Farah A Falix1, Víola B Weeda2, Wilhelmina T Labruyere3, Alexis Poncy4, Dirk R de Waart3, Theodorus B M Hakvoort3, Frédéric Lemaigre4, Ingrid C Gaemers3, Daniël C Aronson5, Wouter H Lamers3. 1. 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. 2. 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. 3. Tytgat Institute for Liver and Intestinal Research, Academic Medical Center, Amsterdam, The Netherlands. 4. Université Catholique de Louvain and the Duve Institute, Brussels, Belgium. 5. Emma Children's Hospital AMC and Pediatric Surgical Center of Amsterdam, Academic Medical Center, Amsterdam, The Netherlands.
Abstract
UNLABELLED: 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. CONCLUSION: 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.
UNLABELLED: 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. CONCLUSION: 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: Juhoon So; Mehwish Khaliq; Kimberley Evason; Nikolay Ninov; Benjamin L Martin; Didier Y R Stainier; Donghun Shin Journal: Hepatology Date: 2018-04-19 Impact factor: 17.425
Authors: Umesh Tharehalli; Michael Svinarenko; Johann M Kraus; Silke D Kühlwein; Robin Szekely; Ute Kiesle; Annika Scheffold; Thomas F E Barth; Alexander Kleger; Reinhold Schirmbeck; Hans A Kestler; Thomas Seufferlein; Franz Oswald; Sarah-Fee Katz; André Lechel Journal: Int J Mol Sci Date: 2018-11-29 Impact factor: 5.923