Figure 1.
Dual immunofluorescence for EPM and GGT in liver frozen sections.
Adult male C57Bl/6 mice were given a single intraperitoneal 10% solution in corn oil injection of CCl4 (10 µl/g body weight) or the same volume corn oil as a control. The CCl4-treated mice (A) or normal control mice (B) were sacrificed to obtain liver tissues at day-7, which were cut into 8 µm frozen liver sections. Biliary epithelial cells were specifically stained with GGT (red). EPM (green) was strongly expressed in the mesenchyme surrounding GGT-positive BD. The diameter of the BD: (A) 11.52–14.04 µm; (B) 33.38–80.24 µm. PV = portal vein. BD = bile ducts. Pictures on the right are magnifications of pictures on the left. The BD was shown in the square region. The diameter of the BD was measured by Image-Pro Plus 6.0 software (Media Cybernetics, MD, USA). Bars = 100 µm.
Figure 2.
The morphogenesis and hepatobiliary genes analysis of the WB cells treated with recombinant EPM.
(A) Confocal images of EPM-induced duct-like structures. The PLL was used as a matrix control, coated with EPM or alone. The duct-like structures appeared 24 hours to 48 hours after cell seeding. The formation of these duct-like structures could be blocked by the EPM-neutralizing antibody with the purified rabbit IgG as a control. Bars = 100 µm. Tubulin, green. F-actin, red. Nuclei, blue. (B) Brightfield images of EPM-induced WB cells. The typical duct-like structures appeared at day-2. The inset shows a magnified view of a duct-like structure and normal WB cells. Individual duct-like structures developed into cord-like structures at day-7 while the WB cells cultured on EPM-free surface remained polygonal and small. Bars = 500 µm (Bars in inset = 100 µm).(C) RT-PCR analysis of WB cells treated with or without EPM at day-7. Lane C: water was substituted for the template as a negative control. Lane 1, cultured on EPM-coated dishes. Lane 2, cultured on EPM-free dishes. Lane3, adult rat liver (liver samples of AFP were from E17.5 rats). The 25 cycle β-actin bands were used as an internal control for semi quantitative RT-PCR. (D) Western blot analysis of the expression of 40-kDa CK19. 42-kDa β-actin was used as an internal control. An increased expression level of CK19 was detected in the EPM-treated cells.
Figure 3.
Microfabrication of lines and the effects of patterned EPM on WB cell orientation during mitosis.
(A) A schematic of the method used to fabricate EPM protein lines on coverslips. Using standard photolithography methods, microarrays of parallel lines (10 µm width, 40 µm spacing)) were manufactured on borosilicate glass wafers. The photoresist was spun onto a borosilicate wafer and exposed to UV light through an optical mask containing the desired pattern to degrade the photoresist (a–f). EPM solution or a mixture of EPM and PLL (for the blocking experiment) was deposited on the patterns (5 µg/cm2) which were dried at room temperature and then stored overnight. The remaining photoresist was dissolved in acetone (g). Bovine serum albumin and PLL on the same micropatterned substrate were used as control. (B) Observations and analyses of direction of division of cells grown on the EPM lines 24 hours after seeding. Before seeding on the micropattern, WB cell division was synchronized using a double-thymidine block at 2 mM. Only the individual growing cells at metaphase and telophase were counted, while cell clusters were excluded from counting. The MO of the cells was determined by the line of nucleus centers or the perpendicular line of the equatorial plate. Dividing cells were visualized by confocal laser scanning microscope. The arrowheads in the merged images and brightfield images indicated EPM line directions. Brightfield images illuminated the cells grown on the micropatterns. These cells were divided into 9 groups (a 10°-wide sector for each group) according to the angles between the MO and the line directions. The polar coordinate charts showed that the MO of the cells on EPM (cell number: n = 192) was predominantly in the direction of the line in contrast to a rather random distribution of MO of the cells on the PLL (cell number: n = 82) patterns. The MO of the La-A-treated cells on EPM patterns was measured by phase-contrast images in which metaphase plates were easily visible. Cells treated by La-A (cell number: n = 83), EPM-neutralizing antibody (cell number: n = 68), and β1 integrin-neutralizing antibody (cell number: n = 54), all demonstrated a rather random distribution in their MO. Bars, brightfield images: 100 μm, confocal images: 50 μm.
Figure 4.
Effects of F-actin, β1 integrin, focal adhesions and MO on duct formation.
(A) Confocal images of stress fiber (F-actin) alignment of WB cells treated with EPM and PLL. Bars = 50 µm. (B) For inhibition experiments, the duct-like structures that formed were treated with La-A at 0.5 µM, which can disrupt microfilament polymerization, for 2 hours before the cells were fixed. For β1 integrin-neutralizing experiments, the WB cells had a pre-incubation with anti-β1 integrin antibody or armenian hamster IgG for 30 minutes at 37°C before they were seeded with antibodies together. Bars = 100 µm. (C) Confocal images of focal adhesions of the WB cells. Focal adhesions were visualized by vinculin (green) 4 hours after cell seeding. Bars = 50 µm. (D) A typical WB cell in division in a duct-like structure induced by EPM. The image in the right is a magnification of the image on the left. The inset shows a magnified view of the spindle. Bars = 50 µm.
Figure 5.
Hypothetical model for mechanisms of action of EPM on duct formation by WB cells in vitro.
EPM: Epimorphin. Unlike the control groups on PLL, cell contact with EPM showed a rapid induction of focal adhesions of which αvβ1 integrin, the EPM receptor, is one of the components. Our results showed that EPM had an effect on stress fiber alignment, MO and bile duct-like structure formation, when compared with the PLL control. Proof of the effects on MO and DF were given in our EPM neutralizing experiments. FA: Focal adhesion. Stress fibers in the cytoplasm are connected to the β1 integrin-containing FA on the cellular membrane. Our data and those of others suggest that blockade of αvβ1 integrin results in disoriented spindles [35] and a loss of key processes in cyst formation [42]. FAK and Src, as components of focal adhesion, are downstream molecules in the EPM biochemical signaling pathway [42]. SF: Stress fiber. The guidance of MO by a patterned EPM protein can be blocked by La-A, which suggests an important role of SFO in guiding the mitosis orientation of WB cells. The fact that the high correlation between MO and the uniaxial static stretch direction can be blocked by La-A provides additional evidence that the SFO determines MO. Disassembly of stress fibers can also block the duct formation induced by EPM. MO: Mitosis orientation. Radisky et al. proposed that EPM might be involved in duct formation by guiding MO [4]. The present study shows that all MO blockers used in our experiments (EPM neutralizing antibody, β1 integrin neutralizing antibody, and La-A) can also suppress the bile duct-like structure formation induced by EPM. DF: Duct formation. In vivo results demonstrated that EPM is involved in bile duct formation in CCl4-injured or healthy adult mice (Fig. 1). Our analysis indicates that these ducts are biliary ducts rather than hepatocytic ducts (Fig. 2).
Table 1.
Primers and conditions used for RT-PCR.