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Sox9b is a key regulator of pancreaticobiliary ductal system development.

Delous M, Yin C, Shin D, Ninov N, Debrito Carten J, Pan L, Ma TP, Farber SA, Moens CB, Stainier DY - PLoS Genet. (2012)

Bottom Line: Analysis of sox9b(fh313) mutant embryos and larvae reveals that the HPD cells appear to mis-differentiate towards hepatic and/or pancreatic fates, resulting in a dysmorphic structure.The defects in the intrahepatic and intrapancreatic ducts of sox9b(fh313) mutants worsen during larval and juvenile stages, prompting the adult phenotype.We further show that Sox9b interacts with Notch signaling to regulate intrahepatic biliary network formation: sox9b expression is positively regulated by Notch signaling, while Sox9b function is required to maintain Notch signaling in the intrahepatic biliary cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Program in Developmental and Stem Cell Biology, Liver Center and Diabetes Center, University of California San Francisco, San Francisco, California, United States of America. marion.delous@ucsf.edu

ABSTRACT
The pancreaticobiliary ductal system connects the liver and pancreas to the intestine. It is composed of the hepatopancreatic ductal (HPD) system as well as the intrahepatic biliary ducts and the intrapancreatic ducts. Despite its physiological importance, the development of the pancreaticobiliary ductal system remains poorly understood. The SRY-related transcription factor SOX9 is expressed in the mammalian pancreaticobiliary ductal system, but the perinatal lethality of Sox9 heterozygous mice makes loss-of-function analyses challenging. We turned to the zebrafish to assess the role of SOX9 in pancreaticobiliary ductal system development. We first show that zebrafish sox9b recapitulates the expression pattern of mouse Sox9 in the pancreaticobiliary ductal system and use a nonsense allele of sox9b, sox9b(fh313), to dissect its function in the morphogenesis of this structure. Strikingly, sox9b(fh313) homozygous mutants survive to adulthood and exhibit cholestasis associated with hepatic and pancreatic duct proliferation, cyst formation, and fibrosis. Analysis of sox9b(fh313) mutant embryos and larvae reveals that the HPD cells appear to mis-differentiate towards hepatic and/or pancreatic fates, resulting in a dysmorphic structure. The intrahepatic biliary cells are specified but fail to assemble into a functional network. Similarly, intrapancreatic duct formation is severely impaired in sox9b(fh313) mutants, while the embryonic endocrine and acinar compartments appear unaffected. The defects in the intrahepatic and intrapancreatic ducts of sox9b(fh313) mutants worsen during larval and juvenile stages, prompting the adult phenotype. We further show that Sox9b interacts with Notch signaling to regulate intrahepatic biliary network formation: sox9b expression is positively regulated by Notch signaling, while Sox9b function is required to maintain Notch signaling in the intrahepatic biliary cells. Together, these data reveal key roles for SOX9 in the morphogenesis of the pancreaticobiliary ductal system, and they cast human Sox9 as a candidate gene for pancreaticobiliary duct malformation-related pathologies.

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sox9b mutant larvae fail to form a complex intrapancreatic ductal network.(A–F) Confocal images of Tg(Tp1bglob:GFP);Tg(Tp1bglob:H2B-mCherry) wild-type (top row) and sox9b mutant (bottom row) pancreata at 80 (A,B), 100 (C,D) and 120 (E,F) hpf. Elastase antibody staining (blue) labels acinar cells. Although acinar and endocrine tissues appear morphologically unaffected in sox9b mutants (data not shown), the intrapancreatic ductal network is less complex and secondary branches are missing in the mutants (D–D′) whereas they start to form by 100 hpf in wild-type larvae (arrowheads and insets, C–C′). (E′″–F′″) Higher magnifications of the area marked by dashed squares in (E–F′) show that at 120 hpf the main duct forms secondary branches (arrowheads) in wild-type larvae (E″–E′″), whereas in the mutants, secondary branches remain absent and clusters of ductal cells are sometimes observed (F″–F′″). (A–F) All images are projections of confocal z-stacks. Ventral views, anterior (A) to the top. Scale bars, 50 µm. (G) Graph representing the number of Tg(Tp1bglob:GFP);Tg(Tp1bglob:H2B-mCherry)-double positive cells (average±SEM) in the intrapancreatic ducts of wild-type and sox9b mutant larvae at different time points. 7 to 11 larvae of each genotype were counted at each stage. (H) Graph representing the area (in arbitrary unit, a.u.) of the primary islet (average±SEM) of TgBAC(neurod:GFP) wild-type and sox9b mutant larvae (7 dpf) and juvenile animals (2, 3 and 4 weeks (wks)). 6 to 11 animals of each genotype were analyzed at each stage. Area of primary islet was determined using ImageJ. (I) Graph representing the number of TgBAC(neurod:GFP)-positive cells/clusters (average±SEM) along the intrapancreatic ducts (IPD) in wild-type and sox9b mutant larvae (10 dpf) and juvenile animals. 7 to 11 animals of each genotype were analyzed at each stage. Asterisks indicate statistical significance: *p<0.05; **p<0.01; ***p<0.0005; ****p<0.0001; ******p<0.000005.
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pgen-1002754-g004: sox9b mutant larvae fail to form a complex intrapancreatic ductal network.(A–F) Confocal images of Tg(Tp1bglob:GFP);Tg(Tp1bglob:H2B-mCherry) wild-type (top row) and sox9b mutant (bottom row) pancreata at 80 (A,B), 100 (C,D) and 120 (E,F) hpf. Elastase antibody staining (blue) labels acinar cells. Although acinar and endocrine tissues appear morphologically unaffected in sox9b mutants (data not shown), the intrapancreatic ductal network is less complex and secondary branches are missing in the mutants (D–D′) whereas they start to form by 100 hpf in wild-type larvae (arrowheads and insets, C–C′). (E′″–F′″) Higher magnifications of the area marked by dashed squares in (E–F′) show that at 120 hpf the main duct forms secondary branches (arrowheads) in wild-type larvae (E″–E′″), whereas in the mutants, secondary branches remain absent and clusters of ductal cells are sometimes observed (F″–F′″). (A–F) All images are projections of confocal z-stacks. Ventral views, anterior (A) to the top. Scale bars, 50 µm. (G) Graph representing the number of Tg(Tp1bglob:GFP);Tg(Tp1bglob:H2B-mCherry)-double positive cells (average±SEM) in the intrapancreatic ducts of wild-type and sox9b mutant larvae at different time points. 7 to 11 larvae of each genotype were counted at each stage. (H) Graph representing the area (in arbitrary unit, a.u.) of the primary islet (average±SEM) of TgBAC(neurod:GFP) wild-type and sox9b mutant larvae (7 dpf) and juvenile animals (2, 3 and 4 weeks (wks)). 6 to 11 animals of each genotype were analyzed at each stage. Area of primary islet was determined using ImageJ. (I) Graph representing the number of TgBAC(neurod:GFP)-positive cells/clusters (average±SEM) along the intrapancreatic ducts (IPD) in wild-type and sox9b mutant larvae (10 dpf) and juvenile animals. 7 to 11 animals of each genotype were analyzed at each stage. Asterisks indicate statistical significance: *p<0.05; **p<0.01; ***p<0.0005; ****p<0.0001; ******p<0.000005.

Mentions: We then addressed the role of Sox9b in intrapancreatic duct formation by using the double transgenic line Tg(Tp1bglob:GFP);Tg(Tp1bglob:H2B-mCherry) that expresses both GFP and H2B-mCherry under the control of a Notch-responsive element [34], [35]. This line allows the visualization of the shape and nuclei of the intrapancreatic duct cells, as indicated by the overlapping expression of these fluorescent proteins with ductal markers such as E-cadherin and 2F11 [34], [35]. Intrapancreatic ducts derive from cells within the ventral pancreatic bud that migrate towards, and eventually surround, the principal islet at 48 hpf [22]. From 60 hpf, ductal progenitors start to migrate caudally to form a row of cells that give rise to the main intrapancreatic duct [22] (Figure 4A–4A′). The migration of the ductal progenitors did not seem to be impaired in sox9b mutants; however, the number of cells within the intrapancreatic ducts was significantly reduced (Figure 4B–4B′, 4G). In wild-type larvae, at 100 hpf, the pancreatic tail keeps elongating, the number of ductal cells has slightly increased (Figure 4C–4C′, 4G) and secondary branches (arrowheads, Figure 4C–4C′) start to form from the main duct. In contrast, in sox9b mutants, the number of ductal cells did not increase from 80 to 100 hpf, and no secondary branches appeared, resulting in a primitive ductal system (Figure 4D–4D′, 4G). At 120 hpf, the ductal network in wild-type larvae has become more complex with numerous secondary branches (arrowheads, Figure 4E″) spreading over the acinar compartment (Figure 4E–4E′″). In contrast, the intrapancreatic ductal system in sox9b mutants remained poorly developed and clusters of cells could be observed along the main duct (Figure 4F–4F′″), which was still devoid of secondary branches. These data indicate that fewer intrapancreatic duct cells differentiate in the mutants and those that do fail to undergo branching morphogenesis. Furthermore, the number of ductal cells in sox9b mutants did not increase as in wild-types. Such a defect is likely due to a problem with cell differentiation as we did not observe any obvious differences in ductal cell proliferation or survival between wild-types and sox9b mutants (data not shown).


Sox9b is a key regulator of pancreaticobiliary ductal system development.

Delous M, Yin C, Shin D, Ninov N, Debrito Carten J, Pan L, Ma TP, Farber SA, Moens CB, Stainier DY - PLoS Genet. (2012)

sox9b mutant larvae fail to form a complex intrapancreatic ductal network.(A–F) Confocal images of Tg(Tp1bglob:GFP);Tg(Tp1bglob:H2B-mCherry) wild-type (top row) and sox9b mutant (bottom row) pancreata at 80 (A,B), 100 (C,D) and 120 (E,F) hpf. Elastase antibody staining (blue) labels acinar cells. Although acinar and endocrine tissues appear morphologically unaffected in sox9b mutants (data not shown), the intrapancreatic ductal network is less complex and secondary branches are missing in the mutants (D–D′) whereas they start to form by 100 hpf in wild-type larvae (arrowheads and insets, C–C′). (E′″–F′″) Higher magnifications of the area marked by dashed squares in (E–F′) show that at 120 hpf the main duct forms secondary branches (arrowheads) in wild-type larvae (E″–E′″), whereas in the mutants, secondary branches remain absent and clusters of ductal cells are sometimes observed (F″–F′″). (A–F) All images are projections of confocal z-stacks. Ventral views, anterior (A) to the top. Scale bars, 50 µm. (G) Graph representing the number of Tg(Tp1bglob:GFP);Tg(Tp1bglob:H2B-mCherry)-double positive cells (average±SEM) in the intrapancreatic ducts of wild-type and sox9b mutant larvae at different time points. 7 to 11 larvae of each genotype were counted at each stage. (H) Graph representing the area (in arbitrary unit, a.u.) of the primary islet (average±SEM) of TgBAC(neurod:GFP) wild-type and sox9b mutant larvae (7 dpf) and juvenile animals (2, 3 and 4 weeks (wks)). 6 to 11 animals of each genotype were analyzed at each stage. Area of primary islet was determined using ImageJ. (I) Graph representing the number of TgBAC(neurod:GFP)-positive cells/clusters (average±SEM) along the intrapancreatic ducts (IPD) in wild-type and sox9b mutant larvae (10 dpf) and juvenile animals. 7 to 11 animals of each genotype were analyzed at each stage. Asterisks indicate statistical significance: *p<0.05; **p<0.01; ***p<0.0005; ****p<0.0001; ******p<0.000005.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3375260&req=5

pgen-1002754-g004: sox9b mutant larvae fail to form a complex intrapancreatic ductal network.(A–F) Confocal images of Tg(Tp1bglob:GFP);Tg(Tp1bglob:H2B-mCherry) wild-type (top row) and sox9b mutant (bottom row) pancreata at 80 (A,B), 100 (C,D) and 120 (E,F) hpf. Elastase antibody staining (blue) labels acinar cells. Although acinar and endocrine tissues appear morphologically unaffected in sox9b mutants (data not shown), the intrapancreatic ductal network is less complex and secondary branches are missing in the mutants (D–D′) whereas they start to form by 100 hpf in wild-type larvae (arrowheads and insets, C–C′). (E′″–F′″) Higher magnifications of the area marked by dashed squares in (E–F′) show that at 120 hpf the main duct forms secondary branches (arrowheads) in wild-type larvae (E″–E′″), whereas in the mutants, secondary branches remain absent and clusters of ductal cells are sometimes observed (F″–F′″). (A–F) All images are projections of confocal z-stacks. Ventral views, anterior (A) to the top. Scale bars, 50 µm. (G) Graph representing the number of Tg(Tp1bglob:GFP);Tg(Tp1bglob:H2B-mCherry)-double positive cells (average±SEM) in the intrapancreatic ducts of wild-type and sox9b mutant larvae at different time points. 7 to 11 larvae of each genotype were counted at each stage. (H) Graph representing the area (in arbitrary unit, a.u.) of the primary islet (average±SEM) of TgBAC(neurod:GFP) wild-type and sox9b mutant larvae (7 dpf) and juvenile animals (2, 3 and 4 weeks (wks)). 6 to 11 animals of each genotype were analyzed at each stage. Area of primary islet was determined using ImageJ. (I) Graph representing the number of TgBAC(neurod:GFP)-positive cells/clusters (average±SEM) along the intrapancreatic ducts (IPD) in wild-type and sox9b mutant larvae (10 dpf) and juvenile animals. 7 to 11 animals of each genotype were analyzed at each stage. Asterisks indicate statistical significance: *p<0.05; **p<0.01; ***p<0.0005; ****p<0.0001; ******p<0.000005.
Mentions: We then addressed the role of Sox9b in intrapancreatic duct formation by using the double transgenic line Tg(Tp1bglob:GFP);Tg(Tp1bglob:H2B-mCherry) that expresses both GFP and H2B-mCherry under the control of a Notch-responsive element [34], [35]. This line allows the visualization of the shape and nuclei of the intrapancreatic duct cells, as indicated by the overlapping expression of these fluorescent proteins with ductal markers such as E-cadherin and 2F11 [34], [35]. Intrapancreatic ducts derive from cells within the ventral pancreatic bud that migrate towards, and eventually surround, the principal islet at 48 hpf [22]. From 60 hpf, ductal progenitors start to migrate caudally to form a row of cells that give rise to the main intrapancreatic duct [22] (Figure 4A–4A′). The migration of the ductal progenitors did not seem to be impaired in sox9b mutants; however, the number of cells within the intrapancreatic ducts was significantly reduced (Figure 4B–4B′, 4G). In wild-type larvae, at 100 hpf, the pancreatic tail keeps elongating, the number of ductal cells has slightly increased (Figure 4C–4C′, 4G) and secondary branches (arrowheads, Figure 4C–4C′) start to form from the main duct. In contrast, in sox9b mutants, the number of ductal cells did not increase from 80 to 100 hpf, and no secondary branches appeared, resulting in a primitive ductal system (Figure 4D–4D′, 4G). At 120 hpf, the ductal network in wild-type larvae has become more complex with numerous secondary branches (arrowheads, Figure 4E″) spreading over the acinar compartment (Figure 4E–4E′″). In contrast, the intrapancreatic ductal system in sox9b mutants remained poorly developed and clusters of cells could be observed along the main duct (Figure 4F–4F′″), which was still devoid of secondary branches. These data indicate that fewer intrapancreatic duct cells differentiate in the mutants and those that do fail to undergo branching morphogenesis. Furthermore, the number of ductal cells in sox9b mutants did not increase as in wild-types. Such a defect is likely due to a problem with cell differentiation as we did not observe any obvious differences in ductal cell proliferation or survival between wild-types and sox9b mutants (data not shown).

Bottom Line: Analysis of sox9b(fh313) mutant embryos and larvae reveals that the HPD cells appear to mis-differentiate towards hepatic and/or pancreatic fates, resulting in a dysmorphic structure.The defects in the intrahepatic and intrapancreatic ducts of sox9b(fh313) mutants worsen during larval and juvenile stages, prompting the adult phenotype.We further show that Sox9b interacts with Notch signaling to regulate intrahepatic biliary network formation: sox9b expression is positively regulated by Notch signaling, while Sox9b function is required to maintain Notch signaling in the intrahepatic biliary cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Program in Developmental and Stem Cell Biology, Liver Center and Diabetes Center, University of California San Francisco, San Francisco, California, United States of America. marion.delous@ucsf.edu

ABSTRACT
The pancreaticobiliary ductal system connects the liver and pancreas to the intestine. It is composed of the hepatopancreatic ductal (HPD) system as well as the intrahepatic biliary ducts and the intrapancreatic ducts. Despite its physiological importance, the development of the pancreaticobiliary ductal system remains poorly understood. The SRY-related transcription factor SOX9 is expressed in the mammalian pancreaticobiliary ductal system, but the perinatal lethality of Sox9 heterozygous mice makes loss-of-function analyses challenging. We turned to the zebrafish to assess the role of SOX9 in pancreaticobiliary ductal system development. We first show that zebrafish sox9b recapitulates the expression pattern of mouse Sox9 in the pancreaticobiliary ductal system and use a nonsense allele of sox9b, sox9b(fh313), to dissect its function in the morphogenesis of this structure. Strikingly, sox9b(fh313) homozygous mutants survive to adulthood and exhibit cholestasis associated with hepatic and pancreatic duct proliferation, cyst formation, and fibrosis. Analysis of sox9b(fh313) mutant embryos and larvae reveals that the HPD cells appear to mis-differentiate towards hepatic and/or pancreatic fates, resulting in a dysmorphic structure. The intrahepatic biliary cells are specified but fail to assemble into a functional network. Similarly, intrapancreatic duct formation is severely impaired in sox9b(fh313) mutants, while the embryonic endocrine and acinar compartments appear unaffected. The defects in the intrahepatic and intrapancreatic ducts of sox9b(fh313) mutants worsen during larval and juvenile stages, prompting the adult phenotype. We further show that Sox9b interacts with Notch signaling to regulate intrahepatic biliary network formation: sox9b expression is positively regulated by Notch signaling, while Sox9b function is required to maintain Notch signaling in the intrahepatic biliary cells. Together, these data reveal key roles for SOX9 in the morphogenesis of the pancreaticobiliary ductal system, and they cast human Sox9 as a candidate gene for pancreaticobiliary duct malformation-related pathologies.

Show MeSH
Related in: MedlinePlus