Abstract
SUMMARYAbnormal Notch signaling in humans results in Alagille syndrome, a pleiotropic disease characterized by a paucity of intrahepatic bile ducts (IHBDs). It is not clear how IHBD paucity develops as a consequence of atypical Notch signaling, whether by a developmental lack of bile duct formation, a post-natal lack of branching and elongation or an inability to maintain formed ducts. Previous studies have focused on the role of Notch in IHBD development, and demonstrated a dosage requirement of Notch signaling for proper IHBD formation. In this study, we use resin casting and X-ray microtomography (microCT) analysis to address the role of Notch signaling in the maintenance of formed IHBDs upon chronic loss or gain of Notch function. Our data show that constitutive expression of the Notch1 intracellular domain in bi-potential hepatoblast progenitor cells (BHPCs) results in increased IHBD branches at post-natal day 60 (P60), which are maintained at P90 and P120. By contrast, loss of Notch signaling via BHPC-specific deletion of RBP-J (RBP KO), the DNA-binding partner for all Notch receptors, results in progressive loss of intact IHBD branches with age. Interestingly, in RBP KO mice, we observed a reduction in bile ducts per portal vein at P60; no further reduction had occurred at P120. Thus, bile duct structures are not lost with age; instead, we propose a model in which BHPC-specific loss of Notch signaling results in an initial developmental defect resulting in fewer bile ducts being formed, and in an acquired post-natal defect in the maintenance of intact IHBD architecture as a result of irresolvable cholestasis. Our studies reveal a previously unappreciated role for Notch signaling in the post-natal maintenance of an intact communicating IHBD structure, and suggest that liver defects observed in Alagille syndrome patients might be more complex than bile duct paucity.
Highlights
Mutations in JAGGED1, a ligand of the Notch pathway, are found in greater than 94% of patients with Alagille syndrome (AGS), with NOTCH2 receptor mutations identified in two families (McDaniell et al, 2006; Warthen et al, 2006)
We previously demonstrated that loss of Notch signaling within the bi-potential hepatoblast progenitor cell (BHPC) lineage results in a dosedependent reduction of peripheral intrahepatic bile ducts (IHBDs) branches, whereas activation of Notch1 within the bi-potential hepatoblast progenitor cells (BHPCs) lineage results in increased peripheral branches at post-natal day 120 (P120) (Sparks et al, 2010)
An Albumin-Cre transgene (Alb-Cre), expressing Cre in the BHPC lineage, was used to delete RBP-J, the common DNA-binding partner required for gene transcription downstream from all Notch receptors, giving Alb-Cre;RBP-Jflox/flox (RBP KO) mice, or to activate Notch1 by conditional expression of the Notch intracellular domain (NICD) (Alb-Cre; ROSA26Notch1; NICD mice)
Summary
Mutations in JAGGED1, a ligand of the Notch pathway, are found in greater than 94% of patients with Alagille syndrome (AGS), with NOTCH2 receptor mutations identified in two families (McDaniell et al, 2006; Warthen et al, 2006). In support of a bile duct maintenance defect, a subset of AGS patients, with clinical indications for progressive liver disease, demonstrate an increase in bile duct paucity from initial to subsequent liver biopsies (Emerick et al, 1999; Libbrecht et al, 2005). The NICD translocates to the nucleus, where it interacts with the common DNA-binding partner for all Notch receptors, recombination signal binding protein for immunoglobulin kappa J region (RBP-J). This association converts RBP-J from a transcriptional co-repressor to a co-activator, resulting in target gene expression. There are two families of canonical Notch ligands (Jagged and 2, and Delta-like-1, -3 and -4) and four Notch receptors (Notch1-4)
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