SUMMARYProtein O-glucosylation is a conserved post-translational modification that occurs on epidermal growth factor-like (EGF) repeats harboring the C 1 -X-S-X-P-C 2 consensus sequence.

Haploinsufficiency for the Notch ligand JAG1 in humans results in an autosomal dominant, multisystem disorder known as Alagille syndrome, which is characterized by a congenital cholangiopathy of variable severity. Here we show that on a C57BL/6 background, jagged1 heterozygous mice (Jag1+/−) exhibit impaired intrahepatic bile duct (IHBD) development, decreased SOX9 expression and thinning of the peri-portal vascular smooth muscle cell (VSMC) layer, which are apparent at embryonic day 18 and the first postnatal week. In contrast, mice double-heterozygous for Jag1 and the glycosyltransferase Poglut1 (Rumi) start showing a significant improvement in IHBD development and VSMC differentiation during the first week. At P30, Jag1+/− mice show widespread ductular reactions and ductopenia in their livers and a mild but statistically significant bilirubinemia. In contrast, P30 Jag1/Rumi double-heterozygous mice show well-developed portal triads around most portal veins, with no elevation of serum bilirubin. Conditional deletion of Rumi in VSMCs results in progressive arborization of the IHBD tree, whereas deletion of Rumi in hepatoblasts frequently results in an increase in the number of hepatic arteries without affecting bile duct formation. Nevertheless, removing one copy of Rumi from either VSMCs or hepatoblasts is sufficient to partially suppress the Jag1+/− bile duct defects. Finally, all Rumi target sites of the human JAG1 are efficiently glucosylated, and loss of Rumi in VSMCs results in increased levels of full-length JAG1 and a shorter fragment of JAG1 without affecting Jag1 mRNA levels. Conclusions On a C57BL/6 background, Jag1 haploinsufficiency results in bile duct paucity in mice. Removing one copy of Rumi suppresses the Jag1+/− bile duct phenotype, indicating that Rumi opposes the JAG1 function in the liver.

Background and Aims Alagille syndrome (ALGS) is a multisystem developmental disorder characterized by bile duct (BD) paucity, caused primarily by haploinsufficiency of the Notch ligand jagged1. The course of the liver disease is highly variable in ALGS. However, the genetic basis for ALGS phenotypic variability is unknown. Previous studies have reported decreased expression of the transcription factor SOX9 (sex determining region Y‐box 9) in late embryonic and neonatal livers of Jag1‐deficient mice. Here, we investigated the effects of altering the Sox9 gene dosage on the severity of liver disease in an ALGS mouse model. Approach and Results Conditional removal of one copy of Sox9 in Jag1+/− livers impairs the biliary commitment of cholangiocytes and enhances the inflammatory reaction and liver fibrosis. Loss of both copies of Sox9 in Jag1+/− livers further worsens the phenotypes and results in partial lethality. Ink injection experiments reveal impaired biliary tree formation in the periphery of P30 Jag1+/− livers, which is improved by 5 months of age. Sox9 heterozygosity worsens the P30 biliary tree phenotype and impairs the partial recovery in 5‐month‐old animals. Notably, Sox9 overexpression improves BD paucity and liver phenotypes in Jag1+/− mice without ectopic hepatocyte‐to‐cholangiocyte transdifferentiation or long‐term liver abnormalities. Notch2 expression in the liver is increased following Sox9 overexpression, and SOX9 binds the Notch2 regulatory region in the liver. Histological analysis shows a correlation between the level and pattern of SOX9 expression in the liver and outcome of the liver disease in patients with ALGS. Conclusions Our results establish Sox9 as a dosage‐sensitive modifier of Jag1+/− liver phenotypes with a permissive role in biliary development. Our data further suggest that liver‐specific increase in SOX9 levels is a potential therapeutic approach for BD paucity in ALGS.

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