RESUMEN
Although the liver of the hagfish, an earliest diverged lineage among vertebrates, has a histological architecture similar to that of mammals, its gene expression has not been explored yet. The present study was undertaken to comparatively characterize gene expression in the liver of the hagfish with that of the mouse, using in situ hybridization technique. Expression of alb (albumin) was detectable in all hepatocytes of the hagfish liver, but was negative in intrahepatic bile ducts. Their expression in abundant periportal ductules was weak. The expression pattern basically resembled that in mammalian livers, indicating that the differential expression of hepatocyte markers in hepatocytes and biliary cells may have been acquired in ancestral vertebrates. alb expression was almost homogeneous in the hagfish liver, whereas that in the mouse liver lobule was zonal. The glul (glutamate-ammonia ligase) expression was also homogeneously detectable in hepatocytes without zonation, and weakly so in biliary cells of the hagfish, which contrasted with its restricted pericentral expression in mouse livers. These findings indicated that the hagfish liver did not have mammalian-type zonation. Whereas tetrapods had Hnf (hepatocyte nuclear factor) 1a and Hnf1b genes encoding the transcription factors, the hagfish had a single gene of their orthologue hnf1. Although HNF1α and HNF1ß were immunohistochemically detected in hepatocytes and biliary cells of the mouse, respectively, hnf1 was expressed in both hepatocytes and biliary cells of the hagfish. These data indicate that gene expression of hnf1 in the hagfish liver may be ancestral with that of alb and glul during vertebrate evolution.
Asunto(s)
Anguila Babosa , Animales , Ratones , Anguila Babosa/genética , Hígado/metabolismo , Hepatocitos , Vertebrados , Mamíferos , Expresión GénicaRESUMEN
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.
RESUMEN
BACKGROUND: The liver architecture of vertebrates can be classified into two types, the portal triad type (having periportal bile ducts) and the non-portal triad type (having bile ducts independent of the course of portal veins). The former is typically detectable in livers of tetrapods and cartilaginous fish, and its ancestral state is found in the hagfish, an earliest diverged lineage among vertebrates. Teleosts other than osteoglossomorphs have the latter. The aim of the present study is to reveal the changes of the hepatic innervation, biliary cilia and smooth muscle distribution, and extracellular matrices along vertebrate evolution with attention to the two types of liver architectures. METHODS: The hepatic innervation, biliary cilia and smooth muscle distribution, and collagen deposition were immunohistochemically and histochemically compared among livers of various vertebrates, using anti-acetylated tubulin and anti-α-smooth muscle actin antibodies, and Sirius red staining. These were also ultrastructurally examined. RESULTS: Although the hagfish liver had periportal intrahepatic bile ducts and ductules as detected in mammalian livers, it lacked smooth muscles around bile ducts and portal veins. Extracellular matrices in their connective tissues had thick collagen fibers. Its innervation was restricted to intrahepatic bile ducts and portal veins in the hilum. In livers of other vertebrates, including teleosts, the innervation was broadly detectable, especially around bile ducts, hepatic arteries and portal veins (afferent vessels), but not around central veins (efferent vessels). The chondrichthyans ultrastructurally had smooth muscle tissue around bile ducts. Cilia distribution was confirmed in intrahepatic bile ducts of tetrapods and basal actinopterygians. Teleosts other than osteoglossomorphs lacked cilia in their intrahepatic bile ducts. CONCLUSIONS: The liver architecture of the hagfish may be unique for innervation and extracellular matrices. Hepatic innervation may not have occurred in vertebrate ancestors. Hepatic innervation in bile ducts, hepatic arteries and portal veins may have been conserved among the extant jawed vertebrates. Cilia distribution in bile ducts may have changed during evolution of actinopterygians.
Asunto(s)
Cilios , Hígado , Animales , Distribución Tisular , Hígado/anatomía & histología , Vertebrados , Matriz Extracelular , Colágeno/metabolismo , MamíferosRESUMEN
The mammalian liver has a lobule structure with a portal triad consisting of the portal vein, hepatic artery, and bile duct, which exhibits zonal gene expression, whereas those of teleosts do not have a portal triad. It remains to be demonstrated what kind of the unit structures they have, including their gene expression patterns. The aims of the present study were to demonstrate the unit structure of the teleost liver and discuss it in terms of evolution and adaptation in vertebrates and the use of teleosts as an alternative model for human disease. The zebrafish liver was examined as a representative of teleosts with respect to its morphological architecture and gene expression. A novel, polygonal lobule structure was detected in the zebrafish liver. In it, portal veins and central veins were distributed at the periphery and center, respectively. Sinusoids connected both veins. Anxa4-positive preductules were incorporated into the tubular lumen of two rows of hepatocytes in sections. Intrahepatic bile ducts resided randomly in the liver lobule. Zebrafish livers did not have zonal gene expression for metabolic pathways examined. The lobules of the zebrafish liver with preductules located in the tubular lumina of hepatocytes may resemble the oval cell reaction of injured livers of mammals and might convey bile to the intestine more safely than mammalian livers. The gene expression pattern in liver lobules and our liver lobule model of the zebrafish may be important to discuss data obtained in experiments using this animal as an alternative model for human disease.
Asunto(s)
Hígado , Pez Cebra , Animales , Hígado/anatomía & histología , MamíferosRESUMEN
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.
Asunto(s)
Quistes , Hepatopatías , Animales , Quistes/metabolismo , Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Hígado/metabolismo , Hepatopatías/metabolismo , Ratones , Ratones Noqueados , Células Madre/patología , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: The liver architecture of vertebrates can be classified into two types, the portal triad type (having periportal bile ducts) and the non-portal triad type (having non-periportal bile ducts). The former is detectable from the hagfish, which is the most ancestral vertebrate, to tetrapod livers whereas many actinopterygian livers have the latter. The aim of the present study is to reveal the distribution of smooth muscle tissue in livers of various vertebrates with attention to their architectures. METHODS: Smooth muscle was immunohistochemically compared in hepatic blood vessels and bile ducts of various vertebrates, using an anti-alpha-smooth muscle actin (ASMA) antibody. RESULTS: Smooth muscle was noted in the gallbladder and hepatic artery in all vertebrates, including the hagfish. Bile ducts having ASMA-positive smooth muscles were absent in the hagfish, but detected in the Chondrichthyes and conserved in actinopterygians with or without portal triads during the evolution of vertebrates. In tetrapods having portal triads, reptiles had a tendency to have strongly ASMA-positive biliary smooth muscle tissues whereas other tetrapods had bile ducts with poor smooth muscle tissues. Although the hagfish livers never had ASMA-positive smooth muscle tissue in the walls of portal and central veins, it was observed in discontinuous distributions or not observed in portal veins and central veins of chondrichthyans and actinopterygians. By contrast, in most tetrapods, ASMA-positive smooth muscle tissue was detectable in portal veins, which supported the adjacent endothelial cells as a circular layer. Central veins did not consistently have smooth muscle tissue in these groups. DISCUSSION AND CONCLUSION: The hagfish liver may retain more ancestral characteristics than other vertebrates in terms of smooth muscle distribution in the vascular and biliary systems. Actinopterygians might have a different mechanism of bile transport from tetrapods from their smooth muscle distribution in intrahepatic bile ducts. The circular smooth muscle distribution in portal veins might be a characteristic acquired by tetrapods.
Asunto(s)
Células Endoteliales , Hígado , Animales , Conductos Biliares Intrahepáticos , Músculo Liso , VertebradosRESUMEN
The liver architecture of vertebrates can be classified into two types, the portal triad type (having periportal bile ducts) and the non-portal triad type (having non-periportal bile ducts). The former is detectable in the tetrapod liver whereas the lungfish liver has the latter. It remains to be revealed which type of hepatic architecture the coelacanth, which together with the lungfish belongs to the Sarcopterygii, possesses. The present study was undertaken to determine the histological characteristics of the coelacanth liver, and to compare with those of other vertebrates. The coelacanth liver had periportal bile ducts and ductules as detected in mammalian livers. The hepatic artery was found around large portal veins. Hagfish, shark, bichir, sturgeon, bowfin and frog livers had periportal bile ducts and bile ductules, whereas most intrahepatic bile ducts of the lungfish were independent of the distribution of the portal veins as seen in the Otocephala and Euteleostei. The lungfish liver developed duct and ductule structures in the parenchyma. These data indicate that the coelacanth liver had a mammalian-type hepatic architecture with a portal triad, and that the ancestors of tetrapods may have had a portal triad-type liver architecture.
Asunto(s)
Hígado/irrigación sanguínea , Hígado/citología , Sistema Porta , Animales , Anguila BabosaRESUMEN
The mammalian liver has a structural and functional unit called the liver lobule, in the periphery of which the portal triad consisting of the portal vein, bile duct and hepatic artery is developed. This type of hepatic architecture is detectable in many other vertebrates, including amphibians and birds, whereas intrahepatic bile ducts run independently of portal vein distribution in actinopterygians such as the salmon and tilapia. It remains to be clarified how the hepatic architectures are phylogenetically developed among vertebrates. The present study morphologically and immunohistochemically analyzed the hepatic structures of various vertebrates, including as many classes and subclasses as possible, with reference to intrahepatic bile duct distribution. The livers of vertebrates belonging to the Agnatha, Chondrichthyes, Amphibia, Aves, Mammalia, and Actinopterygii before Elopomorpha, had the portal triad-type architecture. The Anguilliformes livers developed both periportal bile ducts and non-periportal bile ducts. The Otocephala and Euteleostei livers had independent configuration of bile ducts and portal veins. Pancreatic tissues penetrated the liver parenchyma along portal veins in the Euteleostei. The liver of the lungfish, which shares the same origin with amphibians, did not have the portal triad-type architecture. Teleostei and lungfish livers had ductular development in the liver parenchyma similar to oval cell proliferation in injured mammalian livers. Euteleostei livers had penetration of significant numbers of independent portal veins from their intestines, suggesting that each liver lobe might receive a different blood supply. The hepatic architectures of the portal triad-type changed to non-portal triad-type architecture along the evolution of the Actinopterygii. The hepatic architecture of the lungfish resembles that of the Actinopterygii after Elopomorpha in intrahepatic biliary configuration, which may be an example of convergent evolution.
Asunto(s)
Hígado/anatomía & histología , Vertebrados/anatomía & histología , Animales , Evolución Biológica , FilogeniaRESUMEN
The mouse embryonic yolk sac consists of a visceral yolk sac (VYS) and parietal yolk sac (PYS), and may function as a materno-fetal exchange system for nutrients and wastes, and physical protector for the embryo/fetus. The present study was undertaken to characterize gene expression of the VYS and PYS endodermal cells, and to identify their novel genetic markers from microarray data. Apoa4, Lrp2, Fxyd2, Slc34a3 and Entpd2 were predominantly expressed in VYS epithelial cells. Gkn2 and Pga5 were selected as markers for PYS cells. These genetic markers may be useful for characterization of murine yolk sacs during development.
Asunto(s)
Endodermo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Saco Vitelino/metabolismo , Animales , Endodermo/citología , Femenino , Marcadores Genéticos , Ratones , Embarazo , Análisis de Matrices Tisulares , Saco Vitelino/citologíaRESUMEN
BACKGROUND: Amphibians are able to survive for several months without food. However, it is unclear what molecular mechanisms underlie their survival. To characterize the intestinal responses to fasting and refeeding, we investigated morphological, biochemical, transcriptional and epigenetic changes in the intestine from adult male Xenopus laevis. RESULTS: Frogs were fed for 22 days, fasted for 22 days, or fasted for 21 days and refed for 1 day. Fasting reduced, and refeeding recovered partially or fully, morphological parameters (wet weight of the intestine, circumference of the epithelial layer and number of troughs in a villus-trough unit), activities of digestive enzymes and plasma biochemical parameters (glucose, triglycerides, cholesterol and free fatty acids). Reverse transcription-quantitative polymerase chain reaction analysis revealed overall suppression of the transcript levels by fasting, with various recovery rates on refeeding. Chromatin immunoprecipitation assays on the selected genes whose transcript levels declined with fasting and recovered quickly with refeeding, showed several euchromatin marks in histone (acetylation and methylation) and RNA polymerase II modifications (phosphorylation) with fasting, and returned to the feeding levels by refeeding. The mRNA levels of these genes responded to fasting and refeeding to greater extents than did the pre-mRNA levels, suggesting the involvement of post-transcriptional regulation. CONCLUSIONS: Our results demonstrate that the X. laevis intestine may undergo overall metabolic suppression at least at the transcriptional level to save energy during fasting and quickly recovered to moderate nutritional deficiency by refeeding, and suggest that these dietary responses of the intestine are epigenetically and post-transcriptionally regulated.
RESUMEN
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.
Asunto(s)
Diferenciación Celular/fisiología , Desarrollo Embrionario/fisiología , Células Madre Mesenquimatosas/citología , Vísceras/citología , Saco Vitelino/citología , Animales , Células Cultivadas , Células Madre Mesenquimatosas/fisiología , Ratones , Ratones Endogámicos C3H , Vísceras/fisiología , Saco Vitelino/fisiologíaRESUMEN
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.
Asunto(s)
Conductos Biliares/citología , Ciclo Celular , Células Epiteliales/citología , Hepatectomía , Hepatocitos/citología , Regeneración Hepática/genética , Animales , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , ADN-Topoisomerasas de Tipo II/genética , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Células Epiteliales/metabolismo , Expresión Génica , Hepatocitos/metabolismo , Inmunohistoquímica , Antígeno Ki-67/genética , Antígeno Ki-67/metabolismo , Masculino , Ratones Endogámicos C57BL , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismoRESUMEN
In order to study the freeze-tolerance mechanism in the Japanese tree frog, Hyla japonica, wecloned a eDNA encoding aquaporin (AQP) 9 from its liver. The predicted amino acid sequence ofH. japonica AQP9 (AQP-h9) contained six putative transmembrane domains and two conservedAsn-Pro-Aia motifs, which are characteristic of AQPs. A swelling assay using Xenopus laevisoocytes injected with AQP-h9 cRNA showed that AQP-h9 facilitated water and glycerol permeation,confirming its property as an aquaglyceroporin. Subsequently, glycerol concentrations in serumand tissue extracts were compared among tree frogs that were hibernating, frozen, or thawed afterfreezing. Serum glycerol concentration of thawed frogs was significantly higher than that of hibernatingfrogs. Glycerol content in the liver did not change in the freezing experiment, whereas thatin the skeletal muscle was elevated in thawed frogs as compared with hibernating or frozen frogs. Histological examination of the liver showed that erythrocytes aggregated in the sinusoids during hibernation and freezing, and immunoreactive AQP-h9 protein was detected over the erythrocytes. The AQP-h9 labeling was more intense in frozen frogs than in hibernating frogs, but nearly undetectable in thawed frogs. For the skeletal muscle, weak labels for AQP-h9 were observed in the cytoplasm of myocytes of hibernating frogs. AQP-h9 labeling was markedly enhanced by freezing and was decreased by thawing. These results indicate that glycerol may act as a c;:ryoprotectant in H. japonica and that during hibernation, particularly during freezing, AQP-h9 may be involved in glycerol uptake in erythrocytes in the liver and in intracellular glycerol transport in the skeletal muscle cells.
Asunto(s)
Anuros/genética , Acuagliceroporinas/metabolismo , Clonación Molecular , ADN Complementario/genética , Regulación de la Expresión Génica/fisiología , Secuencia de Aminoácidos , Animales , Acuagliceroporinas/genética , Secuencia de Bases , Congelación , Hibernación , Datos de Secuencia Molecular , Filogenia , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
In mammalian liver development, intrahepatic biliary morphogenesis takes place in periportal, but not in pericentral, regions. Liver progenitor cells transiently form epithelial plate structures and then intrahepatic bile ducts around the portal veins under the influence of the mesenchyme. The present study was undertaken to histochemically examine normal biliary development and its dependence on the action of the thyroid hormone triiodothyronine (T3) in Xenopus laevis tadpoles. In these tadpoles, the development of hepatic ducts and intrahepatic biliary ducts commenced along the portal veins at NF stages 48-50 and stages 50-52, respectively, when the blood concentration of thyroid hormone may be still low. Some periportal hepatocytes expressed carbamoylphosphate synthase I and SOX9, which are hepatocyte and biliary cell markers, respectively, suggesting that periportal hepatocytes give rise to biliary epithelial cells. Periportal biliary cells did not form ductal plates, nor was the periportal mesenchyme well developed as seen in fetal mouse livers. jag1 mRNA was moderately expressed in cells of portal veins and biliary epithelial cells, and notch1 and notch2 mRNAs were weakly detectable in biliary epithelial cells during metamorphosis as seen in developing mammalian livers. These results suggest that Notch signaling plays a decisive role in biliary cell differentiation and morphogenesis of Xenopus tadpoles. Anti-thyroid agent treatment of the tadpoles resulted in delayed biliary morphogenesis, suggesting that biliary development may depend on T3. However, T3 treatment of the tadpoles did not enhance biliary development. Thus, T3 may act positively on biliary development at a very low concentration.
Asunto(s)
Sistema Biliar/crecimiento & desarrollo , Xenopus laevis/crecimiento & desarrollo , Animales , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Genes del Desarrollo , Hepatocitos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteína Jagged-1 , Larva/crecimiento & desarrollo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Metamorfosis Biológica/fisiología , Receptores Notch/genética , Receptores Notch/metabolismo , Proteínas Serrate-Jagged , Proteínas de XenopusRESUMEN
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.
Asunto(s)
Células Endoteliales/citología , Feto/metabolismo , Células Estrelladas Hepáticas/citología , Hepatocitos/citología , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Animales , Células Cultivadas , Medios de Cultivo Condicionados , Células Endoteliales/metabolismo , Expresión Génica , Células Estrelladas Hepáticas/metabolismo , Inmunohistoquímica , Hígado/citología , Hígado/metabolismo , Ratones , Ratones Endogámicos C3H , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
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).
Asunto(s)
Conductos Biliares Intrahepáticos/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/genética , Hepatocitos/citología , Cirrosis Hepática/metabolismo , Factores de Transcripción/metabolismo , Animales , Diferenciación Celular/genética , Modelos Animales de Enfermedad , Células Epiteliales/citología , Células Epiteliales/metabolismo , Hepatocitos/metabolismo , Inmunohistoquímica , Hibridación in Situ , Hígado/crecimiento & desarrollo , Hígado/metabolismo , Cirrosis Hepática/genética , Ratones , Ratones Noqueados , Morfogénesis/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaAsunto(s)
Comunicación Celular/fisiología , Diferenciación Celular , Hepatocitos/citología , Hígado/embriología , Ratones/embriología , Organogénesis , Animales , Conductos Biliares/embriología , Células Cultivadas , Matriz Extracelular/fisiología , Péptidos y Proteínas de Señalización Intercelular/fisiología , Hígado/citología , Receptores Notch/fisiología , Transducción de Señal/fisiologíaRESUMEN
The hagfish liver was histochemically examined with special attention to biliary structures around the portal veins. Hepatocytes were organized into tubular structures surrounded by sinusoids. Biliary ductule structures, which resemble the ductal plates transiently appearing in mammalian liver development, were observed around the portal veins, but they did not appear around central veins. Thus, the hagfish liver demonstrates the same basic structure as the mammalian liver; that is, a vascular system from portal to central veins via sinusoids, and portal triad structures consisting of portal veins, hepatic arteries, and intrahepatic bile ducts. The epithelial cells of the ductal platelike structures strongly expressed cytokeratin, had some lectin-binding sites, and were delineated by the basal lamina, which was reactive for periodic acid-Schiff (PAS) staining and Iectin histochemistry. The lumina of the ductal plate-like structures were comparatively small and heterogeneous in diameter around the portal veins, suggesting that the biliary structures may not be efficient for bile secretion. The epithelial cells of the gall bladder had a simple columnar shape and were a PAS-positive cytoplasm. Those of bile ducts near the hilus, including extrahepatic and hepatic ducts, were simple columnar or cuboidal cells, and had large lumina. The cytoplasm in these cells was PAS-positive. These phenotypes with the expression of lectin-binding sites were clearly different from those of the ductal plate-like structures in the liver proper, suggesting that the extrahepatic and intrahepatic biliary structures may have different developmental origins.
Asunto(s)
Sistema Biliar/anatomía & histología , Sistema Biliar/fisiología , Anguila Babosa/anatomía & histología , Anguila Babosa/fisiología , Hígado/anatomía & histología , Hígado/fisiología , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/metabolismo , Animales , Regulación Enzimológica de la Expresión Génica , Hígado/enzimologíaRESUMEN
Previous studies have shown that various cell-cell interactions between hepatoblasts and nonparenchymal cells, including sinusoidal endothelial cells and stellate cells, are indispensable for the development of fetal murine hepatic architecture. The present study was undertaken to determine the effects of hepatoblasts on the sinusoidal structural formation using a culture system of fetal mouse livers. Primitive sinusoidal structures extensively developed in fetal livers, and were composed of LYVE-1- and PECAM-1-positive endothelial cells, desmin-positive stellate cells and F4/80-positive macrophages. When fetal liver cells at 12.5 days of gestation were cultured in vitro, hepatoblasts spread on glass slides and gave rise to hepatocytes on day 5. Desmin-positive stellate cells also spread on the glass slides. PECAM-1-positive endothelial cells became slender and developed into anastomosing capillary networks. When fetal liver cells were cultured without hepatoblasts, which were excluded by an immunomagnetic method using anti-E-cadherin antibodies, endothelial cells had impaired growth and capillary formation. These results demonstrated that capillary formation of endothelial cells was induced by the presence of hepatoblasts. VEGF and the conditioned medium containing humoral factors produced by hepatoblasts/hepatocytes did not induce capillary formation of endothelial cells in cultures of nonparenchymal cells, although they significantly increased the number of endothelial cells on the glass slides. The presence of hepatoblasts also significantly stimulated expression of CD32b mRNA, which is a sinusoidal endothelial marker. Hepatoblasts may work as a positive stimulator of sinusoid morphogenesis and maturation in liver development, in which a signal other than VEGF may play a decisive role, together with VEGF.
Asunto(s)
Células Endoteliales/metabolismo , Hepatocitos/fisiología , Separación Inmunomagnética , Hígado/embriología , Morfogénesis/fisiología , Animales , Biomarcadores/metabolismo , Cadherinas/metabolismo , Técnicas de Cultivo de Célula , Células Cultivadas , Hepatocitos/citología , Hepatocitos/metabolismo , Inmunohistoquímica , Hígado/citología , Ratones , Ratones Endogámicos C3HRESUMEN
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.