FIGURE 1:

Grhl2 enlarges the central lumen of cysts. (A) Mature cholangiocytes form larger cysts than HPPL. Mature cholangiocytes were isolated from mouse adult liver based on the expression of EpCAM. EpCAM⁺ cholangiocytes and HPPL were cultured in gel containing Matrigel for 7 d. Bar, 50 μm. (B) Effects of transcription factors on the size of a central lumen. HPPL were introduced with retrovirus containing cDNA of Ankrd1, EHF, Hey1, Grhl2, or Scx and used for 3D culture. After 7 d of incubation, pictures were taken for at least 20 areas for measuring the size of cysts. A dot plot with bars of mean ± SEM for a representative culture indicates that the diameter of lumen derived from HPPL expressing Grhl2 (HPPL-Grhl2) is larger than the control with statistical significance. (C) Cyst structures in 3D culture of control HPPL and HPPL-Grhl2. Both HPPL and HPPL-Grhl2 cysts show apical actin bundles (white) and basal β-catenin (green), whereas cysts derived from HPPL-Grhl2 have remarkably larger lumen (4–6) as compared with the control (1–3). The control HPPL and HPPL-Grhl2 were cultured in gel containing Matrigel for 7 d. Cysts were incubated with anti-Grhl2 and anti–β-catenin antibodies, followed by incubation with secondary antibodies and Alexa Fluor 633–conjugated phalloidin. Nuclei were counterstained with Hoechst 34580. BF, bright field. Bars, 50 μm. (D) Immunofluorescence analysis of Grhl2 expression in developing liver. In E17.5 liver (1–3), Grhl2 (red) is not detectable in EpCAM⁺ cholangiocytes (green), whereas it is expressed in cholangiocytes forming tubular structures in P1 (4–6) and P8 (7–9). Boxes in 1, 4, and 7 are enlarged in 2/3, 5/6, and 8/9, respectively. Bars, 50 μm (1, 4, and 7), 10 μm (2, 3, 5, 6, 8, and 9).