More Related Content
What's hot
What's hot (14)
Similar to Aasld boston nov 2010 4
Similar to Aasld boston nov 2010 4 (20)
More from cpbv13
More from cpbv13 (14)
Recently uploaded
Recently uploaded (20)
Aasld boston nov 2010 4
- 1. Genetic Interaction of Hepatocyte Nuclear Factor 6 and Notch Signaling Within the Liver Charles Vanderpool, MD1 Erin Sparks2, Kari Huppert2 Stacey Huppert, PhD2 1D. Brent Polk Division of Pediatric Gastroenterology, Hepatology, and Nutrition 2Department of Cell and Developmental Biology and Center for Stem Cell Biology
- 2. Disclosures I have no financial relationships to disclose within the past 12 months relevant to my presentation My presentation does not include discussion of off-label or investigational use of medications
- 3. Cholangiopathies and Bile Duct Development • Pediatric cholestatic liver disease – Biopsy findings and clinical course can differ amongst patients with similar intrahepatic bile duct (IHBD) defects – Alagille Syndrome: phenotypic variance despite defined genetic alterations in Notch signaling • Clinical variance could be caused by alterations in signaling pathways responsible for various steps in ductal development Hepatocytes Cholangiocytes Hepatoblasts IHBD System Bile Duct Portal vein Specification Morphogenesis Maintenance
- 4. Specification Morphogenesis Maintenance HNF-1β HNF-6 Loss3 Global HNF6 null 75% mortality Notch loss1,2 Liver-specific Conditional RBP-jκflox/flox ✖ ✖ Notch and Hepatocyte Nuclear Factor-6 25% survivors Normal IHBDs Paucity of IHBDs Chronic cholestasis ??? 2,4 HNF-1β ✖ ✖ 1Sparks et al. Hepatology 51(2), 2010 2Zong et al. Development 136, 2009 3Clotman et al. Development 129, 2002 4Tanimizu et al. Journal of Cell Science 117, 2004
- 5. Questions • In setting of chronic cholestasis induced by Notch signaling loss – Does loss of HNF-6 alter the phenotypic severity? • In the setting of loss of both HNF-6 and Notch signaling – Will HNF-1β expression remain altered?
- 6. Mouse Models • HNF-6 KO: Alb:Cre, HNF-6flox/flox (Maureen Gannon) • RBP KO: Alb:Cre, RBP-jκflox/flox (Tasuku Honjo) • DKO: HNF-6 and Notch loss – Alb:Cre, HNF-6flox/flox, RBP-jκflox/flox Hepatocytes Cholangiocytes Hepatoblasts Hepatoblast-specific recombination driven by Albumin-Cre recombinase
- 7. Hepatoblast-specific deletion of HNF-6 AgeP0 HNF-6 Immunostain Control HNF-6 KO
- 8. Deletion of both HNF-6 and RBP results in hepatic fibrosis and severe cholestasis Control HNF-6 KO RBP KO DKO Gomori Trichrome Stain P60
- 9. DKO enhances phenotypic decrease in 3D IHBD density seen with RBP loss alone Intrahepatic Ductal System Resin Cast Benzyl Alcohol - Benzyl Benzoate Tissue Clearing HNF-6 KO P60 Control RBP KO DKO
- 10. Loss of HNF-6 and RBP causes severe impairment in ductal development P15Hilum Control DKO Wide-Spectrum Cytokeratin Immunostain P15Periphery * * * *
- 11. Loss of HNF-6 and RBP alters expression of hepatocyte transcription factor HNF-1β
- 12. Conclusions • Loss of HNF-6 worsens the phenotypic severity of cholestatic liver disease seen with Notch signaling loss • This may result from impaired redundant signaling pathway compensation during IHBD development • HNF-1β may be leading candidate for common downstream mediator1 1Coffinier et al. Development 129, 2002
- 13. Acknowledgements Funding Mouse Lines Albumin-Cre: Mark Magnuson, MD HNF-6: Maureen Gannon, PhD RBP-jκ: Tasuku Honjo, MD, PhD Huppert Lab Stacey Huppert, PhD Kari Huppert Erin Sparks Teagan Walter
Editor's Notes
- The main interest of our lab is the genetic control of intrahepatic bile duct development and maintenance and how this relates to Pediatric chronic cholestatic liver disease. In Pediatric patients with chronic cholestasis related to abnormalities of intrahepatic bile ducts: Biopsy findings and clinical course can differ amongst patients despite having similar intrahepatic bile duct (or IHBD) defects Perhaps the best example of this are patients with Alagille syndrome. Alagille syndrome has been well characterized to arise from defined mutations in Notch signaling. However, phenotypic variance can be seen in familial cohorts with the same genetic defect. This clinical variance in patients with intrahepatic bile duct disease could be caused by alterations in the signaling pathways that control the various steps in ductal maturation and maintenance. Below is a very simplified drawing of the complex process that leads to the mature intrahepatic biliary system. For simplicity, I will refer to specification as the cell fate decision within the bipotential hepatoblasts, morphogenesis as mutiple steps involved in initial bile duct development, and maintenance as the intact mature intrahepatic bile duct system.
- 2 signaling pathways that play an important role in this step-wise process of intrahepatic bile duct development are Notch signaling and HNF-6. There have been various experimental models describing the effects of Notch signaling loss within the liver. One that has been described by our group and others is Notch loss through liver-specific conditional loss of RBP-jk. RBP-jk, which stands for Recombination Binding Protein j kappa, is the DNA binding portion of the Notch transcription complex and is a necessary effector of canonical Notch signaling through all 4 Notch receptors. Depending on timing of deletion, loss of RBP has been shown to affect specification and morphogenesis. This leads to paucity of intrahepatic bile ducts and chronic cholestasis. While there are multiple models for conditional Notch signaling loss within the liver, HNF-6 has largely been studied within the liver in the setting of global loss Global loss of HNF-6 also causes abnormalities in both specification and morphogenesis during bile duct development. With global loss of HNF-6, there is an associated 75% early postnatal mortality related to both pancreatic and liver disease. Also during the embryonic time period, there is down-regulation of HNF-1b levels and HNF-6 was shown to transcriptionally regulate HNF-1b expression. HNF-1b levels begin to return to normal levels during late embryonic time points and postnatally in the global HNF-6 null mouse. Of the 25% of mice that survived into adulthood, they were described to progress to a normal intrahepatic bile duct phenotype. Possible interaction between these two signaling pathways has been suggested through separate experiments. Notch has been shown to upregulate HNF-1b levels in mouse models where Notch is constitutively active. This led us to question if Notch could contribute to HNF-1b levels returning to normal.
- This previous work led us to form the following questions…
- To study these questions, we utilized experimental models with hepatoblast-specific recombination driven by Albumin-Cre recombinase. In this model, cre recombinase is expressed within the hepatoblast population beginning at embryonic day 14.5. Pictured here is rosa-reporter analysis of albumin-cre expression. By embryonic day 16, nearly all hepatoblasts are X-gal positive. Post-natally, both cholangiocytes (seen here in this bile duct) and hepatocytes are positive for X gal staining, indicating that recombination occurred in the hepatoblast population as mature cholangiocytes do not express albumin. The 3 mouse models we have studied are listed here. We have evaluated mice with: -- HNF-6 signaling loss alone, labeled HNF-6 KO -- Notch signaling loss alone, labeled RBP KO -- Loss of both HNF-6 and Notch signaling, labeled DKO
- Conditional deletion of RBP in a similar background has been previously reported. Conditional deletion of HNF-6 was confirmed both by immunostaining and RT-PCR analysis of mRNA levels. As seen here, there is near complete loss of HNF-6 staining in HNF-6 KO mice at postnatal age P0, while it is present both in periportal cholangiocytes and parenchymal hepatocytes. As mentioned in the previous slide, cre expression with Albumin-cre begins at approximately e14.5. Initial hepatoblast specification begins earlier, at approximately embryonic day 13. Given this, we did not see a statistically significant decrease in HNF-6 expression at embryonic day 16.5. However, immunostaining for HNF-6 at postnatal day 0 showed that HNF-6 expression was nearly completely lost in KO animals, and mRNA expression was confirmed to be downregulated postnatally as well.
- Pictures here are representative images of trichrome stain for collagen deposition. Directed deletion of HNF-6 alone failed to demonstrate any change from control mice. At age P60 there was not a consistent increase in collagen deposition with isolated RBP loss. However, mice deficient for both HNF-6 and RBP demonstrated early hepatic fibrosis with areas of bridging visible between portal tracts. The level of cholestasis in DKO mice was seen to be significantly worse at age postnatal day 60 compared to control and single deletion experimental groups. (Unpaired 2 tail T test using Welches correction, P value reached significance for RBP vs DKO)
- These pictures show resin casts of the intrahepatic bile duct system. Synthetic resin is injected retrograde into the common bile duct and allowed to harden within the liver. The liver tissue is then cleared with benzyl alcohol/benzyl benzoate to allow for visualization of the branching pattern within the liver tissue. HNF-6 loss failed to show an appreciable difference in intrahepatic bile duct density compared to control. As has been described, RBP loss is associated with a decrease in peripheral IHBD branching. However, in DKO mice, loss of HNF-6 signaling in the setting of Notch signaling loss resulted in a consistent worsening of this decrease in density of IHBDs.
- At embryonic day e16.5, the amount of cytokeratin positive cells did not differ significantly amongst control and DKO mice at age embryonic day 16.5 (which is not shown). This was similar to what has been seen with isolated RBP loss. Pictured here is wide spectrum cytokeratin staining for postnatal age P15 with control compared to DKO. In control mice, peripheral bile ducts are seen here, occuring in normal number associated with portal veins. As previously published, loss of RBP leads to a decrease in number of peripheral bile ducts. However, with loss of both HNF-6 and RBP, at postnatal day 15 there is near complete loss of peripheral bile ducts, consistent among 5 different animals at this age. Pictured here is a representative image of the hepatic periphery in a DKO animal – there are no visible cytokeratin positive cells. There is widespread areas of hepatic necrosis seen within this age group – shown here, possibly related to cholestatic liver damage. At the hilum, there are formed ducts present in DKO animals. The majority of ducts at the hilum are smaller compared to similar areas in control animals.
- Loss of both HNF-6 and RBP leads to significant downregulation of HNF-1b mRNA expression, not seen with loss of either HNF-6 or RBP alone. At agee16.5, there is a significant increase in HNF-1b expression seen with both loss of HNF-6 and RBP alone. However, this increase is not maintained postnatally compared to control. Loss of both HNF-6 and RBP is seen to cause a significant decrease in expression that is maintained postanatally, suggesting that there is an interaction between the two signaling pathways with regards to HNF-1b expression. There was not a consistent change seen with expression patterns of HNF-4a or OC-2, and we are expanding evaluation of other possible transcription factors in these age groups that may be affected.
- NICD: Doug Melton, PhD