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

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HEP_23431_sm_SuppFig1.tif2298KSupporting Fig. 1. Alb-Cre-mediated recombination causes BHPC-specific recombination. Panels show X-gal detection of Cre-induced recombination of the ROSA26-LacZ reporter allele in frozen sections with a nuclear fast red counterstain. (A) At embryonic day 16, hepatoblasts (arrow) stain blue indicating recombination of the reporter. Cells that are not X-gal positive are hematopoietic cells, as the liver is the major site of hematopoiesis at this age. (B) At postnatal day 30, the two derivatives of the BHPC-lineage, hepatocytes (arrow) and cholangiocytes (indicated BD), are X-gal positive. Cells that are not X-gal positive are non-BHPC-derived, forming the hepatic arteries, portal veins, central veins, mesenchymal cells surrounding the portal triad or the endothelial and stellate cells in the parenchyma. PV, portal vein. BD, bile duct.
HEP_23431_sm_SuppFig2.tif2926KSupporting Fig. 2. Hes1 mRNA expression changes with dosage modulation of Notch signaling at E16 and P15 in whole liver. The relative expression ratio of Hes1 was calculated using real-time PCR efficiency and the deviation in cycle threshold (Ct) of experimental versus control (without Alb-Cre) samples, expressed in comparison to HPRT mRNA levels. (A) At E16, N1/N2 DKO, RBP KO and NICD show increases in Hes1 mRNA expression, further supporting that Alb-Cre is beginning to express around this stage, and cells begin to compensate for the lack of Notch signaling. (B) By P15, the level of Hes1 has decreased in loss-of-function mutants, and increased in the gain-of-function mutant. Statistical analysis was performed using a one-way ANOVA with Tukey's Multiple Comparison Test. Error bars are standard error of the mean. *P<0.05.
HEP_23431_sm_SuppFig3.tif4774KSupporting Fig. 3. Notch receptor mRNA expression changes as Notch signaling dosage decreases at E16. The relative expression ratio of Notch receptors was calculated using real-time PCR efficiency and the deviation in cycle threshold (Ct) of experimental versus control (without Alb-Cre) samples, expressed in comparison to HPRT mRNA levels. (A,C) At E16, N2 KO and RBP KO demonstrate no affect on Notch receptor mRNA expression. (B) In N1/N2 DKO, N1 and N3 are significantly increased, indicating that deletion is beginning around E16. Statistical analysis was performed using a one-way ANOVA with Tukey's Multiple Comparison Test. Error bars are standard error of the mean. *P<0.05. **P<0.01.
HEP_23431_sm_SuppFig4.tif6804KSupporting Fig. 4. Laser capture microdissection of P120 liver. Paraffin sections from the left lobe of control (N1flox/flox;N2flox/flox, A,C) and N1/N2 DKO (Alb-Cre; N1flox/flox; N2flox/flox, B,D) mice were rehydrated, Hematoxylin and Eosin stained, and dehydrated. Bile ducts, identified by morphology, from hilum (A-B) and periphery (C-D) [regions defined in Supporting Figure 5B] were dissected (Veritas Microdissection System, CapSure Macro LCM Cap). Representative tissue region before (A-D) and after (A'-D') capture. Dotted lines indicate specific region of capture. DNA was isolated from captured tissue using the Picopure kit (Arcturus). Three slides of two mice per genotype were captured. An average of 84 hilar and 164 peripheral regions were captured per animal and pooled for PCR analysis. Average capture area was 33.3μm and 36.2μm for hilum and periphery respectively. (E-F) Quantitative genomic PCR results of N1 (E) and N2 (F) deletion at the hilum and periphery demonstrate significant deletion at both regions. Primers used are listed in Supporting Table 1. Statistical analysis was performed using a one-way ANOVA with Tukey's Multiple Comparison Test. Error bars are standard error of the mean. *P < 0.05. **P < 0.01.
HEP_23431_sm_SuppFig5.tif9885KSupporting Fig. 5. Morphologic defects of wsCK+ cells in hilar and peripheral liver tissue upon BHPC-specific deletion of N1 and N2. (A) Schematic of the left lobe from the ventral surface. The major branches of the biliary system are represented in orange. Dotted line illustrates a section through the lobe. (B) The representative schematic section of the left lobe is further defined by region, hilum (blue) and periphery (red). These regions indicate where hilar (C-J) and peripheral (K-R) images were obtained. D, dorsal. V, ventral. (C-R) Paraffin sections from control [Alb-Cre] (left panels in hilum and periphery) and N1/N2 DKO (right panels in hilum and periphery) tissue, over a developmental time course, was immunostained with wsCK to mark cholangiocytes and counterstained with Hematoxylin and Eosin. Arrowheads, unresolved ductal plate remnants. Asterisk, necrosis. PV, portal vein. DP, ductal plate. BD, bile duct. Scale bar = 100 μm.
HEP_23431_sm_SuppFig6.tif8971KSupporting Fig. 6. Morphologic changes of wsCK+ cells in hilar liver tissue upon BHPC-specific deletion of RPB-JK or activation of N1. Paraffin sections from control [Alb-Cre] (A,D,G,J), RBP KO (B,E,H,K) and two copies of NICD (C,F,I,L) tissue, over a developmental time course, was immunostained with wsCK to mark cholangiocytes and counterstained with Hematoxylin and Eosin. Images were obtained from the hilum of the left liver lobe as illustrated in Supporting Fig. 5B. (A-F) At E16 and P3, each genotype appears grossly identical, though NICD mice (C,F) have an increase in portal vein associated wsCK+ cells at this time. (I) By P15 an excess of wsCK+ cells become apparent in the parenchyma of NICD animals, which persist at P30 (L). Conversely, in the hilum of RBP KO mice, at P15 (H) and P30 (K), there is mild to moderate inflammation and bile ducts appear tortuous and distorted. Arrowheads, unresolved ductal plate remnants. PV, portal vein. DP, ductal plate. BD, bile duct. Scale bar = 100 μm.
HEP_23431_sm_SuppFig7.tif1966KSupporting Fig. 7. Focal regions of necrosis are seen in N1/N2 DKO and RBP KO mice at P30. Paraffin sections from the left lobe of P30 control [Alb-Cre] (A), N1/N2 DKO (B) and RBP KO (C) mice were immunostained with wsCK and counterstained with Hematoxylin and Eosin. Images were obtained from the periphery as indicated in Supporting Fig. 5B. Arrows, wsCK+ cells. Asterisks, regions of necrotic tissue. Scale bar = 100 μm.
HEP_23431_sm_SuppTab1.doc85KSupporting Table 1. Primers used in this study
HEP_23431_sm_SuppTab2.doc28KSupporting Table 2. Primary and Secondary Antibodies used in Immunohistochemistry
HEP_23431_sm_SuppTab3.doc38KSupporting Table 3. Proliferation of wsCK+ cells at Postnatal day 15 and 30. BrdU was either injected 1-hour prior to sacrifice (P15, top) or administered in the drinking water 4 days prior to sacrifice (P30, bottom). N equals the number of mice analyzed by double immunofluorescence of paraffin sections for wsCK and BrdU. Regions of analysis were identified as in Supporting Fig. 5, and total indicates the combination of hilum and periphery. The number of fields counted at 40x is indicated as “Regions analyzed”. The number of double-positive BrdU/wsCK cells was divided by the total number of wsCK-positive cells for each region to obtain the percent proliferation. These regional percentages were averaged to obtain a mean proliferation index (%).

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