Inversin-deficient (inv) mice do not establish a polarized duct system in the liver and pancreas.

Inversin-deficient (inv) mice have anomalies in liver and pancreatic development in addition to an inverted left-right axis of the body. The present study was undertaken to unveil mechanisms of bile and pancreatic duct development from immunohistochemical analyses of anomalies in inv mice. Intrahepatic bile ducts having proximodistal polarity in size and the height of their epithelia, and ductules were formed in livers of wild-type neonates. By contrast, in inv mice, ductal plates, precursor structures of intrahepatic bile ducts and ductules, persisted without the proximodistal polarity. Their epithelial cells did not acquire planar cell polarity (PCP) in terms of expression of tight junction proteins although they expressed bile duct markers, HNF1ß and SOX9. They had an apicobasal polarity from expression of basal laminar components. Enlargement of the hepatic artery and poor connective tissue development, including the abnormal deposition of the extracellular matrices, were also noted in inv mice, suggesting that bile duct development was coupled to that of the hepatic artery and portal vein. In pancreata of inv neonates, neither the main pancreatic duct was formed, nor dilated duct-like structures had the morphological polarity from the connecting point with the common bile duct. Lumina of acini was dilated, and centroacinar cells changed their position in the acini to their neck region. Immunohistochemical analyses of tight junction proteins suggested that epithelial cells of the duct-like structures did not have a PCP. Thus, Invs may be required for the establishment of the PCP of the whole duct system in the liver and pancreas.

Liver progenitor cells may construct cysts having heterogeneous gene expression of liver-enriched transcription factors in mice with conditional knockout of the Hhex gene.

The deletion of the Hhex (Hematopoietically expressed homeobox) gene causes agenesis of the liver and polycystic liver disease depending on its timing. The present study was undertaken to determine the role of the Hhex gene in not only signaling cascades to cyst and abnormal bile duct formation but also the liver progenitor contribution to cystic development. Liver-specific Hhex knockout mice (Alb-Cre/HhexloxP/loxP) in adult stages were used. Wild-type and conditional knockout (cKO) livers were immunohistologically compared for cell growth, and gene expression of liver functions, biliary markers and cystic markers. In Hhex cKO livers, cyst formation and dilated intrahepatic bile ducts were noted, which resembled the histology of the von Meyenburg complex. Ki67 immunohistochemistry showed that the growth activity in bile ducts and cysts of cKO livers was elevated compared with that of wild-type livers. There were far fewer liver progenitor cells or bile ductule cells around portal veins of cKO livers than in wild-type livers. Several liver-enriched transcription factors, including Foxa1 and Foxa2, were heterogeneously expressed in bile ducts and cysts of cKO livers whereas their expression in wild-type bile ducts was comparatively homogeneous. PC1 and PC2 immunohistochemistry revealed their up-regulation in cysts of cKO livers. These data indicate that Hhex is not only required for proper bile duct morphogenesis, but is also involved in cyst formation through promoted cell growth. Liver progenitor cells may form cysts. Unbalanced expression of liver-enriched transcription factors might be involved in cyst formation. Hhex cKO mice may be a good animal model for hepatic cystic diseases.

Transdifferentiation of mouse visceral yolk sac cells into parietal yolk sac cells in vitro.

The mouse embryonic yolk sac is an extraembryonic membrane that consists of a visceral yolk sac (VYS) and parietal yolk sac (PYS), and functions in hematopoietic-circulation in the fetal stage. The present study was undertaken to examine the normal development of both murine VYS and PYS tissues using various molecular markers, and to establish a novel VYS cell culture system in vitro for analyzing differentiation potentials of VYS cells. RT-PCR and immunohistochemical analyses of gene expression in VYS and PYS tissues during development revealed several useful markers for their identification: HNF1ß, HNF4α, Cdh1 (E-cadherin), Krt8 and Krt18 for VYS epithelial cells, and Stra6, Snail1, Thbd and vimentin for PYS cells. PYS cells exhibited mesenchymal characteristics in gene expression and morphology. When VYS cells at 11.5 days of gestation were cultured in vitro for 7 days, the number of HNF1ß-, HNF4α-, E-cadherin- and cytokeratin-positive VYS epithelial cells was significantly reduced and, instead, Stra6-and vimentin-positive PYS-like cells increased with culture. RT-PCR analyses also demonstrated that gene expression of VYS markers decreased, whereas that of PYS markers increased in the primary culture of VYS cells. These data indicate that VYS epithelial cells rapidly transdifferentiate into PYS cells having mesenchymal characteristics in vitro, which may provide a culture system suitable for studying molecular mechanisms of VYS transdifferentiation into PYS cells and also epithelial-mesenchymal transition.

Immunohistochemical analyses of cell cycle progression and gene expression of biliary epithelial cells during liver regeneration after partial hepatectomy of the mouse.

The liver has a remarkable regeneration capacity, and, after surgical removal of its mass, the remaining tissue undergoes rapid regeneration through compensatory growth of its constituent cells. Although hepatocytes synchronously proliferate under the control of various signaling molecules from neighboring cells, there have been few detailed analyses on how biliary cells regenerate for their cell population after liver resection. The present study was undertaken to clarify how biliary cells regenerate after partial hepatectomy of mice through extensive analyses of their cell cycle progression and gene expression using immunohistochemical and RT-PCR techniques. When expression of PCNA, Ki67 antigen, topoisomerase IIα and phosphorylated histone H3, which are cell cycle markers, was immunohistochemically examined during liver regeneration, hepatocytes had a peak of the S phase and M phase at 48-72 h after resection. By contrast, biliary epithelial cells had much lower proliferative activity than that of hepatocytes, and their peak of the S phase was delayed. Mitotic figures were rarely detectable in biliary cells. RT-PCR analyses of gene expression of biliary markers such as Spp1 (osteopontin), Epcam and Hnf1b demonstrated that they were upregulated during liver regeneration. Periportal hepatocytes expressed some of biliary markers, including Spp1 mRNA and protein. Some periportal hepatocytes had downregulated expression of HNF4α and HNF1α. Gene expression of Notch signaling molecules responsible for cell fate decision of hepatoblasts to biliary cells during development was upregulated during liver regeneration. Notch signaling may be involved in biliary regeneration.

Immunomagnetic exclusion of PECAM-1-positive endothelial cells in fetal mouse liver cell cultures causes impaired growth and gene expression of hepatoblasts and stellate cells.

Previous studies using mice having defective VEGF signaling have demonstrated that vascular development is indispensable for early hepatic organogenesis. However, not only whether its action lasts during later hepatic development, but also what molecules are involved in that action remains to be determined. The present study was undertaken to examine the effects of primitive sinusoidal endothelial cells on hepatic growth and maturation in primary culture of fetal mouse liver cells, and to determine their molecular mechanisms. When endothelial cells were excluded from E12.5 liver cell cultures by using PECAM-1-antibody-coated magnetic beads, the growth of hepatoblasts and stellate cells was conspicuously reduced and hepatic maturation was also suppressed. Conditioned medium prepared from fetal liver cell cultures containing almost all hepatic cell types stimulated the growth and gene expression of hepatoblasts and stellate cells similarly to the cultures in the presence of endothelial cells. HGF mRNA expression was downregulated in endothelial cellfree cultures of fetal liver cells, and the addition of HGF to the culture medium rescued the cells from the effects of endothelial cell depletion. These data suggest that humoral factors, including HGF, which are produced by endothelial cells or stellate cells, are involved in fetal hepatocyte growth and maturation.

Impaired hepatocyte maturation, abnormal expression of biliary transcription factors and liver fibrosis in C/EBPα(Cebpa)-knockout mice.

Inactivation of the C/EBPα gene (Cebpa) in the mouse not only causes impaired hepatocyte maturation, but also induces pseudoglandular structures in the liver parenchyma. The present study was undertaken to determine how the expression of other transcription factors controlling differentiation into hepatocytes and biliary epithelial cells is affected, and how the hepatic architecture, including the bile and vascular systems, is disordered in the fetal knockout liver. Histochemical analyses demonstrated that the expression of HNF1α and HNF4α was heterogeneous in the knockout liver, and that not all parenchymal cells (pseudoglandular) expressed these transcription factors, whereas parenchymal cells in the wild-type liver homogeneously expressed these transcription factors. SOX9, which was expressed only in biliary cells in the wild-type liver, was detectable in many pseudoglandular cells of the knockout liver. Although the pseudoglandular cells often coexpressed SOX9 and HNF1α/HNF4α, cells expressing SOX9 but not expressing HNF1α/HNF4α (biliary cells) were sometimes detectable in the parenchyma. Periportal biliary structures were abnormal in their segregation from the parenchyma and in their expression of the transcription factors and Ep-CAM, a biliary adhesion molecule. These results suggest that the inactivation of the Cebpa gene causes unstable expression of liver-enriched transcription factors or biliary transcription factors and elevated expression of Ep-CAM, which may lead to abnormal biliary morphogenesis in the knockout liver. The impaired maturation of the parenchyma caused elevated expression of PECAM-1, desmin and Foxf1, suggesting that the maturation of the parenchyma plays an important role in the normal histogenesis of nonparenchymal cells (stellate cells and sinusoidal endothelial cells).

Purification and culture of fetal mouse hepatoblasts that are precursors of mature hepatocytes and biliary epithelial cells.

To investigate cell-cell interactions during mammalian liver development, it is essential to separate hepatoblasts (fetal liver progenitor cells) from nonparenchymal cells, including stellate cells, endothelial cells, and hemopoietic cells. Various factors, which may be produced by nonparenchymal cells, could be assayed for their effects on the growth and maturation of separated hepatoblasts. The protocol using immunomagnetic beads coated with anti-mouse E-cadherin antibody is described for efficient isolation of hepatoblasts from cell suspensions of fetal mouse livers. The purity and recovery rate are larger than 95% and approximately 30%, respectively. The protocol may be useful for various studies focusing on the fetal liver progenitor cells.

Developmental changes of cell adhesion molecule expression in the fetal mouse liver.

Developmental changes of cell adhesion molecule expression, especially in nonparenchymal cells, have hardly ever been analyzed in the murine liver. The present study was undertaken to immunohistochemically examine the expression of NCAM, ICAM, VCAM, and N-cadherin during mouse liver development and in fetal liver cell cultures. NCAM was transiently expressed in mesenchymal cells of the septum transversum and sinusoidal cells in liver development. In vitro studies demonstrated that desmin-positive stellate cells expressed this cell adhesion molecule. NCAM expression in periportal biliary epithelial cells and connective tissue cells also coincided well with bile duct remodeling processes in the perinatal periods. Expression of ICAM and VCAM was transiently restricted to hepatoblasts, hepatocytes and hemopoietic cells in fetal stages. N-cadherin was expressed not only in hepatoblasts and hepatocytes, but also in nonparenchymal cells such as endothelial cells, stellate cells and connective tissue cells, however the expression was weak. These results suggest that each cell adhesion molecule may play an important role during development in hepatic histogenesis, including hepatoblast/hepatocyte-stellate cell interactions, hemopoiesis, and bile duct morphogenesis.

Sinusoid development and morphogenesis may be stimulated by VEGF-Flk-1 signaling during fetal mouse liver development.

Early morphogenesis of hepatic sinusoids was histochemically and experimentally analyzed, and the importance of VEGF-Flk-1 signaling in the vascular development was examined during murine liver organogenesis. FITC-gelatin injection experiments into young murine fetuses demonstrated that all primitive sinusoidal structures were confluent with portal and central veins, suggesting that hepatic vessel development may occur via angiogenesis. At 12.5-14.5 days of gestation, VEGF receptors designated Flk-1, especially their mature form, were highly expressed in endothelial cells of primitive sinusoidal structures and highly phosphorylated on their tyrosine residues. At the same time, VEGF was also detected in hepatoblasts/hepatocytes, hemopoietic cells, and megakaryocytes of the whole liver parenchyma. Furthermore, the addition of VEGF to E12.5 liver cell cultures significantly induced the growth and branching morphogenesis of sinusoidal endothelial cells. Therefore, VEGF-Flk-1 signaling may play an important role in the growth and morphogenesis of primitive sinusoids during fetal liver development.

Expression of neuritin during liver maturation and regeneration.

Cell surface molecules are not only important for cell-cell interactions but also useful for a marker to define cell types and differentiation stages. Unlike hematopoietic system in which numerous such antigens have been identified, only a few cell surface molecules have been used to define differentiation stage of hepatocytes. In order to identify such cell surface molecules, we performed DNA microarray analysis using mRNA from fetal hepatocytes in E12.5 and E17.5 mice and cDNAs encoding a membrane protein were selected. Northern blot analysis was employed to confirm the genes upregulated during maturation of fetal hepatocytes and neuritin, a GPI-anchored protein, was found as a membrane protein expressed in hepatocytes, but not in nonparenchymal cells. Its expression increased along with liver development and the maximum expression was achieved from the neonatal to adult stage. The neuritin protein was localized in sinusoidal lumen of hepatocytes in adult liver. Partial hepatectomy transiently downregulated the expression of neuritin. The expression of neuritin mRNA in C/EBPalpha deficient liver was reduced to about 50% of that of wild type mice. Thus, neuritin expression is well correlated to the maturation of hepatocytes and can be a useful tool to define the differentiation stage of hepatocytes.

Suppression of C/EBP alpha expression in biliary cell differentiation from hepatoblasts during mouse liver development.

BACKGROUND/AIMS: Intrahepatic biliary cell differentiation takes place in periportal hepatoblasts under the influence of the subjacent mesenchyme, which leads to the suppression of mature hepatocyte marker expression. This study was undertaken to analyze C/EBP alpha and beta expression, which may govern transcription of mature hepatocyte marker genes, during mouse liver development with special attention given to biliary differentiation. METHODS: Expression of C/EBP alpha and beta was immunohistochemically examined. Expression of alpha-fetoprotein, albumin and urea cycle enzymes, the genes of which have CCAAT motifs in their upstream regulatory sequences, was examined immunohistochemically or by using in situ hybridization. RESULTS: C/EBP alpha started to be expressed in endodermal cells of 9.5-day liver primordium, and continued to be expressed in hepatoblasts and hepatocytes throughout development. Although biliary cell progenitors transiently expressed mature hepatocyte markers, their expression of C/EBP alpha was weak or totally absent. The signals of C/EBP beta in hepatocytes were weak in fetal liver, but became stronger with postnatal development. Differentiated epithelial cells of intrahepatic biliary structures did not express C/EBP alpha. CONCLUSIONS: These data suggest that the suppression of C/EBP alpha expression may be prerequisite to biliary cell differentiation in the hepatoblast population and one of its earliest signs.

Immunolocalization of extracellular matrix components and integrins during mouse liver development.

Intrahepatic biliary cell differentiation takes place in periportal hepatoblasts under the influence of the subjacent connective tissue, the mechanism of which is still unclear. This study was undertaken to analyze the immunolocalization of extracellular matrix components and their cellular receptors during mouse liver development, with special attention given to biliary differentiation and vascular development. In young fetal mouse liver, primitive structures of sinusoids were developed between hepatic cords associated with hematopoietic cells demonstrated by immunohistochemistry of basal laminar components, the alpha6 integrin subunit, and PECAM-1. Portal veins and hepatic veins showed different staining intensities of alpha2, alpha3, and alpha6 integrin subunits from early stages of development. Anti-beta4 integrin subunit antibodies reacted with portal veins, but not with hepatic veins after perinatal stages. Their different phenotypes may be related to the preferential differentiation of periportal bile ducts. In intrahepatic bile duct development, periportal hepatoblasts adjacent to the connective tissue were immunostained for each basal laminar component on the basal side at almost the same time; alpha3, alpha5, alpha6, and beta4 integrin subunits were immunohistochemically detectable later than the basal laminar components. These staining patterns of intrahepatic bile duct cells clearly differed from those of extrahepatic bile duct cells from the beginning of their development, suggesting that these ducts are of different origins. In conclusion, the vascular structures, including sinusoids, portal veins, and hepatic veins, develop from early stages of liver development, and the extracellular matrix components may play important roles in biliary differentiation and vascular development. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html).