev 121:599–604.

Teutsch HF, Schuerfeld D, Groezinger E. 1999. Three-dimensionalreconstruction of parenchymal units in the liver of the rat. Hepa-tology 29:494–505.

Theise ND, Saxena R, Portmann BC, Thung SN, Yee H, ChiribogaL, Kumar A, Crawford JM. 1999. The canals of Hering and he-patic stem cells in humans. Hepatology 30:1425–1433.

Turcotte D, Pelletier J, Newman W. 1998. Networks with sidebranching in biology. J Theor Biol 193:577–592.

Van Eyken P, Sciot R, Desmet VJ. 1989. A cytokeratin immunohisto-chemical study of cholestatic liver disease: evidence that hepatocytescan express ‘bile duct-type’ cytokeratins. Histopathology 15:125–135.

Vandersteenhoven AM, Burchette J, Michalopoulos G. 1990. Char-acterization of ductular hepatocytes in end-stage cirrhosis. ArchPathol Lab Med 114:403–406.

Wakamatsu Y, Pristyazhnyuk S, Kinoshita M, Tanaka M, Ozato K.2001. The see-through medaka: a fish model that is transparentthroughout life. Proc Natl Acad Sci USA 98:10046–10050.

Weaire D, Phelan R. 1994. Optimal design of honeycombs. Nature367:123.

Wittbrodt J, Shima A, Schartl M. 2002. Medaka--a model organismfrom the far East. Nat Rev Genet 3:53–64.

Yamamoto K, Itoshima T, Tsuji T, Murakami T. 1990. Three-dimen-sional fine structure of the biliary tract: scanning electron micros-copy of biliary casts. J Electron Microsc Tech 14:208–217.

782 HARDMAN ET AL.

an in vivo look at vertebrate liver

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