Additional Supporting Information may be found in the online version of this article.

HEP_26254_sm_SuppFig1.tif273KSupporting Information Figure 1. Schematic illustration of the mouse strains used.
HEP_26254_sm_SuppFig2.tif8554KSupporting Information Figure 2. Additional information for R26N2ICAlbCre mice with conditional expression of N2IC in hepatoblasts. (A) At P0 N2IC expression in hepatoblasts of R26N2ICAlbCre animals leads to architectural disruption of the entire liver (a, inset enlarged in b). While N2IC-expressing cells form predominantly tubular structures in the center of the liver lobe (b, left inset enlarged in c), tubular-cystic and microcystic structures are observed in the periphery of the liver lobe (b, right inset enlarged in d). (B) HNF4α expression is dramatically reduced in R26N2ICAlbCre livers at P0 with only few HNF4α-positive cells with typical hepatocyte morphology remaining (arrowheads). (C) Co-immunofluorescence for Sox9 and HNF4α demonstrates loss of HNF4α in nearly all Sox9-expressing cells, while co-expression of Sox9/HNF4α is a rare event (asterisk). Few HNF4α-positive/ Sox9-negative cells were observed that have presumably escaped Cre-driven recombination (arrowheads). Scale bar in panel (A) = 1000 μm, scale bar in panel (B) = 100 μm and 25 μm for magnification, scale bar in panel (C) = 25 μm.
HEP_26254_sm_SuppFig3.tif9708KSupporting Information Figure 3. Conditional expression of N2IC in embryonic hepatoblasts results in persistence of ectopic bile ducts with cholangiocytic tumor formation in surviving R26N2ICAlbCre animals. Crossing heterozygous AlbCre animals with R26N2IC animals yielded two double transgenic animals (R26N2ICAlbCre) out of 80 animals genotyped after P20 (= 2,5 %, expected Mendelian frequency 25 %). (A) Left panel: Macroscopic view at a surviving R26N2ICAlbCre mouse at P30 displaying hepatomegaly and widespread yellowish irregular lesions. Right panel: HE staining of the liver reveals abnormal bile ducts with multicystic cholangiocellular tumor formation reminiscent of biliary hamartoma. (B) Upper panels: Immunostaining for N2IC, Sox9 and E-cadherin shows strong reactivity for biliary markers. Lower panels: Biliary cystic tumors with surrounding reactive stroma (asterisk) displayed both regular cuboidal (right inset) but also dysplastic epithelial cells with nuclear crowding and pseudostratification (left inset, enlarged with E-cadherin and Ki67 staining). (C) In some parts of the liver there are also areas with apparently normal hepatocytes that do not express N2IC (arrows) and Sox9. (D) Surviving R26N2ICAlbCre mice at P30 display hepatomegaly (n=5 for control, n=2 for surviving R26N2ICAlbCre). Scale bar in panel (A) = 1000 μm, scale Bar in panel (B) = 50 μm (upper) and 200 μm (lower) for HE stainings, 25 μm for IHC stainings, Scale bar in panel (C) = 200 μm for HE staining and 25 μm for IHC-stainings.
HEP_26254_sm_SuppFig4.tif18306KSupporting Information Figure 4. Time-dependent portal-to-central development of biliary tubular-cystic structures in R26N2ICMxCre animals. R26N2ICMxCre were treated with 10 μg or 2.5 μg (“low dose pIC”) /g BW i.p. for 6 and 10 days as indicated in the Figures. (A) HNF4α and N2IC immunostainings in serial sections reveal time-dependent progression of the N2IC-expressing tubular-cystic structures towards the central vein in R26N2ICMxCre animals with progressive loss of the hepatocyte marker HNF4α (panels “pIC 6 days” and “pIC 10 days”). After “low dose pIC” treatment for 10 days (right panels) N2IC-positive tubular and tubular-cystic structures appearing both in the periportal and pericentral area have almost exclusively lost HNF4α (arrowheads). (B) 6 days after pIC-treatment many cells co-stain for Sox9 and HNF4α while 10 days of pIC treatment results in loss of HNF4α in nearly all Sox9-positive cells as assessed by co-immunofluorescence for Sox9 (green) and HNF4α (red). PV, portal vein. CV, central vein. BD, bile duct. Scale bar in (A) = 100 μm and 25 μm for higher magnification.
HEP_26254_sm_SuppFig5.tif4930KSupporting Information Figure 5. Additional analysis establishing the HNF1?CreERT2 mouse strain to specifically drive Cre expression to the biliary compartment. (A) Representative immunofluorescence co-staining of a 6 week-old WT liver for HNF1β and Sox9 as indicated. HNF1? co-localizes with Sox9 in bile ducts and periportal ductules (arrows). The outlined area is magnified in the lower panels. (B) Co-immunofluorescence for td-Tomato (arrows), Sox9 and HNF4α in 6 week-old R26TomHNF1?CreERT2 reporter mice 7 days after tamoxifen injection indicate Cre expression to be strictly confined to the biliary compartment. PV, portal vein. BD, bile duct. Scale bar = 20 μm.
HEP_26254_sm_SuppFig6.tif3325KSupporting Information Figure 6. Additional information for mice with conditional deletion of Hes1 in Hes1F/FAlbCre and RBP-J in RbpjF/FAlbCre animals. (A) Immunostaining for HNF1β in Hes1F/FAlbCre mice at P1 reveals normal remodeling of the ductal plate with regular tubule formation indistinguishable from control animals. The outlined areas are magnified in the lower panels. (B) Effective recombination of floxed Hes1 alleles in Hes1F/FAlbCre mice was confirmed by real-time RT-PCR at P1 and does not indicate compensatory increase of transcripts of other Notch target genes. (C) Representative panCK staining of livers of RbpjF/FAlbCre mice at P20 accentuates cells of intermediate morphology with immature irregular tubule formation. Real time RT-PCR data shown in (B) are mean ± SD (n= 4 for each genotype). Scale bar in panel (A) = 100 μm and 25 μm in the magnified panels. Scale bar in (B) = 50 μm.
HEP_26254_sm_SuppFig7.tif1699KSupporting Information Figure 7. Cre-driven deletion of Hes1 precedes morphogenetic changes in R26N2ICHes1F/FMxCre animals. (A) 4 days after pIC injection into R26N2ICHes1F/FMxCre animals broad ectopic Sox9 expression as a surrogate for N2IC expression is observed in hepatocytes preceding formation of tubular-cystic structures. (B) At this time Hes1F/F alleles are effectively deleted by Cre-driven recombination as assessed by real-time RT-PCR (n = 2) and compared to equally treated Cre-negative controls (n=4). Scale bar in panel (A) = 100 μm and 25 μm respectively.
HEP_26254_sm_SuppFig8.tif37KSupporting Information Figure 8. Expression of recognized Notch-target genes in N2IC-expressing livers after concomitant inactivation Hes1. mRNA expression levels of the Notch-target genes Hey1, HeyL, and Hes5 were analyzed by real-time RT-PCR analysis of P0 liver tissue from control, R26N2ICAlbCre, and R26N2ICHes1F/FAlbCre animals (n= 4-5). mRNA levels in R26N2ICAlbCre and R26N2ICHes1F/FAlbCre livers are expressed as relative expression changes over control.
HEP_26254_sm_SuppInfo.doc40KSupporting Information
HEP_26254_sm_SuppTables.doc77KSupporting Information Tables: Mouse strains used in this study

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.