Thyroid Epithelial Morphogenesis in Vitro: A Role for Bumetanide-Sensitive Cl Secretion during Follicular Lumen Development

The structural and functional unit of the thyroid gland is the follicle, consisting of a closed lumen surrounded by a single layer of polarized epithelial cells. In this paper we have attempted to characterize the process of lumenal development when primary cultures of porcine thyroid cells reorganized to form follicles. Cells incubated with the loop diuretic, bumetanide, an inhibitor of NaK2Cl cotransport, aggregated but failed to form normal follicles. Laser scanning confocal microscopy combined with immunohistochemical markers of thyroid cell-surface proteins demonstrated that in the presence of bumetanide cells polarized and assembled ZO-1-containing tight junctions separating their apical and basolateral membrane domains. Cultures formed small lumena but their subsequent growth was inhibited by bumetanide. Electrophysiological studies confirmed that bumetanide-sensitive Cl^- transport was the major contributor to the transepithelial electrical potential difference across the follicular wall after 48 h incubation. Other potential mechanisms did not contribute significantly to follicular lumenal growth. In particular, bumetanide did not affect cell proliferation and, in contrast to tissue follicles, thyroglobulin could not be detected within the lumena of cultured follicles. We conclude that thyroid follicular reorganization involves two distinct and separate phases of lumenal development: initial lumen formation which probably reflects the assembly of a specialized apical membrane domain; and subsequent lumenal growth which is mediated by the inward transport of Cl^- by polarized epithelial cells.     

Functional differentiation and alveolar morphogenesis of primary mammary cultures on reconstituted basement membrane

An essential feature of mammary gland differentiation during pregnancy is the formation of alveoli composed of polarized epithelial cells, which, under the influence of lactogenic hormones, secrete vectorially and sequester milk proteins. Previous culture studies have described either organization of cells polarized towards lumina containing little or no demonstrable tissue-specific protein, or establishment of functional secretory cells exhibiting little or no glandular architecture. In this paper, we report that tissue-specific vectorial secretion coincides with the formation of functional alveoli-like structures by primary mammary epithelial cells cultured on a reconstituted basement membrane matrix (derived from Engelbreth-Holm-Swarm murine tumour). Morphogenesis of these unique three-dimensional structures was initiated by cell-directed remodelling of the exogenous matrix leading to reorganization of cells into matrix-ensheathed aggregates by 24 h after plating. The aggregates subsequently cavitated, so that by day 6 the cells were organized into hollow spheres in which apical cell surfaces faced lumina sealed by tight junctions and basal surfaces were surrounded by a distinct basal lamina. The profiles of proteins secreted into the apical (luminal) and basal (medium) compartments indicated that these alveoli-like structures were capable of an appreciable amount of vectorial secretion. Immunoprecipitation with a broad spectrum milk antiserum showed that more than 80% of caseins were secreted into the lumina, whereas iron-binding proteins (both lactoferrin and transferrin) were present in comparable amounts in each compartment. Thus, these mammary cells established protein targeting pathways directing milk-specific proteins to the luminal compartment. A time course monitoring secretory activity demonstrated that establishment of tissue-specific vectorial secretion and increased total and milk protein secretion coincided with functional alveolar-like multicellular architecture. This culture system is unique among models of epithelial cell polarity in that it demonstrates several aspects of epithelial cell polarization: vectorial secretion, apical junctions, a sequestered compartment and formation of a basal lamina. These lumina-containing structures therefore reproduce the dual role of mammary epithelia to secrete vectorially and to sequester milk proteins. Thus, in addition to maintaining tissue-specific cytodifferentiation and function, a basement membrane promotes the expression of tissue-like morphogenesis.     

HAS3-induced accumulation of hyaluronan in 3D MDCK cultures results in mitotic spindle misorientation and disturbed organization of epithelium

The amount of hyaluronan (HA) is low in simple epithelia under normal conditions, but during tumorigenesis, trauma or inflammation HA is increased on the epithelial cells and surrounding stroma. Excessive HA in epithelia is suggested to interfere with cell–cell adhesions, resulting in disruption of the epithelial barrier function. In addition, stimulated HA synthesis has been correlated with epithelial-to-mesenchymal transition and invasion of cancer cells. However, the effects of HA overload on normal epithelial morphogenesis have not been characterized in detail. Madin-Darby canine kidney (MDCK) cells form polarized epithelial cysts, when grown in a 3-dimensional (3D) matrix. These cells were used to investigate whether stimulated HA synthesis, induced by stable overexpression of GFP-HAS3, influences cell polarization and epithelial morphogenesis. GFP-HAS3 expression in polarized MDCK cells resulted in active HA secretion at apical and basolateral membrane domains. HA-deposits interfered with the formation of cell–cell junctions, resulting in impaired barrier function. In 3D cyst cultures, HA accumulated into apical lumina and was also secreted from the basal side. The HAS3-expressing cysts failed to form a single lumen and instead displayed multiple small lumina. This phenotype was correlated with aberrant mitotic spindle orientation in dividing cells. The results of this study indicate that excess pericellular HA disturbs the normal cell–cell and cell–ECM interactions in simple epithelia, leading to aberrant epithelial morphogenesis. The morphological abnormalities observed in 3D epithelial cultures upon stimulated HAS3 expression may be related to premalignant changes, including intraluminal invasion and deregulated epithelialization, probably mediated by the mitotic spindle orientation defects.     

Polarity reversal of inside-out thyroid follicles cultured within collagen gel: an ultrastructural study

Inside-out porcine thyroid follicles in culture undergo polarity reversal after being embedded in collagen gel. The newly-formed follicles reexpress some specific thyroid functions lost in inside-out follicles (Chambard et al., 1984. We present here an ultrastructural study of the inversion of polarity in this model system. This process takes place within 24 to 48 hr, without any opening of the original tight junctions, as shown by fixation in the presence of ruthenium red. A general shrinkage of cellular aggregates was noted soon after embedding. At the apical pole, three different modifications were observed: structural changes appeared in the kinocilium, microvilli and underlying cytoskeleton as early as 10 min after embedding, mainly when the apical pole of the cells was in close contact with the collagen fibers; large cytoplasmic lamellipod- or pseudopod-like extensions, covering the adjacent apical domain, protruded from outer apical regions; some other apical areas invaginated and formed channels inside the aggregates. The last two processes prevented close contact between apical cell surfaces and collagen fibers and allowed a persistence of the initial polarity in some of the cells. Newly-formed lumens were closed 24 hr after embedding in gel and the outer surface of the cellular aggregates in close contact with collagen fibers looked like a basal membrane. These mechanisms proceeded at different rates and involved different numbers of cells, but they all appeared to be related to the transformation of inside-out follicles into follicular structures.     

Sealing of the follicular lumen of the anterior pituitary gland of the male rat

It is commonly accepted that follicular lumina of the adult rat anterior pituitary gland are tightly sealed by junctional complexes, especially tight junctions. In this report, we describe the presence of follicular lumina that are unsealed. Peroxidase (HRP) was used to study such structures and when injected through the femoral vein, was observed in association with a few follicular lumina, on their microvilli and around the cilia of folliculo-stellate cells. The existence of peroxidase-positive follicles clearly shows that follicles of the hypophysis are not always firmly sealed by tight junctions. The folliculo-stellate cells which faced the peroxidase-positive follicles displayed HRP deposits which were membrane bound within their cytoplasm. These findings suggest an absorptive function for the folliculo-stellate cells.     

Influenza virus hemagglutinin expression is polarized in cells infected with recombinant SV40 viruses carrying cloned hemagglutinin DNA

Primary cell cultures of African Green monkey kidney (AGMK) contain polarized epithelial cells in which influenza virus matures predominantly at the apical surfaces above tight junctions. Influenza virus glycoproteins were found to be localized at the same membrane domain from which the virus budded. When polarized primary AGMK cells were infected with recombinant SV40 viruses containing DNA coding for either an influenza virus H1 or H2 subtype hemagglutinin (HA), the HA proteins were preferentially expressed at the apical surface in a manner identical to that observed in influenza virus-infected cells. Thus, cellular mechanisms for sorting membrane glycoproteins recognize some structural feature of the HA glycoprotein itself, and other viral proteins are not necessary for this process.     

Determination of apical membrane polarity in mammary epithelial cell cultures: The role of cell-cell, cell-substratum, and membrane-cytoskeleton interactions

The membrane glycoprotein, PAS-O, is a major differentiation antigen on mammary epithelial cells and is located exclusively in the apical domain of the plasma membrane. We have used 734B cultured human mammary carcinoma cells as a model system to study the role of tight junctions, cell-substratum contacts, and submembraneous cytoskeletal elements in restricting PAS-O to the apical membrane. Immunofluorescence and immunoelectronmicroscopy experiments demonstrated that while tight junctions demarcate PAS-O distribution in confluent cultures, apical polarity could be established at low culture densities when cells could not form tight junctions with neighboring cells. In such cultures the boundary between apical and basal domains was observed at the point of cell contact with the substratum. Immunocytochemical analysis of these cell-substratum contacts revealed the absence of a characteristic basement membrane containing laminin, collagen (IV), and heparan sulfate proteoglycan. However, serum-derived vitronectin was associated with the basal cell surface and the cells were shown to express the vitronectin receptor on their basolateral membranes. Additionally, treatment of cultures with antibodies against the vitronectin receptor caused cell detachment. We suggest, then, that interactions between vitronectin and its receptor, are responsible for establishment of membrane domains in the absence of tight junctions. The role of cytoskeletal elements in restricting PAS-O distribution was examined by treating cultures with cytochalasin D, colchicine, or acrylamide. Cytochalasin D led to a redistribution of PAS-O while colchicine and acrylamide did not. We hypothesize that PAS-O is restricted to the apical membrane by interactions with a microfilament network and that the cytoskeletal organization is dependent upon cell-cell and cell-substratum interactions.     

Distribution of microtubules and microfilaments in thyroid follicular epithelial cells of normal,TSH-treated,aged, and hypophysectomized rats

Summary We investigated the distribution of microtubules and microfilaments in rat thyroid follicular epithelial cells by applying an immunofluorescence technique with monoclonal antibodies against tubulin and by staining sections with rhodamine-phalloidin. In normal thyroid cells, microtubules run longitudinally from the apical region to the basal region intersecting with each other. In addition, intense labelling with tubulin antibodies was observed in the apical part of the cell. The ultrastructural examinations showed that microtubules often run along the apical plasma membrane. Dot-like labelling with anti-tubulin antibodies was often observed in the perinuclear space, but no microtubules were recognized in the nucleus. Microfilaments bound to rhodamine-phalloidin were distributed mainly beneath the apical plasma membrane, and the portion along the basolateral membrane was scarcely positive. The apical pole of the follicle cell was also decorated by anti-microtubule-associated protein-2 (MAP-2). After TSH stimulation, the intensity of immunocytochemical staining against tubulin was remarkably increased in the cytoplasm. Simultaneously, at the apical region, the staining intensity of rhodamine-phalloidin was increased. Microtubules and microfilaments appeared in the pseudopods after TSH injection. In hypophysectomized or aged rats, thyroid follicular epithelial cells decreased in height, and both immunofluorescent labelling against tubulin and rhodamine-phalloidin labelling were markedly decreased. These results indicate that the distribution and polymerization of microtubules and microfilaments in thyroid follicular epithelial cells vary with the functional stage.     

Microtubule organization and function in epithelial cells

Microtubules are essential for many aspects of polarity in multicellular organisms, ranging from the asymmetric distribution of cell-fate determinants in the one-cell embryo to the transient polarity generated in migrating fibroblasts. Epithelial cells exhibit permanent cell polarity characterized by apical and basolateral surface domains of distinct protein and lipid composition that are segregated by tight junctions. They are also endowed with a microtubule network that reflects the asymmetry of their cell surface: microtubule minus-ends face the apical- and microtubule plus-ends the basal domain. Strikingly, the formation of distinct surface domains during epithelial differentiation is accompanied by the re-organization of microtubules from a uniform array focused at the centrosome to the noncentrosomal network that aligns along the apico-basolateral polarity axis. The significance of this coincidence for epithelial morphogenesis and the signaling mechanisms that drive microtubule repolymerization in developing epithelia remain major unresolved questions that we are only beginning to address. Studies in cultured polarized epithelial cells have established that microtubules serve as tracks that facilitate targeted vesicular transport. Novel findings suggest, moreover, that microtubule-based transport promotes protein sorting, and even the generation of transport carriers in the endo- and exocytic pathways.     

A small rab GTPase is distributed in cytoplasmic vesicles in non polarized cells but colocalizes with the tight junction marker ZO-1 in polarized epithelial cells

Small rab/Ypt1/Sec4 GTPase family have been involved in the regulation of membrane traffic along the biosynthetic and endocytic pathways in eucaryotic cells. Polarized epithelial cells have morphologically and functionally distinct apical and basolateral surfaces separated by tight junctions. The establishment and maintenance of these structures require delivery of membrane proteins and lipids to these domains. In this work, we have isolated a cDNA clone from a human intestinal cDNA library encoding a small GTPase, rab13, closely related to the yeast Sec4 protein. Confocal microscopy analysis on polarized Caco-2 cells shows that rab13 protein colocalized with the tight junction marker ZO- 1. Cryostat sections of tissues confirm that rab13 localized to the junctional complex region of a variety of epithelia, including intestine, kidney, liver, and of endothelial cells. This localization requires assembly and integrity of the tight junctions. Disruption of tight junctions by incubation in low Ca2+ media induces the redistribution of rab13. In cells devoid of tight junctions, rab13 was found associated with vesicles dispersed throughout the cytoplasm. Cell- cell contacts initiated by E-cadherin in transfected L cells do not recruit rab13 to the resulting adherens-like junction complexes. The participation of rab13 in polarized transport, in the assembly and/or the activity of tight junctions is discussed.     

Studies on the development and maintenance of epithelial cell surface polarity with monoclonal antibodies

We examined epithelial cell surface polarity in subconfluent and confluent Madin-Darby canine kidney (MDCK) cells with monoclonal antibodies directed against plasma membrane glycoproteins of 35,000, 50,000, and 60,000 mol wt. The cell surface distribution of these glycoproteins was studied by immunofluorescence and immunoelectron microscopy. At the ultrastructural level, the electron-dense reaction product localizing all three glycoproteins was determined to be uniformly distributed over the apical and basal cell surfaces of subconfluent MDCK cells as well as on the lateral surfaces between contacted cells; however, after formation of a confluent monolayer, these glycoproteins could only be localized on the basal-lateral plasma membrane. The development of cell surface polarity was followed by assessing glycoprotein distribution with immunofluorescence microscopy at selected time intervals during growth of MDCK cells to form a confluent monolayer. These results were correlated with transepithelial electrical resistance measurements of tight junction permeability and it was determined by immunofluorescence that polarized distributions of cell surface glycoproteins were established just after electrical resistance could be detected, but before the development of maximal resistance. Our observations provide evidence that intact tight junctions are required for the establishment of the apical and basal- lateral plasma membrane domains and that development of epithelial cell surface polarity is a continuous process.     

Orientation of spindle axis and distribution of plasma membrane proteins during cell division in polarized MDCKII cells

MDCKII cells differentiate into a simple columnar epithelium when grown on a permeable support; the monolayer is polarized for transport and secretion. Individual cells within the monolayer continue to divide at a low rate without disturbing the function of the epithelium as a barrier to solutes. This presents an interesting model for the study of mitosis in a differentiated epithelium which we have investigated by confocal immunofluorescence microscopy. We monitored the distribution of microtubules, centrioles, nucleus, tight junctions, and plasma membrane proteins that are specifically targeted to the apical and basolateral domains. The stable interphase microtubule cytoskeleton was rapidly disassembled at prophase onset and reassembled at cytokinesis. As the interphase microtubules disassembled at prophase, the centrioles moved from their interphase position at the apical membrane to the nucleus and acquired the ability to organize microtubule asters. Orientation of the spindle parallel to the plane of the monolayer occurred between late prophase and metaphase and persisted through cytokinesis. The cleavage furrow formed asymmetrically perpendicular to the plane of the monolayer initiating at the basolateral side and proceeding to the apical domain. The interphase microtubule network reformed after the centrioles migrated from the spindle poles to resume their interphase apical position. Tight junctions (ZO-1), which separate the apical from the basolateral domains, remained assembled throughout all phases of mitosis. E-cadherin and a 58-kD antigen maintained their basolateral plasma membrane distributions, and a 114- kD antigen remained polarized to the apical domain. These proteins were useful for monitoring the changes in shape of the mitotic cells relative to neighboring cells, especially during telophase when the cell shape changes dramatically. We discuss the changes in centriole position during the cell cycle, mechanisms of spindle orientation, and how the maintenance of polarized plasma membrane domains through mitosis may facilitate the rapid reformation of the polarized interphase cytoplasm.     

The subcellular organization of Madin-Darby canine kidney cells during the formation of a polarized epithelium

Studies of the developing trophectoderm in the mouse embryo have shown that extensive cellular remodeling occurs during epithelial formation. In this investigation, confocal immunofluorescence microscopy is used to examine the three-dimensional changes in cellular architecture that take place during the polarization of a terminally differentiated epithelial cell line. Madin-Darby canine kidney cells were plated at a low density on permeable filter supports. Antibodies that specifically recognize components of the tight junction, adherens junction, microtubules, centrosomes, and the Golgi complex were used to study the spatial remodeling of the cytoarchitecture during the formation of the polarized cell layer. The immunofluorescence data were correlated with establishment of functional tight junctions as measured by transepithelial resistance and back-exchange of the cell surface, labeled with metabolites of the fluorescent lipid analogue N-(7-[4- nitrobenzo-2-oxa-1,3-diazole]) aminocaproyl sphingosine. 1 d after plating, single cells had microtubules, radiating from a broad region, that contained the centrosomes and the Golgi complex. 2 d after plating, the cells had grown to confluence and had formed functional tight junctions close to the substratum. The centrioles had split and no longer organized the microtubules which were running above and below the nucleus. The Golgi complex had spread around the nucleus. By the fifth day after plating, the final polarized state had been achieved. The junctional complex had moved greater than 10 microns upward from its basal location. The centrioles were together below the apical membrane, and the Golgi complex formed a ribbon-like convoluted structure located in the apical region above the nucleus. The microtubules were organized in an apical web and in longitudinal microtubule bundles in the apical-basal axis of the columnar cell. The longitudinal microtubules were arranged with their minus ends spread over the apical region of the cell and their plus ends toward the basal region. These findings show that there is an extensive remodeling of epithelial cytoarchitecture after formation of cell-cell contacts. Reorganization of the microtubule network results in functional polarization of the cytoplasm.     

Four-dimensional imaging of filter-grown polarized epithelial cells

Understanding how epithelial cells generate and maintain polarity and function requires live cell imaging. In order for cells to become fully polarized, it is necessary to grow them on a permeable membrane filter; however, the translucent filter obstructs the microscope light path required for quantitative live cell imaging. Alternatively, the membrane filter may be excised but this eliminates selective access to apical and basolateral surfaces. Conversely, epithelial cells cultured directly on glass exhibit different phenotypes and functions from filter grown cells. Here, we describe a new method for culturing polarized epithelial cells on a Transwell filter insert that allows superior live cell imaging with spatial and temporal image resolution previously unachievable using conventional methods. Cells were cultured on the underside of a filter support. Epithelial cells grown in this inverted configuration exhibit a fully polarized architecture, including the presence of functional tight junctions. This new culturing system permits four-dimensional (three spatial dimension over time) imaging of endosome and Golgi apparatus dynamics, and permits selective manipulation of the apical and basolateral surfaces. This new technique has wide applicability for visualization and manipulation of polarized epithelial cells.     

Suspension culture reveals a morphogenetic property of a thyroid epithelial cell line

It is known that freshly dissociated thyroid cell clusters form follicles in suspension culture. Thyroid epithelial cell lines, grown for many generations in vitro, fail to show colloid-containing lumina when cultured as monolayers. Several thyroid cell lines, some transformed, have been tested with respect to their ability to form extracellular lumina when transferred from monolayer to suspension culture. One cell line in particular, the T78 cell line, showed this property when cultured in suspension. Lumina formed within 3 days even in the absence of added thyrotropin (TSH). The ultrastructure of lumina within cell aggregates resembled that of the thyroid follicle in vivo. The ability to undergo morphogenesis may therefore be an intrinsic property of thyroid epithelial cells which is retained for a large number of generations in vitro and is revealed by proper culture conditions. The shift from monolayer to suspension culture may thus lead to the expression of a thyroid differentiated function such as the formation of follicle-like structures.     

Dbl3 drives Cdc42 signaling at the apical margin to regulate junction position and apical differentiation

Epithelial cells develop morphologically characteristic apical domains that are bordered by tight junctions, the apical–lateral border. Cdc42 and its effector complex Par6–atypical protein kinase c (aPKC) regulate multiple steps during epithelial differentiation, but the mechanisms that mediate process-specific activation of Cdc42 to drive apical morphogenesis and activate the transition from junction formation to apical differentiation are poorly understood. Using a small interfering RNA screen, we identify Dbl3 as a guanine nucleotide exchange factor that is recruited by ezrin to the apical membrane, that is enriched at a marginal zone apical to tight junctions, and that drives spatially restricted Cdc42 activation, promoting apical differentiation. Dbl3 depletion did not affect junction formation but did affect epithelial morphogenesis and brush border formation. Conversely, expression of active Dbl3 drove process-specific activation of the Par6–aPKC pathway, stimulating the transition from junction formation to apical differentiation and domain expansion, as well as the positioning of tight junctions. Thus, Dbl3 drives Cdc42 signaling at the apical margin to regulate morphogenesis, apical–lateral border positioning, and apical differentiation.     

Differential trafficking of transforming growth factor-beta receptors and ligand in polarized epithelial cells

Epithelial cells in vivo form tight cell-cell associations that spatially separate distinct apical and basolateral domains. These domains provide discrete cellular processes essential for proper tissue and organ development. Using confocal imaging and selective plasma membrane domain activation, the type I and type II transforming growth factor-beta (TGFbeta) receptors were found to be localized specifically at the basolateral surfaces of polarized Madin-Darby canine kidney (MDCK) cells. Receptors concentrated predominantly at the lateral sites of cell-cell contact, adjacent to the gap junctional complex. Cytoplasmic domain truncations for each receptor resulted in the loss of specific lateral domain targeting and dispersion to both the apical and basal domains. Whereas receptors concentrate basolaterally in regions of direct cell-cell contact in nonpolarized MDCK cell monolayers, receptor staining was absent from areas of noncell contact. In contrast to the defined basolateral polarity observed for the TGFbeta receptor complex, TGFbeta ligand secretion was found to be from the apical surfaces. Confocal imaging of MDCK cells with an antibody to TGFbeta1 confirmed a predominant apical localization, with a stark absence at the basal membrane. These findings indicate that cell adhesion regulates the localization of TGFbeta receptors in polarized epithelial cultures and that the response to TGFbeta is dependent upon the spatial distribution and secretion of TGFbeta receptors and ligand, respectively.     

Gp135/podocalyxin and NHERF-2 participate in the formation of a preapical domain during polarization of MDCK cells

Epithelial polarization involves the segregation of apical and basolateral membrane domains, which are stabilized and maintained by tight junctions and membrane traffic. We report that unlike most apical and basolateral proteins in MDCK cells, which separate only after junctions have formed, the apical marker gp135 signifies an early level of polarized membrane organization established already in single cells. We identified gp135 as the dog orthologue of podocalyxin. With a series of domain mutants we show that the COOH-terminal PSD-95/Dlg/ZO-1 (PDZ)-binding motif is targeting podocalyxin to the free surface of single cells as well as to a subdomain of the terminally polarized apical membrane. This special localization of podocalyxin is shared by the cytoplasmic PDZ-protein Na+/H+ exchanger regulatory factor (NHERF)-2. Depleting podocalyxin by RNA interference caused defects in epithelial polarization. Together, our data suggest that podocalyxin and NHERF-2 function in epithelial polarization by contributing to an early apical scaffold based on PDZ domain-mediated interactions.     

Factors affecting the differentiation of epithelial transport and responsiveness to hormones

Under standard culture conditions, epithelial cells grow with their basal surface attached to the culture dish and their apical surface facing the medium. Morphological and functional markers are located in the appropriate plasma membrane, and transepithelial transport occurs in a variety of cultured epithelia. As a result of the polarity of the cells and the presence of tight junctions between cells, on standard tissue culture dishes there is restricted access of growth medium to the basolateral surface of the epithelium, which is the surface at which nutrient exchange normally occurs. Greater differentiation of epithelial cultures can be achieved by growing primary cultures or continuous cell lines on permeable surfaces such as porous bottom cultures dishes in which the porous bottom is formed by a filter or membrane of collagen, or on floating collagen gels. In many cultures, differentiation varies with the time after the culture was seeded. Certain chemicals that accelerate differentiation in nonepithelial cells also accelerate the differentiation of epithelial cultures. Ultimately, defined media and specific substrates for cell attachment should lead to further differentiation of epithelia in culture.     

The unique polarity phenotype of hepatocytes

Hepatocytes, the main epithelial cell type of the liver, function like all epithelial cells to mediate the vectorial flow of macromolecules into and out of the organ they encompass. They do so by establishing polarized surface domains and by restricting paracellular flow via their tight junctions and cell–cell adhesion. Yet, the cell and tissue organization of hepatocytes differs profoundly from that of most other epithelia, including those of the digestive and urinary tracts, the lung or the breast. The latter form monolayered tissues in which the apical domains of individual cells align around a central continuous luminal cavity that constitutes the tubules and acini characteristic of these organs. Hepatocytes, by contrast, form capillary-sized lumina with multiple neighbors resulting in a branched, tree-like bile canaliculi network that spreads across the liver parenchyme. I will discuss some of the key molecular features that distinguish the hepatocyte polarity phenotype from that of monopolar, columnar epithelia.