A faithful JAGGED1 haploinsufficiency mouse model of arteriohepatic dysplasia (Alagille syndrome) after all
Authors: Stacey S. Huppert
Journal: Hepatology (2015)

Potential conflict of interest: Nothing to report. See Article on Page 550 The association of human JAGGED1 (JAG1) mutations in more than 94% of definitive Alagille syndrome (ALGS) cases reveals an involvement of the Notch pathway.1 Studies in model organisms from invertebrates to vertebrates have established that Notch signaling is used reiteratively to provide communication between two cells, actively influencing cell fate decisions. Therefore, it is not surprising that ALGS is a pleiotropic disease, characterized by developmental abnormalities presenting in organs such as liver, heart, eye, kidney, and pancreas, as well as skeletal and craniofacial abnormalities. A common and distinguishing feature of this sporadic genetic disorder is cholestasis due to paucity of intrahepatic bile ducts (IHBDs).2 Mouse models investigating the loss or gain of Notch pathway components in specific hepatic cell lineages have elucidated the importance of Notch signal reception within the hepatoblast lineage for IHBD morphogenesis. The Notch ligand JAG1 is presented by portal vein (PV) mesenchyme to activate Notch receptors on juxtaposed bipotential hepatoblasts establishing IHBD architecture.3 However, two very perplexing questions remain. First, given that ALGS is a dominantly inherited disease, why does the loss of one copy of Jag1 in mice not recapitulate ALGS IHBD paucity?3 Reports analyzing the phenotype of the null Jag1dDSL allele,4 generated by targeted deletion of the C‐terminal portion of the DELTA/SERRATE/LAG‐2 (DSL) domain required for interaction with NOTCH receptors, have not detected an IHBD phenotype. Second, why do patients with ALGS inheriting the same Jag1 gene mutation display a wide spectrum of disease severity?6 The report by Thakurdas et al.7 provides new data imparting insight into each of these questions. To examine the long‐standing question of whether a haploinsufficiency of JAG1 in mice produces a hepatic phenotype similar to patients with ALGS, the investigators performed a spatial and temporal analysis of IHBD markers implementing the Jag1dDSL allele5 backcrossed >10 generations onto a C57BL/6 strain background. Mice heterozygous for the Jag1dDSL allele (Jag1+/–) had decreased survival, mild growth retardation, and a statistically significant elevation in serum total bilirubin levels. At embryonic day 18, Jag1+/– ductal plates assessed by cytokeratin (CK)–positive immunostaining are less organized than controls. Interestingly, the disorganized CK‐positive Jag1+/– ductal plates are not expressing one of the earliest markers of ductal plate formation and known Notch target gene, SOX9.3 At postnatal day 0, Jag1+/– mice have a paucity of patent formed IHBDs in the hilar region and absence of IHBDs in peripheral regions compared to controls. By postnatal day 7, SOX9‐positive and CK‐positive cells begin appearing around PVs, but there is still a paucity of patent ducts. To determine if the IHBD paucity observed in Jag1+/– mice is due to a developmental delay of tubulogenesis, as observed in hepatoblasts deficient for SOX9,3 the hepatic phenotype of postnatal day 30 Jag1+/– mice was evaluated. A variable phenotype ranging from focal necroinflammatory lesions with ductular reactions, ductular reactions without necroinflammatory lesions, or ductopenia was observed. Armed with current knowledge of IHBD formation and bile duct epithelial markers, the authors were able to identify the variable phenotypic defects not previously perceived. This result is very reminiscent of the variable presentation of the hepatic phenotype observed in patients with ALGS.2 These observations indicate that IHBD development in mice is sensitive to the dosage of JAG1, as is observed in humans. The variability in the severity of ALGS phenotypes is not unusual for parents and siblings of a patient with ALGS.6 These observations led to the hypothesis that genetic modifiers play a major role in ALGS phenotypic expression. The Notch pathway is unique in its sensitivity to the stoichiometric gene dosage of ligands and receptors, highlighted by the lack of an amplification step as is present in most signaling pathways.8 Therefore, candidate modifiers are predicted to be genes encoding proteins that modulate the ligand and receptor interactions, thereby influencing Notch activity level. Different types of carbohydrates modify the Notch extracellular domain.9 Thakurdas et al.7 investigate a glycosyltransferase, Rumi/Protein O‐glucosyltransferase (POGLUT1), for its ability to modify the outcome of the JAG1 haploinsufficient phenotype. Examination of mice double heterozygous for JAG1 and Rumi strongly demonstrates that decreasing the level of Rumi improves the Jag1+/– viability, growth, and hepatic phenotype. Ductal plates at embryonic day 18 are SOX9‐positive and CK‐positive. Portal triads are visible in the first postnatal week, and there is a statistically significant increase in the number of IHBDs per PV compared to Jag1+/–. At postnatal day 30, there is an absence of the focal necroinflammatory lesions and ductular reactions observed in mice double heterozygous for JAG1 and Rumi compared to Jag1+/– mice. This result is very exciting, not only implicating another glycosyltransferase regulating the level of Notch activity but also identifying a genetic modifier that alleviates the JAG1 haploinsufficiency hepatic phenotype. In addition to the IHBD phenotype, a decrease in the region of α‐smooth muscle actin–positive cells surrounding the PV and hepatic arteries (HAs) in Jag1+– mice is observed. This supports a role for JAG1 in the differentiation of vascular smooth muscle cells (VSMCs)10 and provides an explanation for why SOX9‐positive ductal plate cells are not efficiently induced. Although not well characterized, paucity of IHBDs in patients with ALGS is associated with a concomitant reduction in the number of portal tracts per area and potentially reduced HA branches.11 Removal of one copy of Rumi in Jag1+– mice improves the deficient VSMC phenotype. Together, these data highlight multiple inherent defects potentially contributing to IHBD paucity due to JAG1 haploinsufficiency. To investigate the cell lineage that requires Rumi function for IHBD formation, the investigators deleted Rumi in the mesenchyme using SM22α‐Cre and in the hepatoblasts using albumin‐Cre. No difference in IHBD development was observed when the hepatoblast lineage was deficient for Rumi (RumiAlbKO). Intriguingly, the HA/PV ratio was significantly increased, suggesting that Rumi is not necessary in the hepatoblast lineage for IHBD formation but is involved in the regulation of HA architecture. These results suggest that Notch receptors in the hepatoblast lineage do not depend on Rumi glycosylation to regulate IHBD formation. In contrast, there is an increase in the number of IHBDs per PV when the mesenchymal lineage was deficient for Rumi (RumiSMKO) with a concomitant increase of α‐smooth muscle actin–positive cells in the periportal area. These results suggest Rumi is required in the VSMCs for proper IHBD and HA formation. Again, removal of one copy of Rumi in the hepatoblasts or mesenchyme alleviates the JAG1 haploinsufficient hepatic phenotype. Overall, the genetic experiments indicate that reducing Rumi levels promotes the signal sending ability of JAG1 in the liver. To address this hypothesis, VSMCs from adult control and RumiSMKO inferior vena cava were harvested and cultured. Protein and RNA analysis reveals an increase of JAG1 protein (total and soluble) without a change in Jag1 messenger RNA levels. Therefore, the increase of JAG1 in the absence of Rumi may have the ability to activate Notch signaling and support the hypothesis that Rumi plays an antagonistic role in Notch activation. To determine if Rumi has the potential to directly influence JAG1 activity, mass spectrometric analysis was performed on the human JAG1 extracellular domain. All four predicted Rumi C1XSX(A/P)C2 consensus sites in JAG1 EGF repeats 4, 8, 11, and 16 were efficiently O‐glucosylated, indicating that JAG1 is a bona fide target for O‐glycosylation by Rumi. Importantly, this study not only provides a Jag1+/– mouse that recapitulates the IHBD paucity observed in patients with ALGS but also identifies Rumi as a new molecular player genetically opposing the function of JAG1 in VSMCs. The results support a mechanism by which Rumi opposes JAG1 function by glycosylating the JAG1 extracellular domain. ALGS is mainly caused by a haploinsufficiency of JAG, indicating that one functional gene copy is not sufficient to produce enough ligand‐mediated Notch receptor activation to reach the critical threshold for proper target gene expression in all cells. The observation that reducing the dose of Rumi alleviates the JAG1 haploinsufficient hepatic phenotype provides a potential opportunity to uncover novel mechanisms to block the progression of ALGS chronic liver disease by enhancing the JAG1 signaling activity of the protein produced from the remaining functional allele. Authors names in bold designate shared co‐first authorship.