Lumen morphogenesis in hepatocytes is accompanied by specific actin structures that interconnect the two lumen-forming cells. (a) Schematic overview of primary Dlk1⁺ hepatoblasts in culture differentiating into hepatocytes and recapitulating BC formation. (b) Differentiated hepatocytes form branched interconnected BC lumina. Immunofluorescence microscopy images of differentiated hepatocytes (Protocol 2, Materials and methods) stained for F-actin with phalloidin–Alexa 488, and for apical markers CD13 and ZO-1. Scale bar: 10 µm. (c) In vitro differentiated (Diff.) hepatocytes express mature hepatocyte markers and down-regulate the hepatoblast marker Dlk1. Heatmap comparing the expression of selected hepatocyte marker genes in primary Dlk1⁺ hepatoblasts (Hepatoblasts), in vitro differentiated hepatocytes (Diff. hepatocytes, Protocol 1, Materials and methods), and control mature hepatocytes isolated from adult mouse livers (Mature hepatocytes). RNA-seq experiment in four biological replicates. (d) Images from live-cell time-lapse microscopy documenting the formation of BC between two differentiating hepatoblasts expressing LifeAct-EGFP. During imaging, the extending tubular lumen displayed a bulge at 27 h from the start of imaging, which was subsequently “reabsorbed.” The insert d’ documents the recovery of the tubule (white star) in temporal resolution 10 min/frame. Note the transverse striped pattern in brightfield and actin channels, which is apparent when the lumen is tubular but not observed within the bulge. Scale bar: 10 µm. See also Video 1. (e) SMLM image of a lumen between two differentiated hepatocytes, actin labeled with phalloidin–Alexa 647. Note the transverse striped actin pattern. Scale bar: 5 µm.