DEV029140 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Adjacent sections showing continuity of primitive ductal structures. Four sequential 5 µm sections were stained for Ck19 and Hnf4α. Asterisks mark two asymmetric tubules that converge over the span of this 20 µm series. pv, portal vein.

• Supplemental Figure S2A -

• Supplemental Figure S2B -

  Fig. S2. EM analysis of ductal morphogenesis. (A) E17.5. The outer and inner layer cells lining asymmetric tubules have distinct ultrastructural features, although the two cell types exhibit a strong overall similarity. The outer layer cells (h) are characterized by high glycogen content (arrowheads, top panels) and rounded nuclei, whereas inner layer cells (b) lack glycogen and tend to have more irregularly shaped nuclei. Tight junctions (arrow) were observed between adjacent inner layer cells and between inner and outer layer cells (not shown). e, endothelial cell; g, glycogen. Scale bars: black, 4 µm; white, 1 µm. (B) P2. Symmetrical bile ducts are seen at this stage (b, top left panel), along with some lumens that remain asymmetrically lined by hepatocytes and BECs (asterisk, upper right panel). BECs exhibiting degenerative changes can be seen (arrows, top panels). Hepatocytes are easily distinguished from BECs at this stage by their larger cell size, larger and more rounded nucleus, and high mitochondrial (m) content. BECs exhibit tight junctions (arrowheads), basal bodies of cilia (white arrow), irregularly shaped nuclei and abundant free ribosomes. These images are representative of multiple areas. pv, portal vein. Scale bars: black, 2 µm; white, 1 µm.

• Supplemental Figure S3 -

  Fig. S3. Expression of Notch signaling components as detected by real-time PCR. Total RNA was isolated from livers at the indicated time points and first-strand cDNA was used as template for real-time PCR amplification using specific primer pairs from a panel of Notch ligands, receptors and Hes/Hey target genes. Standard curves of high quality were generated from all primer pairs except for Dll3, which was hence considered non-expressed (not shown). Data are presented as mean (±s.e.m.) normalized to Hprt; the relative expression level at E14.5 was assigned a value of 1. Data were collected from three livers at each time point.

• Supplemental Figure S4 -

  Fig. S4. Hes1 immunofluorescence in liver from wild-type mice at the indicated time points. Hes1 is expressed broadly in the ductal plate at E16.5. Its expression is confined to bile ducts postnatally, where it remains expressed in adult liver. Scale bar: 50 µm.

• Supplemental Figure S5 -

  Fig. S5. Timing and specificity of Cre strains used in this study. All Cre strains were mated to Rosa^YFP mice and labeling was detected using an anti-YFP antibody. Numbers next to panels indicate the efficiency of recombination in hepatoblasts, measured as the percentage of Hnf4α⁺ cells that were YFP-positive at the indicated time point. Insets show Hnf4α⁺ (green) and YFP (red) co-immunofluorescence for selected pairs; note the presence of numerous Hnf4α-negative hematopoietic cell nuclei. (A) Foxa3-Cre mediates early and robust loxP deletion in the embryonic liver (35% of hepatoblasts labeled at E13.5; 81% of hepatoblasts labeled at E15.5). (B) In AFP-Cre; Rosa^YFP mice, only 36% of hepatoblasts were labeled at E15.5; however, by E16.5, 88% of hepatoblasts were labeled. By P2, 95% of hepatocytes (98% of Ck19⁺ BECs; arrowheads) were labeled in AFP-Cre; Rosa^YFP mice, underscoring their hepatoblast origins. (C) Albumin-CreER mediates hepatocyte-specific recombination postnatally. P6 Albumin-CreER; Rosa^YFP mice treated with TM exhibit widespread and specific labeling of hepatocytes but no BEC labeling (arrowhead).

• Supplemental Figure S6 -

  Fig. S6. Additional characterization of Foxa3-RBP and Foxa3-NICD mutants. (A) Foxa3-RBP livers have a biliary phenotype at E16.5 and P0. The number of Ck19⁺ cells per portal vein (pv) was quantified as described for Sox9⁺ cells in Fig. 3. Foxa3-RBP animals exhibit a trend towards a reduced number of Ck19⁺ cells at E16.5 and P0, although inter-animal variability was observed (P=NS; four animals examined from each genotype at each time point). (B) Compared with controls (a), Foxa3-NICD embryos exhibit widespread ectopic BECs in the hepatic lobules at E17.5 (b,c). In some areas, ectopic cells are arranged in a ductular conformation (arrowheads, d-f). Scale bars: 100 µm.

• Supplemental Figure S7 -

  Fig. S7. Additional characterization of dose-dependent tubulogenesis in AFP-NICD and AFP-N/N animals. (A-C) Portal tract images from animals of the indicated genotype at E16.5. Increased Notch dosage at this stage is associated with increased tubulogenesis, particularly in the ductal plate region (C, arrowheads), but not with ectopic BECs. (D-F) Images from the lobules of P15 animals of the indicated genotypes. In control livers (D), Ck19 expression is limited to the portal tract (inset). In AFP-NICD mice (E), small cords of BECs or isolated Ck19⁺ cells are found in the lobule, replacing the lobular tubules present perinatally (Fig. 6). By contrast, tubules persist in the lobules of AFP-N/N mice, forming mature-appearing bile duct structures (F). Scale bars: 200 µm in A-C; 100 µm in D-F.

• Supplemental Figure S8 -

  Fig. S8. AFP-NICD (AFP-N) and AFP-NICD/NICD (AFP-N/N) mice exhibit preserved liver chemistries. Serum was obtained from 2-month-old AFP-N, AFP-N/N, or control mice (lacking Cre) and tested for the indicated analytes. Each dot represents an individual animal. Dashed lines indicate the upper limit of normal (for mice) provided by the laboratory (Analytics). Note that the two AFP-N/N mice are the only animals observed to have an undetectable total bilirubin.

• Movie 1 -

  Movie 1. Confocal images of asymmetric tubules. A 30 µm section of liver from a wild-type E16.5 mouse was stained for Ck19 (red) and WGA lectin (Vector) to outline cell boundaries (green). Images from 0.8 µm optical sections were collected to a total depth of 19 µm (when signal intensity became faint) and compiled as a z-stack movie. A single asymmetric primitive ductal structure can be seen in the center of the field.

• Movie 2 -

  Movie 2. Confocal images of asymmetric tubules. A 30 µm section of liver from a wild-type E16.5 mouse was prepared as described for Movie 1. An asymmetric tubule can be seen in the center of the field in early sections; a second asymmetric tubule becomes visible in the upper left corner later in the z-stack. Total depth: 17 µm.