Retina-less eye-cups were prepared as described previously (Susaki and Chiba, 2007; also see Figure 2A). Briefly, eyeballs were placed, cornea side up, on a membrane filter (Millipore, Billerica, MA, USA) in a 35-mm plastic dish (Becton Dickinson, Franklin Lakes, NJ, USA) at one eyeball per dish. The eyeball was cut along the equator, its anterior half was carefully removed, the posterior half (i.e. the eye-cup) was soaked in PBS for ∼1 h, and finally the neural retina was carefully removed to make a RLEC. The RLEC on the filter was transferred into a chamber – a cap of a 1.5-ml sample tube (Assist, Tokyo, Japan) – containing 200 μl of culture medium. After the chamber was closed with the sample-tube body, it was incubated at 25°C. The culture was transferred into another chamber to which fresh medium was added every 5 days. The newt MEM was composed of 80% L-15 (Invitrogen, Carlsbad, CA, USA), 7.5 μg/ml heparin (heparin, sodium salt; 081-00136, Wako, Osaka, Japan), and 5 μg/ml BrdU (Sigma-Aldrich, St. Louis, MO, USA) and was used as the standard culture medium.
In some experiments, the RLEC was cut in half along a longitudinal axis through the optic disk with a razor blade immediately before culture (Figure 3A). In other experiments, a piece of the epithelium was removed from the central region of the RPE in the RLEC before culture as follows: The tip of a micropipette (0.2–10 μl, outer diameter: ∼800 μm; BIO-BIK No. 1088, INA-OPTIKA, Osaka, Japan) was carefully placed on the apical surface of the RPE under a stereomicroscope, a small negative pressure was applied, and then lifted up together with a piece of the epithelium attached (Figure 10B).
To investigate the participation of cell-to-ECM and cell-to-cell contact in the cell-cycle entry, RLECs were incubated in either 0.1% (w/v) elastase solution at 25°C for 1 h or 10 mM EGTA solution at 25°C for 1.5 h, rinsed twice (10 and 35 min) in 80% L-15, and then cultured in the basal medium. The elastase solution was prepared by diluting 1% stock [Elastase – high purity, porcine, EC134; Elastin Products Co., Owensville, MO, USA; stored in 10 mM Tris-HCl (pH = 8.4) at 4°C and used at room temperature (pH = 7.8)] in 80% L-15 and sterilized through a syringe filter (DISMIC-25cs, cellulose acetate, 0.2 μm pore size; Toyo Roshi Kaisha, Ltd., Tokyo, Japan) right before use. The EGTA solution contains (in mM) 115 NaCl, 3.7 KCl, 10 EGTA, 18 d-glucose, 10 HEPES, and 0.001% phenol red (pH was adjusted to 7.5 with 0.3 N NaOH). For mock control of each treatment, 80% L-15 containing 1 mM Tris-HCl, or a newt saline [(in mM) 115 NaCl, 3.7 KCl, 3 CaCl2, 1 MgCl2, 18 d-glucose, 10 HEPES, 0.001% phenol red; pH = 7.5] was used.
To investigate signaling pathways involved in the cell-cycle entry of RPE cells, the following reagents were tested: 50 ng/ml FGF2 (a synthetic C. pyrrhogaster FGF2; stock solution: 10 μg/ml in PBS, −20°C; Susaki and Chiba, 2007), 5 μM U0126 [V1121; Promega, Madison, WI, USA; stock solution: 2 mM in DMSO (D2650; Sigma-Aldrich), −20°C], 10 ng/ml Dkk-1 [1096-DK; R&D Systems, Minneapolis, MN, USA; stock solution: 10 μg/ml in PBS containing 0.1% bovine serum albumin (BSA, A3294; Sigma-Aldrich), −20°C], 500 ng/ml Shh [Human Sonic Hedgehog (C24II), amino terminal peptide, 1845-SH; R&D systems; stock solution: 20 μg/ml in PBS containing 0.1% BSA, −20°C], 40 μM KAAD (KAAD-cyclopamine, K171000; Toronto Research Chemicals, North York, ON, Canada; stock solution: 10 mM in ethanol, −80°C), 5 U/ml thrombin (a bovine thrombin, 605157; EMD Chemicals, Gibbstown, NJ, USA; stock solution: 500 U/ml in PBS, −20°C), and 20 μM PPACK (520222; Calbiochem, Darmstadt, Germany; stock solution: 9.54 mM in 5% acetic acid solution, 4°C; pH was adjusted to 7.4 with 2 N NaOH before administration). These reagents were added to the culture medium from the beginning of culture except for U0126 which was administered from the time at which the eye-cup was soaked in PBS. For mock control of each drug, only the solution to store the drug was administered at the corresponding concentration, pH, and timing.
Counting the BrdU+ cells
Retina-less eye-cup cultures were fixed in 4% paraformaldehyde (PFA) in PBS for 15 h at 4°C and washed thoroughly (PBS, 15 min 0.5% Triton X-100 in PBS, 15 min PBS, 15 min). They were incubated in 0.3% H2O2 diluted with PBS for 20 min, rinsed twice in PBS (5 min each), and then incubated in 2 N HCl for 2 h. After washing thoroughly, they were incubated in a blocking solution [3% goat normal serum (S-1000; Vector, Burlingame, CA, USA)/0.5% TritonX-100 in PBS] containing Avidin D (1:50; Avidin/Biotin blocking kit, SP-2001, Vector) for 2 h. After rinsing twice in PBS, they were incubated in a mouse anti-BrdU antibody (1:400; B2531-2ML, Sigma-Aldrich) diluted with the blocking solution containing Biotin (1:50; Avidin/Biotin blocking kit; Vector) for 15 h at 4°C. After washing thoroughly, they were incubated in a biotinylated goat anti-mouse IgG antibody (1:400; BA-9200; Vector) diluted with the blocking solution for 4 h. After rinsing twice in PBS, they were incubated in a mixture of Avidin and Biotin Complex (Vectastain ABC Elite kit, PK-6100; Vector) for 2 h. After washing thoroughly, they were incubated in a DAB solution (DAB substrate kit, SK-4100; Vector) for 3 min. Finally, the reaction was stopped by washing them in distilled water (DW) for 15 min.
The total RPE cell number in a RLEC culture was counted as follows: the culture was refixed in 4% PFA in PBS for 20 min and rinsed in DW; the RPE-choroid tissue was separated from the sclera by fine forceps and pins under a stereomicroscope and transferred onto a glass slide; the tissue was immersed into 90% glycerol in PBS and mounted under a cover slip; the preparation was placed on the stage of a fluorescence microscope (BX50; Olympus, Tokyo, Japan) and viewed through a filter set (excitation: 460–495 nm, emission: 510–550 nm; U-MWIBA/GFP; Olympus); and RPE cells, which could be identified by their characteristic morphology observed over green autofluorescence of the choroid (see Figure 2C), were counted.
BrdU+ nuclei in the RPE were counted as follows: The cover slip mounted on the RPE-choroid tissue was removed, and the tissue was transferred into DW and rinsed well, incubated in 15% H2O2/1.5% sodium azide (197-11091, Wako) in PBS overnight to bleach their melanin pigments, and rinsed twice in DW; they were transferred into 90% glycerol in PBS on a glass slide and mounted under a cover slip; and the preparation was placed on a microscope stage and the number of brown nuclei (BrdU+) was counted under transmitted light (Figure 2D, E).
Retina-less eye-cup cultures were collected in a tube containing PBS on ice. After the solution in the tube was removed carefully, a lysis buffer [25 mM Tris-HCl (pH = 7.5), 150 mM NaCl, 1 mM EDTA-2Na, 1% Igepal CA-630 (56741, Sigma-Aldrich), 1% protease inhibitor cocktail (P8340, Sigma-Aldrich), 1% sodium deoxycholate (190-08313, Wako), 0.1% SDS (191-07145, Wako)] chilled on ice was poured into the tube at 7 μl/RLEC. The sample was frozen in liquid nitrogen and sonicated at 46 kHz, while thawed, in chilled water for 5 min. After the freeze/thaw/sonication cycle was repeated again, the suspension was centrifuged at 7000 g (4°C) for 10 min. The supernatant was mixed with the same amount of SDS sample buffer [0.5 M Tris-HCl (pH = 6.8), 10% (w/v) SDS, 10% 2-mercaptoethanol (M3148, Sigma-Aldrich), 20% glycerol, 0.5% (w/v) bromophenol blue (021-02911, Wako)], heat-denatured for 5 min, and stored at −20°C until use.
SDS-PAGE and immunoblotting were carried out as described previously (Susaki and Chiba, 2007). The primary antibodies were rabbit anti-MEK1/2 polyclonal antibody (1:400; 9122, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-phospho-MEK1/2 monoclonal antibody (1:500; 9154S, Cell Signaling Technology), rabbit anti-ERK1/2 polyclonal antibody (1:300; p44/42 MAP Kinase antibody, 9102, Cell Signaling Technology), rabbit anti-phospho-ERK1/2 polyclonal antibody (1:500; Phospho-p44/42 MAP Kinase antibody, 9101S; Cell Signaling Technology), and mouse anti-β-actin monoclonal antibody (1:3000; ab6276, Abcam, Cambridge, UK). The secondary antibodies were biotinylated goat anti-rabbit IgG antibody (1:300; BA-1000, Vector) and biotinylated goat anti-mouse IgG antibody (1:300; BA-9200, Vector).
For semiquantitative analysis of protein expression and phosphorylation/activation, the values were measured using a function of Photoshop Extended CS5 (Adobe, San Jose, CA, USA): The mean signal intensity of the protein band against the background was measured in a gray scale image of the immunoblotted membrane with inverted gradation.
Cell viability assays
For the MTT assay, RLEC cultures were incubated in 80% L-15 medium containing 500 μg/ml 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium (MTT; Nacalai Tesque, Tokyo, Japan) at 25°C for 3 h, transferred into a sample tube containing 200 μl DMSO, and sonicated for 3 min. The supernatant containing formazan was collected by centrifugation at 12 000 rpm for 5 min and examined for its absorbance at 570 nm by a spectrophotometer (D640; Beckman Coulter, Brea, CA, USA). The TUNEL assay, used to label apoptotic cells, was carried out using the Neuro TACS II kit (4823-30-K, Trevigen, Gaithersburg, MD, USA) according to the manufacturer’s instructions.