Sox9 Is a Modifier of the Liver Disease Severity in a Mouse Model of Alagille Syndrome

Abstract

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.

Fig. 1.. Reducing the gene dosage of Sox9 in the liver worsens the Jag1 haploinsufficient liver phenotypes.

(A) Jag1^+/–;Albumin-Cre;Sox9^+/flox animals show growth retardation compared to their siblings at P30. f: flox allele without Cre, Δ: flox allele with Cre. Horizontal lines show mean ± standard deviation. Each circle represents an animal (n=20, 20, 23, 26; left to right). (B) Immunofluorescent staining of P30 mouse liver sections with wide-spectrum cytokeratin (wsCK) antibody and DAPI counterstaining indicate that the ductular reactions and hypercellularity observed in Jag1^+/–;Sox9^+/flox animals is worsened in Jag1^+/–; Sox9^+/Δ animals. Asterisks mark PVs, arrows represent ductular reactions, arrowhead mark patent BDs. Scale bar: 100 µm. (C) The BD to PV ratio was reduced in P30 Jag1^+/–; Sox9^+/Δ animals. The phenotype was further enhanced upon removal of both copies of Sox9. (D) Half of the Jag1^+/–;Albumin-Cre;Sox9^f/f (Jag1^+/–;Sox9^Δ/Δ) animals did not survive beyond P25 (n=9, 7, 5, 10; top to bottom). (E,F) Serum total bilirubin and serum bile acids are significantly increased in Jag1^+/–;Sox9^Δ/+ compared to controls. Both numbers are significantly higher in Jag1^+/–;Sox9^Δ/Δ animals. One-way ANOVA with Tukey’s multiple comparisons test was performed in A,C,D. Circles represent males, triangles represent females. Overall, male and female mice showed comparable phenotypes (C,E,F). For E and F, since the number of animals for the four original genotypes was significantly higher than Jag1^+/–;Sox9^Δ/Δ and because the values for this genotype were dramatically different from other genotypes, we performed ANOVA on the four original phenotype and then performed two-sample t-test to compare Jag1^+/–;Sox9^+Δ and Jag1^+/–;Sox9^Δ/Δ values. ns: not significant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Fig. 2.. Removing one copy of Sox9 significantly worsens periportal inflammation and fibrosis in Jag1 haploinsufficient animals.

(A) H&E staining indicates inflammatory cell infiltration in P30 Jag1^+/–;Sox9^+/flox and Jag1^+/–; Sox9^+/Δ livers (n=3). (B-D) P30 mouse liver sections stained with αSMA antibody, Desmin antibody and Sirius Red (n=3 for each genotype) show enhanced fibrosis in Jag1^+/–; Sox9^+/Δ livers compared to Jag1^+/–;Sox9^+/flox. Arrows in B show bridging of αSMA⁺ cells between neighboring portal vessels, which is seen occasionally in Jag1^+/–;Sox9^+/f livers and commonly in Jag1^+/–;Sox9^+/∆ livers. Scale bar: 100 µm. (E) Quantification of percent area of fibrosis confirms increased fibrosis in Jag1^+/–; Sox9^+/Δ livers compared to Jag1^+/–;Sox9^+/flox animals. (E,F) Consistent with elevated fibrosis, Jag1^+/–; Sox9^+/Δ animals show significantly elevated liver to body weight ratios, although their liver weights are not statistically different from other genotypes. One-way ANOVA with Tukey’s multiple comparisons test was performed in E and F. *P<0.05, ****P<0.0001.

Fig. 3.. JAG1 and SOX9 play a partially redundant role to ensure the biliary commitment of developing cholangiocytes.

(A,B) Immunofluorescent staining of P0 liver sections for CK8 demonstrates reduced cholangiocyte numbers in Jag1^+/+; Sox9^Δ/Δ animals. Jag1^+/–; Sox9^+/f animals show significantly reduced cholangiocyte numbers that are not worsened with loss of one or both copies of Sox9. Scale bar: 100 µm. (C) Immunofluorescent staining of P7 liver sections for wsCK and HNF4A shows double-positive cells in Jag1^+/–; Sox9^+/Δ livers. Insets show close-up images. Scale bar: 100 µm (n=4, 3, 3, 4; left to right). (D) The number of double-positive cells around PVs is significantly increased in P7 Jag1^+/–; Sox9^+/Δ livers (n=4, 3, 3 and 4, left to right). Mean and standard deviation are shown. (E) The BD to PV ratio was decreased in P7 Jag1^+/–; Sox9^+/Δ livers. Note that while Jag1^+/+;Sox9^+/∆ livers have a normal BD to PV at P30, they show a small but significant decrease in BD to PV at P7, suggesting that BD development is slightly delayed even in a Sox9 heterozygous state. One-way ANOVA with Tukey’s multiple comparisons test was performed in B,C. ns: not significant, *P<0.05, ***P<0.001, ****P<0.0001.

Fig. 4.. Jag1^+/– animals show impaired formation of the biliary tree in liver periphery at P30 which is worsened upon Sox9 heterozygosity.

(A,B) Retrograde ink injection into the common bile duct of P30 animals shows impaired formation of biliary tree in the periphery of Jag1^+/–;Sox9^+/f livers, with worsening in Jag1^+/–; Sox9^+/Δ livers (n=7, 5, 5, 4; left to right). In two of four double-heterozygous animals analyzed by ink injection, none of the ink-filled ducts reach beyond the hilum. Whole left lobes and close-up of left lobes are shown. Arrowheads in A mark fine networks of biliary conduits that replace the smaller bile duct branches in Jag1^+/–;Sox9^+/f livers. Red lines mark the liver edge. (C) The ratio of OPN⁺ cells in P30 liver sections which are also Ki67⁺ (proliferation index) is not decreased in Jag1^+/–;Sox9^+/f and Jag1^+/–; Sox9^+/Δ livers, indicating that reduced proliferation does not lead to impaired biliary tree arborization in these animals. One-way ANOVA with Tukey’s multiple comparisons test was performed. ns, not significant. (D) Immunofluorescent staining for OPN in rehydrated and sectioned ink-injected livers at P30. Arrowheads represent OPN⁺ cholangiocytes without ink-filling (n=3 for each genotype).

Fig. 5.. Sox9 heterozygosity in the liver impairs biliary recovery observed in 5-month old Jag1^+/– animals.

(A) Jag1^+/–;Sox9^+/f biliary tree shows improvement in 5-month-old animals but removing one copy of Sox9 impairs the improvement (n=4 for each genotype). Left lobes are shown. (B) Immunofluorescent staining with anti-wsCK counterstained with DAPI shows improvement in Jag1^+/–;Sox9^+/f but not in Jag1^+/–; Sox9^+/Δ liver phenotypes in 5-month-old animals (n=3 for each genotype). Asterisks mark PVs. Scale bar: 100 µm. (C) The BD to PV ratio was improved in 5-month-old Jag1^+/–;Sox9^+/f livers but not in Jag1^+/–; Sox9^+/Δ. One-way ANOVA with Tukey’s multiple comparisons test was performed in C. *P<0.05.

Fig. 6.. SOX9 and NCAM1 staining can distinguish between mild and severe ALGS cases and suggest ongoing BD formation in mild cases.

(A) Shown is the information about the ALGS subjects analyzed in this work, including age at biopsy, history of transplant and JAG1 mutations. Those with a serum total bilirubin below 6.6 mg/dL were classified as mild. Note that a review of the medical charts of the two mild cases who were less than one year of age at the time of biopsy (Subjects 2 and 3) showed that they never required transplant and their clinical symptoms and laboratory measurements subsided over time. Also, based on the pathology report, fibrosis in subject #3 was confined to some portal tracts without any bridging. All severe subjects in this study underwent transplant and had a serum bilirubin of greater than 6.6 mg/dL, all had some degree of cholestasis on pathological examination and all but one were reported to have liver fibrosis. Mutations in JAG1 were identified in all tested subjects. y, year(s); m, months; w, weeks; NT, not tested. (B) Shown are immunohistochemical staining of human liver samples obtained from biopsies of mild and severe ALGS cases, biliary atresia (BA) and a control age-matched liver (n=3 for mild ALGS and 7 for severe ALGS). Antibodies against CK7, CK19, SOX9 and NCAM1 (CD56) were used. Groups of SOX9⁺, CK7⁺, CK19⁺ cells resembling BD structures are seen in the mild ALGS liver but not in the severe one. NCAM1 expression in two BDs in the mild case (close-up) suggest ongoing BD formation. NCAM1 expression is found in BA (close-up) but not normal liver samples. Arrowheads mark mature or developing BDs; arrows indicate individual SOX9⁺ cells in the severe ALGS case. All staining was performed on consecutive sections. Note that the NCAM1 staining for BA is rotated compared to other BA images. The arrow points to the same location in all BA samples. Green asterisks in each sample mark the same PV. Scale bar: 100 µm.

Fig. 7.. Sox9 overexpression in the liver rescues Jag1^+/– BD phenotypes without inducing cytokeratin expression in hepatocytes.

(A,B) Immunofluorescent staining of P30 liver sections with anti-wsCK counterstained with DAPI and BD quantification show a rescue of Jag1^+/– phenotypes upon SOX9 overexpression. Arrowheads mark patent BDs. Alb^Cre: Albumin-Cre, SOX9^OE: Albumin-Cre;CAG-Sox9 (n=3, 3, 4, 6; left to right). (C) H&E staining of a P30 liver section of a Jag1^+/–;Sox9^OE animal show rescue of hypercellularity (n=3). Compare to Fig. 1E. (D) Sirius Red staining in P30 mouse liver sections show rescue of Jag1^+/–periportal fibrosis upon SOX9 overexpression. Scale bars: 100 µm. (E) H&E staining on 10–12-month-old mouse liver sections reveals no evidence of tumor formation or altered hepatocyte morphology in Jag1^+/+; Sox9^OE and Jag1^+/–; Sox9^OE animals. (F–H) Serum chemistry from 10–12-month-old mice reveals no sign of altered liver function in animals overexpressing Sox9. One-way ANOVA with Tukey’s multiple comparisons test was performed in B and F-H. ns: not significant, **P<0.01, ***P<0.001.

Fig 8.. Sox9 overexpression improves biliary specification in Jag1^+/– animals through reduced hepatocyte differentiation from hepatoblasts and increased Notch2 expression.

(A-B) Sox9 overexpression in the liver does not increase cholangiocyte numbers in P0 Jag1^+/+ animals but restores cholangiocyte numbers in P0 Jag1^+/– animals. Scale bar: 100 µm. (C) Dox-induced SOX9 expression in BMEL cells cultured in differentiation medium results in the formation of tubular structures (arrows) by 96 hours of culture/induction. In the absence of Dox, the cells show a typical hepatocyte-like cobblestone appearance (inset). Scale bars: 1 mm for bright field images, 100 µm for fluorescent images. (D) Western blots on BMEL cell extracts with or without Dox show increased SOX9 and OPN but decreased Albumin levels. (E) qRT-PCR assays on whole liver extracts show increased Notch2 and Hes1 expression upon SOX9 overexpression (n=5, 6; left to right). (F) Schematic of the mouse Notch2 promoter and its upstream region with the two consensus SOX9-binding sites identified within 3 kb of the Notch2 transcription start site (red vertical lines). The weighted JASPAR matrix used to identify the sites is shown (http://jaspar.genereg.net/matrix/MA0077.1/). Chromatin immunoprecipitation shows enrichment of the SOX9 antibody compared to control IgG. The positive control (+ Ctrl) is anti-H3. Scale bars: 100 µm., Two-sample t-test was performed in E,F. ns: not significant, *P<0.05, **P<0.01.