title: Hepatic Notch2 deficiency leads to bile duct agenesis perinatally and secondary bile duct formation after weaning.
abstract: Notch signaling plays an acknowledged role in bile-duct development, but its involvement in cholangiocyte-fate determination remains incompletely understood. We investigated the effects of early Notch2 deletion in Notch2(fl/fl)/Alfp-Cre(tg/-) ("Notch2-cKO") and Notch2(fl/fl)/Alfp-Cre(-/-) ("control") mice. Fetal and neonatal Notch2-cKO livers were devoid of cytokeratin19 (CK19)-, Dolichos-biflorus agglutinin (DBA)-, and SOX9-positive ductal structures, demonstrating absence of prenatal cholangiocyte differentiation. Despite extensive cholestatic hepatocyte necrosis and growth retardation, mortality was only ~15%. Unexpectedly, a slow process of secondary cholangiocyte differentiation and bile-duct formation was initiated around weaning that histologically resembled the ductular reaction. Newly formed ducts varied from rare and non-connected, to multiple, disorganized tubular structures that connected to the extrahepatic bile ducts. Jaundice had disappeared in ~30% of Notch2-cKO mice by 6 months. The absence of NOTCH2 protein in postnatally differentiating cholangiocyte nuclei of Notch2-cKO mice showed that these cells had not originated from non-recombined precursor cells. Notch2 and Hnf6 mRNA levels were permanently decreased in Notch2-cKO livers. Perinatally, Foxa1, Foxa2, Hhex, Hnf1β, Cebpα and Sox9 mRNA levels were all significantly lower in Notch2-cKO than control mice, but all except Foxa2 returned to normal or increased levels after weaning, coincident with the observed secondary bile-duct formation. Interestingly, Hhex and Sox9 mRNA levels remained elevated in icteric 6 months old Notch2-cKOs, but decreased to control levels in non-icteric Notch2-cKOs, implying a key role in secondary bile-duct formation.
  
  
  CONCLUSIONS
  Cholangiocyte differentiation becomes progressively less dependent on NOTCH2 signaling with age, suggesting that ductal-plate formation is dependent on NOTCH2, but subsequent cholangiocyte differentiation is not.
authors.affiliation: Tytgat Institute for Liver and Intestinal Research, Academic Medical Center, Amsterdam, The Netherlands; Emma Children's Hospital AMC and Pediatric Surgical Center of Amsterdam, Academic Medical Center, Amsterdam, The Netherlands. Electronic address: f.a.falix@amc.nl.
authors.forename: Farah A
authors.initials: FA
authors.lastname: Falix
authors.affiliation: Tytgat Institute for Liver and Intestinal Research, Academic Medical Center, Amsterdam, The Netherlands; Emma Children's Hospital AMC and Pediatric Surgical Center of Amsterdam, Academic Medical Center, Amsterdam, The Netherlands.
authors.forename: Víola B
authors.initials: VB
authors.lastname: Weeda
authors.affiliation: Tytgat Institute for Liver and Intestinal Research, Academic Medical Center, Amsterdam, The Netherlands.
authors.forename: Wilhelmina T
authors.initials: WT
authors.lastname: Labruyere
authors.affiliation: Université Catholique de Louvain and the Duve Institute, Brussels, Belgium.
authors.forename: Alexis
authors.initials: A
authors.lastname: Poncy
authors.affiliation: Tytgat Institute for Liver and Intestinal Research, Academic Medical Center, Amsterdam, The Netherlands.
authors.forename: Dirk R
authors.initials: DR
authors.lastname: de Waart
authors.affiliation: Tytgat Institute for Liver and Intestinal Research, Academic Medical Center, Amsterdam, The Netherlands.
authors.forename: Theodorus B M
authors.initials: TB
authors.lastname: Hakvoort
authors.affiliation: Université Catholique de Louvain and the Duve Institute, Brussels, Belgium.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Lemaigre
authors.affiliation: Tytgat Institute for Liver and Intestinal Research, Academic Medical Center, Amsterdam, The Netherlands.
authors.forename: Ingrid C
authors.initials: IC
authors.lastname: Gaemers
authors.affiliation: Emma Children's Hospital AMC and Pediatric Surgical Center of Amsterdam, Academic Medical Center, Amsterdam, The Netherlands.
authors.forename: Daniël C
authors.initials: DC
authors.lastname: Aronson
authors.affiliation: Tytgat Institute for Liver and Intestinal Research, Academic Medical Center, Amsterdam, The Netherlands.
authors.forename: Wouter H
authors.initials: WH
authors.lastname: Lamers
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D001652:Bile Ducts / Q000002:abnormalities* / Q000254:growth & development*; D017931:DNA Primers / Q000235:genetics; D051559:Hepatocyte Nuclear Factor 6 / Q000378:metabolism; D006652:Histological Techniques; D007150:Immunohistochemistry; D008099:Liver / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D038081:Organogenesis / Q000235:genetics* / Q000502:physiology; D016133:Polymerase Chain Reaction; D051883:Receptor, Notch2 / Q000172:deficiency*; D012044:Regression Analysis; D014886:Weaning
chemical_list: D017931:DNA Primers; D051559:Hepatocyte Nuclear Factor 6; C497765:Notch2 protein, mouse; C496606:Onecut1 protein, mouse; D051883:Receptor, Notch2
keywords: Cholangiocytes; Ductal plate; Ductular reaction; Liver; Notch2