Intrahepatic Bile Duct Regeneration in Mice Does Not Require Hnf6 or Notch Signaling through Rbpj

The potential for intrahepatic bile duct (IHBD) regeneration in patients with bile duct insufficiency diseases is poorly understood. Notch signaling and Hnf6 have each been shown to be important for the morphogenesis of IHBDs in mice. One congenital pediatric liver disease characterized by reduced numbers of IHBDs, Alagille syndrome, is associated with mutations in Notch signaling components. Therefore, we investigated whether liver cell plasticity could contribute to IHBD regeneration in mice with disruptions in Notch signaling and Hnf6. We studied a mouse model of bile duct insufficiency with liver epithelial cell–specific deficiencies in Hnf6 and Rbpj, a mediator of canonical Notch signaling. Albumin-Cre Hnf6^flox/floxRbpj^flox/flox mice initially developed no peripheral bile ducts. The evolving postnatal liver phenotype was analyzed using IHBD resin casting, immunostaining, and serum chemistry. With age, Albumin-Cre Hnf6^flox/floxRbpj^flox/flox mice mounted a ductular reaction extending through the hepatic tissue and then regenerated communicating peripheral IHBD branches. Rbpj and Hnf6 were determined to remain absent from biliary epithelial cells constituting the ductular reaction and the regenerated peripheral IHBDs. We report the expression of Sox9, a marker of biliary epithelial cells, in cells expressing hepatocyte markers. Tissue analysis indicates that reactive ductules did not arise directly from preexisting hilar IHBDs. We conclude that liver cell plasticity is competent for regeneration of IHBDs independent of Notch signaling via Rbpj and Hnf6.Studies have demonstrated that bipotential progenitors may be present in the liver that are capable of giving rise to hepatocytes and biliary epithelial cells (BECs).^1–4 However, the in vivo capacity of specific stem, progenitor, or liver epithelial cells to regenerate a functional intrahepatic bile duct (IHBD) system, and whether endogenous hepatic cells hold potential therapeutic benefit to treat bile duct insufficiency or ductopenic liver diseases, remains unknown.In addition to human studies of Alagille syndrome (ALGS) etiology, mouse models have elucidated the importance of Notch signaling in IHBD morphogenesis.^5–9 The Jagged1 ligand, expressed in portal vein mesenchyme, activates Notch receptors on bipotential hepatoblasts to promote IHBD formation.⁶ The DNA-binding co-factor recombination signaling binding protein immunoglobulin kappa J (Rbpj) is required for canonical Notch signaling within hepatoblasts. Along with Notch signaling, hepatocyte nuclear factor 6 (Hnf6) is also important for BEC specification of bipotential hepatoblasts during embryonic development.¹⁰Previous work has demonstrated that the Albumin-Cre–mediated¹¹ hepatoblast-specific deletion of Rbpj¹² and Hnf6¹³ produces a synergistic defect whereby Rbpj and Hnf6 double knockout (DKO) mice are developmentally unable to form peripheral IHBDs.¹⁴ Early in life, IHBD paucity causes severe cholestasis, hepatic necrosis, and fibrosis in these mice. However, DKO mice eventually generate BECs in reactive ductules.¹⁴Herein, we demonstrate that DKO mice are indeed capable of forming communicating peripheral IHBDs subsequent to the emergence of a ductular reaction. This finding indicates that Notch and Hnf6 are together dispensable for IHBD regeneration in adult mice, which may explain the recovery of cholestasis that occurs in some patients with ALGS while providing a new concept for investigation of potential Notch-independent IHBD regenerative therapies.     

Ductular network formation by rat biliary epithelial cells in the dynamical culture with collagen gel and dimethylsulfoxide stimulation

Formation of bile ducts in culture is important for reconstructing hepatic organoids with bile drainage systems. However, morphogenic factors of biliary epithelial cells (BECs) have been poorly understood because of the lack of experimental models. Here, we demonstrated that rat BECs formed bile ductular networks in dynamic culture, when culture conditions were sequentially controlled. BEC morphogenesis was achieved through two-dimensional culture on collagen gel, collagen gel sandwich configuration, and 1% dimethylsulfoxide stimulation. In this culture system, BECs developed into large bile duct structures (LBDs) that formed interconnected networks of continuous lumens. LBD luminal surfaces possessed well developed microvilli, consisted of 7 to 10 BECs, and their inner diameters measured 20 to 50 microm. Quantitative PCR analysis revealed that the cells in LBDs expressed apical and basal domain markers of BECs. Immunofluorescent staining identified apical domain markers such as Cl(-)/HCO(3)(-) anion exchanger 2 and cystic fibrosis transmembrane regulator on the luminal surface of LBDs, responding to secretin stimulation as well as laminin protein surrounding LBDs. Furthermore, the cells in LBDs transported metabolized fluorescein from the basal side to the luminal space, further demonstrating that the reconstructed LBDs were functionally and morphologically similar to the bile ducts in vivo. The culture model described here will be useful in reconstructing hepatic tissues as well as in understanding the mechanism of bile duct development and its disruption in disease.     

BMP4 modulates fibroblast growth factor-mediated induction of proximal and distal lung differentiation in mouse embryonic tracheal epithelium in mesenchyme-free culture.

Lung morphogenesis and differentiation require interaction between the epithelium and mesenchyme, which is mediated by diffusible molecules such as fibroblast growth factors (FGFs), bone morphogenetic protein 4 (BMP4), and Shh. We have used mesenchyme-free culture to study the effects of these molecules on lung epithelial differentiation. We have tested the individual abilities of FGF1, FGF2, FGF7, FGF9, FGF10, and FGF18, as well as BMP4 and Shh to promote growth and specify distal lung differentiation in mouse tracheal epithelium. The different FGFs exhibited distinct abilities to induce epithelial growth and the expression of the distal lung epithelial marker, surfactant protein C (SP-C), although all FGFs were able to induce expression of BMP4. Tracheal epithelium treated with FGF10 showed little growth and failed to express SP-C as measured by whole-mount in situ hybridization and quantitative real-time polymerase chain reaction. FGF1 treatment resulted in the strongest induction of SP-C. Treatment with BMP4 inhibited epithelial growth and differentiation and antagonized the stimulatory effects of FGF1. In contrast, inhibition of endogenous BMP4 signaling with Noggin protein did not inhibit growth or expression of SP-C but did increase the expression of the proximal lung markers CCSP and HFH4. Expression of Shh was not affected by any of the conditions tested. These results suggest that BMP4 does not signal epithelial cells to adopt a distal fate but may regulate the expansion of proximal epithelial cells in the lung.     

Replicative senescence of biliary epithelial cells precedes bile duct loss in chronic liver allograft rejection: increased expression of p21(WAF1/Cip1) as a disease marker and the influence of immunosuppressive drugs

Early chronic liver allograft rejection (CR) is characterized by distinctive cytological changes in biliary epithelial cells (BECs) that resemble cellular senescence, in vitro, and precede bile duct loss. If patients suffering from early CR are treated aggressively, the clinical and histopathological manifestations of CR can be completely reversed and bile duct loss can be prevented. We first tested whether the senescence-related p21(WAF1/Cip1) protein is increased in BECs during early CR, and whether treatment reversed the expression. The percentage of p21+ BECs and the number of p21+ BECs per portal tract is significantly increased in early CR (26 +/- 17% and 3.6 +/- 3.1) compared to BECs in normal liver allograft biopsies or those with nonspecific changes (1 +/- 1% and 0.1 +/- 0.3; P: < 0.0001 and P: < 0.02), chronic hepatitis C (2 +/- 3% and 0.7 +/- 1; P: < 0.0001 and P: < 0.04) or obstructive cholangiopathy (7 +/- 7% and 0.7 +/- 0.6; P: < 0.006 and P: = 0.04). Successful treatment of early CR is associated with a decrease in the percentage of p21+ BECs and the number of p21+ BECs per portal tract. In vitro, nuclear p21(WAF1/Cip1) expression is increased in large and multinucleated BECs, and is induced by transforming growth factor (TGF)-beta. TGF-beta1 also increases expression of TGF-beta receptor II, causes phosphorylation of SMAD-2 and nuclear translocation of p21(WAF1/Cip1), which inhibits BEC growth. Because conversion from cyclosporine to tacrolimus is an effective treatment for early CR, we next tested whether these two immunosuppressive drugs directly influenced BEC growth in vitro. The results show that cyclosporine, but not tacrolimus, stimulates BEC TGF-beta1 production, which in turn, causes BEC mito-inhibition and up-regulation of nuclear p21(WAF1/Cip1). In conclusion, expression of the senescence-related p21(WAF1/Cip1) protein is increased in BECs during early CR and decreases with successful recovery. Replicative senescence accounts for the characteristic BEC cytological alterations used for the diagnosis of early CR and lack of a proliferative response to injury. The ability of cyclosporine to inhibit the growth of damaged BECs likely accounts for the relative duct sparing properties of tacrolimus.     

Postnatal epithelium and mesenchyme stem/progenitor cells in bioengineered amelogenesis and dentinogenesis

Rodent incisors provide a classic model for studying epithelial–mesenchymal interactions in development. However, postnatal stem/progenitor cells in rodent incisors have not been exploited for tooth regeneration. Here, we characterized postnatal rat incisor epithelium and mesenchyme stem/progenitor cells and found that they formed enamel- and dentin-like tissues in vivo. Epithelium and mesenchyme cells were harvested separately from the apical region of postnatal 4–5 day rat incisors. Epithelial and mesenchymal phenotypes were confirmed by immunocytochemistry, CFU assay and/or multi-lineage differentiation. CK14+, Sox2+ and Lgr5+ epithelium stem cells from the cervical loop enhanced amelogenin and ameloblastin expression upon BMP4 or FGF3 stimulation, signifying their differentiation towards ameloblast-like cells, whereas mesenchyme stem/progenitor cells upon BMP4, BMP7 and Wnt3a treatment robustly expressed Dspp, a hallmark of odontoblastic differentiation. We then control-released microencapsulated BMP4, BMP7 and Wnt3a in transplants of epithelium and mesenchyme stem/progenitor cells in the renal capsule of athymic mice in vivo. Enamel and dentin-like tissues were generated in two integrated layers with specific expression of amelogenin and ameloblastin in the newly formed, de novo enamel-like tissue, and DSP in dentin-like tissue. These findings suggest that postnatal epithelium and mesenchyme stem/progenitor cells can be primed towards bioengineered tooth regeneration.     

Interaction between FGF and BMP signaling pathways regulates development of metanephric mesenchyme

Nephrogenesis in the mouse kidney begins at embryonic day 11 and ends approximately 10 days postpartum. During this period, new nephrons are continually being generated from a stem-cell population-the nephrogenic mesenchyme-in response to signals emanating from the tips of the branching ureter. Relatively little is known about the mechanism by which the nephrogenic mesenchyme cell population is maintained at the tips of the ureter in the presence of signals promoting tubulogenesis. Previous studies have shown that a loss of Bmp7 function leads to kidney defects that are a likely result of progressive loss of nephrogenic mesenchyme by apoptosis. The studies presented here demonstrate that BMP7 signaling can prevent apoptosis in explants of metanephric mesenchyme. The surviving mesenchyme cell population, however, is not competent to respond to signals promoting tubulogenesis. In conjunction with FGF2, BMP7 promotes growth and maintains competence of the mesenchyme in vitro. In addition, FGF2 and BMP7 signaling, both independently and in combination, inhibit tubulogenesis. Interestingly, FGF2 and BMP7 also promote expansion of the stromal progenitor cell population in whole kidney culture. Because BMP7 is not produced by stromal progenitor cells, these data suggest a novel interaction between the nephrogenic mesenchyme and stromal progenitor cell populations. A model for the regulation of nephrogenesis by FGF and BMP signaling is presented.     

Biliary Epithelial Cells Are Not the Predominant Source of Hepatic CXCL12

Hepatic expression levels of CXCL12, a chemokine important in inflammatory and stem cell recruitment, and its receptor, C-X-C chemokine receptor 4, are increased during all forms of liver injury. CXCL12 is expressed by both parenchymal and nonparenchymal hepatic cells, and on the basis of immunohistochemistry, biliary epithelial cells (BECs) are thought to be a predominant source of hepatic CXCL12, thereby promoting periportal recruitment of C-X-C chemokine receptor 4–expressing lymphocytes. Our study aims to show that BECs may, in fact, not be the predominant source of hepatic CXCL12. We measured CXCL12 secretion and expression from human and murine BECs using enzyme-linked immunosorbent assay and Western blot analysis from cell culture supernatants and whole cell lysates, respectively, whereas CXCL12 expression in murine livers was analyzed in a Cxcl12-Gfp reporter mouse. Cell culture supernatants and whole cell lysates from BECs failed to demonstrate their expression of CXCL12. Furthermore, we confirmed these results with a Cxcl12-Gfp reporter mouse in which green fluorescent protein expression is notably absent from BECs. Interestingly, on the basis of green fluorescent protein expression, we demonstrate a population of CXCL12-expressing cells within the portal tract that are distinct, yet intimately associated with BECs. These findings indicate that BECs are not a predominant source of CXCL12.CXCL12, a chemokine important in hematopoietic stem cell homeostasis, and its receptor, C-X-C chemokine receptor 4 (CXCR4), are up-regulated in many disease pathologies and promote inflammation and tumor metastasis.¹ Specifically, in patients with liver disease, CXCL12 expression is increased in both serum and hepatic tissue proportional to the extent of injury.² CXCL12 expression has been documented in stellate cells, sinusoidal endothelial cells, and biliary epithelial cells (BECs) and is thought to drive inflammation and fibrogenesis, although its role, particularly in normal liver, remains largely unknown.³ During fetal development, B-cell lymphopoiesis is dependent on hepatic CXCL12,⁴ and in the adult, hepatic CXCL12 may support a hepatic hematopoietic stem cell niche.⁵ Finally, with injury, immunohistochemical data demonstrate robust CXCL12 expression by proliferating bile ductules in all forms of liver injury, and biliary CXCL12 expression is further supported by the accumulation of CXCR4-positive lymphocytes in the periportal region.^2,6A careful review of the literature, however, reveals that most data supporting BEC expression of CXCL12 are based on immunohistochemistry using a single monoclonal CXCL12 antibody (murine anti-human/mouse CXCL12, clone 79018). We believe that differentiated BECs may not express CXCL12 and that the observed pattern of BEC expression is a result of antibody cross-reactivity to an epitope found in BECs. Lack of CXCL12 expression by BECs has previously been alluded to by Mavier et al⁷ using in situ hybridization, where CXCL12 RNA expression in biliary cells lining interlobular bile ducts seemed to be absent. Herein, we show that despite previous studies demonstrating robust CXCL12 expression by BECs, their role in hepatic CXCL12 production may be more limited.^2,6,8     

Systemic minor histocompatibility antigen expression in blood endothelial cells prevents T cell‐mediated vascular immunopathology

Attenuation of T cell‐mediated damage of blood endothelial cells (BECs) in transplanted organs is important to prevent transplant vasculopathy (TV) and chronic rejection. Here, we assessed the importance of minor histocompatibility antigen (mHA) distribution and different coinhibitory molecules for T cell‐BEC interaction. A transgenic mHA was directed specifically to BECs using the Tie2 promoter and cellular interactions were assessed in graft‐versus‐host disease‐like and heterotopic heart transplantation settings. We found that cognate CD4⁺ T‐cell help was critical for the activation of BEC‐specific CD8⁺ T cells. However, systemic mHA expression on BECs efficiently attenuated adoptively transferred, BEC‐specific CD4⁺ and CD8⁺ T cells and hence prevented tissue damage, whereas restriction of mHA expression to heart BECs precipitated the development of TV. Importantly, the lack of the coinhibitory molecules programed death‐1 (PD‐1) and B and T lymphocyte attenuator fostered the initial activation of BEC‐specific CD4⁺ T cells, but did not affect development of TV. In contrast, TV was significantly augmented in the absence of PD‐1 on BEC‐specific CD8⁺ T cells. Taken together, these results indicate that antigen distribution in the vascular bed determines the impact of coinhibition and, as a consequence, critically impinges on T cell‐mediated vascular immunopathology.     

Fibroblast growth factor enriches the embryonic liver cultures for hepatic progenitors

Fibroblast growth factors (FGFs) play an important role in hepatic induction during development. The aim of our study was to investigate the effect of exogenous FGFs on ex vivo liver development. We begin our analysis by examining FGF signaling during early mouse liver development. Phospho-FGF receptor (Tyr653/654) was detected in embryonic day 10 (E10) to E12 livers only. Next, E10 livers were cultured in the presence of FGF1, FGF4, or FGF8 for 72 hours and examined for histology, proliferation, apoptosis, and differentiation. FGFs especially FGF8 promoted sheet-like architecture, cell proliferation, and survival as compared to the control. All FGFs induced a striking increase in the number of c-kit and alpha-fetoprotein-positive progenitors, without altering albumin staining. However these progenitors were CK-19-positive (biliary and bipotential progenitor marker) only in the presence of FGF1 or FGF4 and not FGF8. FGFs also induced beta-catenin, a stem cell renewal factor in these cultures. In conclusion, the presence of activated FGFR indicates a physiological role of FGF during early liver development. FGF1 and FGF4 enrich the embryonic liver cultures for bipotential hepatic progenitors. FGF8 promotes such enrichment and induces a one-step differentiation toward a unipotential hepatocyte progenitor. Thus, FGFs might be useful for enrichment and propagation of developmental hepatic progenitors.     

Dynamic patterns of expression of BMP isoforms 2, 4, 5, 6, and 7 during chicken heart development

Bone morphogentic proteins (BMPs) play an important role in cardiac development. Using an in vitro explant analysis, we show that BMPs are crucial for myocardium formation. As a first approach to identify which BMP may be involved in myocardium formation in intra- and extracardiac mesenchyme in vivo, a survey of the expression patterns of BMP2, -4, -5, -6, and -7 mRNA is prepared by in situ hybridization in chicken embryonic hearts from HH5 to 44. During recruitment of mesodermal cells to the outflow tract myocardium (HH10-23), BMP2, -4, -5, and -7 mRNA are expressed in the distal myocardial border and the flanking mesenchyme. After completion, BMP2 and -4 mRNA become restricted to the mesenchyme and BMP5 and -7 mRNA to the myocardium. At the venous pole, BMP2, -5, and -7 mRNA are expressed in the distal myocardial border of the caval vein, while BMP2, -5, -6, and -7 mRNA are expressed in the distal myocardium around the pulmonary vein. BMP4 mRNA is expressed in the adjacent mesenchyme at both sides. During muscularization of the atrioventricular cushions and the tricuspid valve, the cardiomyocytes that protrude into the mesenchyme express BMP2, -4, -5, and -7 mRNA, whereas BMP6 mRNA is expressed in the cushion mesenchyme. The myocardial protrusions formed in the mesenchymal proximal outlet septum express BMP4, -5, and -7 mRNA, while BMP2 and -6 mRNA are expressed in the mesenchyme. The spatiotemporal expression patterns of these BMPs in relation to myocardium formation at the distal ends and within the heart suggest a role for BMPs in myocardium formation. During delamination of the valves, BMP4 and -6 mRNA are expressed at the ventricular side of the forming mitral valve, BMP4 mRNA at the ventricular side of the forming tricuspid valve, and BMP2, -4, and -6 mRNA at the vascular side of the forming semilunar valves.     

Cholestasis in a rat model of graft-versus-host disease is accompanied by alteration of the expression and distribution of tight-junction-associated proteins

Intrahepatic cholestasis has been recognized as one of the characteristic features of the hepatic manifestations in graft-versus-host disease (GVHD). Tight junctions (TJs) play crucial roles in bile formation and alterations of TJs in hepatocytes and/or biliary epithelial cells (BECs) that cause intrahepatic cholestasis. To assess the changes of TJs in hepatocytes and BECs in a rat model of GVHD, we examined the localization of TJ-associated proteins, 7H6 and zonula occludens (ZO)-1. GVHD was induced by injecting spleen cells of parental strain rats (Brown Norway) into non-irradiated (Brown Norway x Lewis) F1 hybrid rats. Untreated F1 hybrid rats served as controls. Double-labeled immunofluorescent staining for 7H6 and ZO-1 was performed in liver sections. In control rats, immunostaining for 7H6 and ZO-1 colocalized to the apical site of BECs and was continuous along the bile canaliculi. In GVHD, 7H6 expression was decreased in BECs and was discontinuous along the bile canaliculi. On the other hand, the intensity of ZO-1 staining in hepatocytes increased and did not change in BECs compared with that of control rats. Changes in TJ-associated proteins of both hepatocytes and BECs may reflect the immunopathogenesis of GVHD-associated intrahepatic cholestasis.     

Embryonic myogenesis pathways in muscle regeneration.

Embryonic myogenesis involves the staged induction of myogenic regulatory factors and positional cues that dictate cell determination, proliferation, and differentiation into adult muscle. Muscle is able to regenerate after damage, and muscle regeneration is generally thought to recapitulate myogenesis during embryogenesis. There has been considerable progress in the delineation of myogenesis pathways during embryogenesis, but it is not known whether the same signaling pathways are relevant to muscle regeneration in adults. Here, we defined the subset of embryogenesis pathways induced in muscle regeneration using a 27 time-point in vivo muscle regeneration series. The embryonic Wnt (Wnt1, 3a, 7a, 11), Shh pathway, and the BMP (BMP2, 4, 7) pathway were not induced during muscle regeneration. Moreover, antagonists of Wnt signaling, sFRP1, sFRP2, and sFRP4 (secreted frizzled-related proteins) were significantly up-regulated, suggesting active inhibition of the Wnt pathway. The pro-differentiation FGFR4 pathway was transiently expressed at day 3, commensurate with expression of MyoD, Myogenin, Myf5, and Pax7. Protein verification studies showed fibroblast growth factor receptor 4 (FGFR4) protein to be strongly expressed in differentiating myoblasts and newly formed myotubes. We present evidence that FGF6 is likely the key ligand for FGFR4 during muscle regeneration, and further suggest that FGF6 is released from necrotic myofibers where it is then sequestered by basal laminae. We also confirmed activation of Notch1 in the regenerating muscle. Finally, known MyoD coactivators (MEF2A, p/CIP, TCF12) and repressors (Twist, Id2) were strongly induced at appropriate time points. Taken together, our results suggest that embryonic positional signals (Wnt, Shh, and BMP) are not induced in postnatal muscle regeneration, whereas cell-autonomous factors (Pax7, MRFs, FGFR4) involving muscle precursor proliferation and differentiation are recapitulated by muscle regeneration.     

A method for culturing and transplanting biliary epithelial cell from syrian golden hamster

The present paper describes the establishment of a method for simultaneous culturing of biliary epithelial cells (BECs) from the gall bladder (GB), extrahepatic bile duct (EBD) and intrahepatic bile duct (IBD) of the hamster. GB, EBD and IBD were cut from the biliary tree after collagenase perfusion of the liver. These biliary segments were minced into fragments. The fragments were embedded in collagen gel and cultured in Dulbecco’s modified Eagle medium / HamF12 medium containing 10% fetal bovine serum. The various cells subsequently spread from the fragments and formed cellular sheets. After the fragments and flattened cells were removed with the aid of a Pasteur pipette under phase-contrast microscopy, the sheets remaining were found to be composed of cuboidal cells. These cuboidal cells were shown to express gamma glutamyl transpeptidase and cytokeratin 7, which are known to be specific markers of BECs. Ultrastructurally, a large number of microvilli were observed on the luminal surface and junctional complex and interdigitation was identifiable on the lateral surfaces. BEC cultures were subcultured by digestion with collagenase and dispase and then dissociated by subsequent digestion in trypsin and ethylenediaminetetraacetic acid and then maintained on collagen gel for up to 8 weeks. After several passages, the BECs in culture eventually increased in size and showed vacuoles in the cytoplasm. They demonstrated irreversible growth arrest at 9 weeks. The BECs tended to form cystic structures when the BECs with collagen gel were transplanted into the interscapular fat pads of syngeneic hamsters. We established a method for culturing and transplanting biliary cells from syrian golden hamsters. This method may help to clarify the mechanism of hepatobiliary diseases. Received: 31 May 1999 / Accepted: 25 August 1999     

Fibroblast growth factor signaling regulates Dach1 expression during skeletal development.

Dach1 is a mouse homologue of the Drosophila dachshund gene, which is a key regulator of cell fate determination during eye, leg, and brain development in the fly. We have investigated the expression and growth factor regulation of Dach1 during pre- and postnatal skeletal development in the mouse limb to understand better the function of Dach1. Dach1 was expressed in the distal mesenchyme of the early embryonic mouse limb bud and subsequently became restricted to the tips of digital cartilages. Dach1 protein was localized to postmitotic, prehypertrophic, and early hypertrophic chondrocytes during the initiation of ossification centers, but Dach1 was not expressed in growth plates that exhibited extensive ossification. Dach1 colocalized with Runx2/Cbfa1 in chondrocytes but not in the forming bone collar or primary spongiosa. Dach1 also colocalized with cyclin-dependent kinase inhibitors p27 (Kip1) and p57 (Kip2) in chondrocytes of the growth plate and in the epiphysis before the formation of the secondary ossification center. Because fibroblast growth factors (FGF), bone morphogenetic proteins (BMP), and hedgehog molecules (Hh) regulate skeletal patterning of the limb bud and chondrocyte maturation in developing endochondral bones, we investigated the regulation of Dach1 by these growth and differentiation factors. Expression of Dach1 in 11 days postcoitus mouse limb buds in organ culture was up-regulated by implanting beads soaked in FGF1, 2, 8, or 9 but not FGF10. BMP4-soaked beads down-regulated Dach1 expression, whereas Shh and bovine serum albumin had no effect. Furthermore, FGF4 or 8 could substitute for the apical ectodermal ridge in maintaining Dach1 expression in the limb buds. Immunolocalization of FGFR2 and FGFR3 revealed overlap with Dach1 expression during skeletal patterning and chondrocyte maturation. We conclude that Dach1 is a target gene of FGF signaling during limb skeletal development, and Dach1 may function as an intermediary in the FGF signaling pathway regulating cell proliferation or differentiation.     

Increased expression of nuclear envelope gp210 antigen in small bile ducts in primary biliary cirrhosis

The sustained antibody response to nuclear envelope gp210 antigen indicates a group of primary biliary cirrhosis (PBC) patients at high risk for the progression to end-stage hepatic failure. To address this issue, we immunohistochemically studied the expression of gp210 antigen in needle liver biopsy specimens from PBC patients using a monoclonal antibody specific for gp210 antigen. The specimens from autoimmune hepatitis (AIH), chronic viral hepatitis B (CHB) and C (CHC) patients served as disease controls. The expression of gp210 antigen was apparently increased on the nuclear envelope of biliary epithelial cells (BECs) of small bile ducts in almost all specimens from PBC. In contrast, the expression of gp210 antigen was negative in BECs of small bile ducts in normal liver, while relatively weak anti-gp210 immunostaining was observed in AIH, CHC and CHB. In addition, the degree of gp210 expression in BECs of small bile ducts was positively correlated to that of portal inflammation, interface hepatitis and lobular inflammation in PBC. These results indicate that the increased expression of gp210 in small bile ducts, which is probably associated with damage to BECs by inflammation, is possibly involved in autoimmune response to gp210 leading to the progression to end-stage hepatic failure in PBC.     

Gene expression profiling in biliary epithelial cells of primary biliary cirrhosis using laser capture microdissection and cDNA microarray

Primary biliary cirrhosis (PBC) is a chronic, cholestatic liver disease characterized by progressive destruction of interlobular bile ducts that leads to biliary cirrhosis. To elucidate the etiology of PBC, the gene expression profile in biliary epithelial cells (BECs) was analyzed. Liver specimens of 5 PBC, 3 chronic hepatitis C (CHC), and 3 normal subjects were obtained. BECs were selectively collected by laser capture microdissection (LCM), RNA were obtained by extraction and amplification with T7 RNA polymerase, and a cDNA microarray analysis was performed. The following genes exhibited increased expression in BEC of PBC, as compared with CHC or normal subjects: human leukocyte antigen DQ alpha 1 (HLA-DQA-1), carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), and vascular cell adhesion molecule 1 (VCAM-1). The immunohistochemistry for HLA-DQA-1, CEACAM1, TRAIL, and VCAM-1 confirmed these results. Furthermore, two-way cluster analysis showed that the gene expression profiling in BEC of PBC were categorized into a separate cluster, distinct from CHC or normal subjects. Conclusions: The gene expression profiling in BEC of PBC differed from those of CHC and normal subjects, and the genes concerning local immune response, such as HLA-DQA-1, CEACAM1, TRAIL, and VCAM-1, exhibited increased expression, indicating that they were involved in the development of bile duct injury.