Genetic Interaction of HepatocyteNuclear 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 Nutrition2Department of Cell and Developmental Biology and Center for Stem Cell Biology

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

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

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% survivorsNormal IHBDs

Paucity of IHBDsChronic cholestasis

??? 2,4

HNF-1β

✖ ✖

1Sparks et al. Hepatology 51(2), 20102Zong et al. Development 136, 20093Clotman et al. Development 129, 20024Tanimizu et al. Journal of Cell Science 117, 2004

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?

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 recombinationdriven by Albumin-Cre recombinase

Hepatoblast-specific deletion of HNF-6A

ge P

0

HNF-6 Immunostain

Control HNF-6 KO

Deletion of both HNF-6 and RBP results in hepatic fibrosis and severe cholestasisControl HNF-6 KO RBP KO DKO

Gomori Trichrome Stain

P6

0

DKO enhances phenotypic decrease in 3D IHBD density seen with RBP loss alone

Intrahepatic Ductal System Resin CastBenzyl Alcohol - Benzyl Benzoate Tissue Clearing

HNF-6 KO

P6

0

Control RBP KO DKO

Loss of HNF-6 and RBP causes severe impairment in ductal development

P1

5 H

ilum

Control DKO

Wide-Spectrum Cytokeratin Immunostain

P1

5 P

erip

her

y

**

**

Loss of HNF-6 and RBP alters expression of hepatocyte transcription factor HNF-1β

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

Acknowledgements

Funding Mouse LinesAlbumin-Cre: Mark Magnuson, MD

HNF-6: Maureen Gannon, PhDRBP-jκ: Tasuku Honjo, MD, PhD

Huppert LabStacey Huppert, PhD

Kari HuppertErin Sparks

Teagan Walter