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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?
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
*
*
*
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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
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.
