21 Human samples (five normal at 11 weeks of gestation [W], two ARPKD at 13W, one ARPKD at 22W, and one patient with HNF1B mutation at 4 days after birth) were formalin-fixed and paraffin-embedded. Mouse liver preparation and immunofluorescence analysis of 5-μm-thick (human) or 9-μm-thick (mouse) sections were as described22 (Supporting Table 1). RNA from mouse liver was extracted

with Tripure RNA Isolation reagent (Roche, Vilvoorde, this website Belgium), and quantitative reverse-transcriptase polymerase chain reaction (Supporting Table 2) was performed with SYBR Green Master Mix Reagent (Invitrogen, Merelbeke, Belgium). For quantification of Sec63, Pkhd1, and Cys1, copy number for each messenger RNA (mRNA) was normalized to β-actin mRNA copy number using standard calibration curves, and reported by reference to control values set at 100%. To NVP-LDE225 cell line investigate how DPMs develop in the absence of HNF6 or HNF1β, we first determined the differentiation status of the ductal cells lining DPMs in livers of Hnf6−/− mice and of mice with liver-specific

inactivation of Hnf1b (Hnf1bloxP/loxP-Alfp-Cre). Because differentiation progresses from the hilum to the periphery, all livers were analyzed near the hilum. At E17.5, the cells lining biliary structures in Hnf6−/− mice did not express the biliary marker sex-determining region Y–related HMG box transcription factor 9 (SOX9), but most cells expressed the hepatocyte/hepatoblast marker

HNF4. Higher medchemexpress E-cadherin (Ecad) expression in biliary cells as compared to parenchymal cells is typical for mouse fetal liver (Fig. 1A). The ectopic expression of HNF4 in biliary structures (arrowheads) was in line with our earlier observation that the lack of HNF6 generates hybrid hepatobiliary cells.23 Such cells were observed on the portal and parenchymal sides of the biliary structures, which suggests that HNF6 is required for differentiation of the two biliary layers. In control mice, expression of the transforming growth factor receptor type II (TβRII) is absent from the portal side of PDS at E15.5 but is detected on their parenchymal side.3 At E17.5, the expression on the parenchymal side progressively waned, leading to ducts with a limited number TβRII-positive cells (open arrowheads, Supporting Fig. 1) and devoid of TβRII at E18.5.3 In the absence of HNF6, expression of TβRII persisted on both sides of the biliary structures at E17.5 (arrowheads, Supporting Fig. 1), and this was again in line with our earlier data at E15.5 which showed elevated expression of TβRII in the liver.23 In the absence of HNF1β at E17.5, differentiation of cells that lined the portal side of the DPM was normal throughout the liver: these cells were SOX9+/HNF4−/Ecadhigh.