Panel A shows a schematic timeline of mouse liver development from hepatoblasts to hepatocytes across embryonic stages. Panel B shows a schematic illustration of bile canaliculus formation, highlighting adherens junctions, tight junctions, and apical membrane organization. Panel C shows violin plots of E-cadherin and N-cadherin gene expression levels during different stages of liver development. Panel D shows immunofluorescence micrographs of liver tissue stained for E-cadherin, N-cadherin, ZO-1, and nuclei, with enlarged views highlighting cadherin colocalization at cellular junctions. Panel E shows immunoblot images of cadherin protein expression across developmental stages with beta-actin controls. Panel F shows bar graphs comparing normalized E-cadherin and N-cadherin expression levels and their expression ratios during bile canaliculus biogenesis.
Dual expression of E-cadherin and N-cadherin in hepatoblasts and hepatocytes during mouse liver development. (A) Schematic of mouse hepatocyte differentiation from hepatoblasts during liver development. Each circle shows the distribution of the PCA plot at each embryonic day based on the data from scRNA-seq analysis (Yang et al., 2017). Hepatoblast to hepatocyte differentiation occurs around E14.5 (Yang et al., 2017). (B) Schematic of tubular BC formation. Oriented cell division of a cell with a primordial BC partitions the preexisting lumen into two daughter cells, resulting in a tubular BC surrounded by three or more cells. (C) ScRNA-seq analysis of cadherin genes during mouse liver development. Violin plots represent the expression of CDH1/E-cadherin and CDH2/N-cadherin in hepatoblasts and hepatocytes from the scRNA-seq data (Yang et al., 2017). Each dot represents a single cell. The black line within each violin plot indicates the median expression level. (D) Expression of E- and N-cadherin in hepatoblasts and hepatocytes during liver development. Left: liver sections from different developmental stages were immunostained with antibodies against E-cadherin, N-cadherin, and ZO-1, along with DAPI staining. Right: magnified views of the E-cad/N-cad/ZO-1–positive structures indicated by yellow arrows in the left panels. Yellow arrows denote colocalization of E-cadherin and N-cadherin at cellular junctions, whereas yellow arrowheads indicate that E-cadherin extends beyond N-cadherin at these junctions. Double arrows indicate E-cadherin–positive nascent cell–cell contacts between two daughter cells. See also Fig. S1, A–E. (E and F) Immunoblot analysis of hepatoblasts and hepatocytes at different developmental stages of the mouse liver. (E) Immunoblotting was performed using antibodies against E-, N-, and pan-cadherin, as well as β-actin. Arrowheads indicate E-cadherin corresponding to the lower band of pan-cadherin. MWs of marker proteins are indicated in kDa. (F) Left: normalized expression of E- and N-cadherin during BC biogenesis; the values at E13.5 are set to 1.0. Right: expression ratio of E-cadherin to N-cadherin during BC biogenesis after normalizing pan-cadherin antibody affinities to E- and N-cadherin. Data represent means ± SD from three independent samples. Protein expression levels of E-cadherin and N-cadherin, as well as their expression ratio (E-cadherin/N-cadherin), were analyzed by pairwise t tests with Holm’s correction. See also Fig. S1, F and G. Scale bars, 1 µm (right panels in D except zoomed 2 at E17.5), 5 µm (zoomed 2 at E17.5 in D), 10 µm (left panels in D). P values are indicated in each graph; n.s., not significant. MWs, molecular weights. Source data are available for this figure: SourceData F1.