Materials and Methods

Mouse strains and genotyping.
Mice were treated according to institutional and national guidelines. α-Cre mice (P. Gruss), Chx10-Cre mice (C. Cepko), RbloxP/loxP mice (A. Berns), E2f1–/– mice, E2f2–/– mice, E2f3loxP/loxP mice, and E2f3a−/− mice were maintained on a mixed (NMRI × C57/Bl × FVB/N × 129sv) background. A detailed description of E2f3a−/− mice will be published elsewhere. Mice of different genotypes were compared within the same litter and across a minimum of three litters. We have not noted any phenotypic differences in separate litters. Genotyping was performed as before [2,5], and the primers used for genotyping E2f3a−/− mice were E2f3a KL (5′-CTCCAGACCCCCGATTATTT-3′), E2f3a KR1 (5′-TCCAGTGCACTACTCCCTCC-3′), and E2f3a KM (5′-GCTAGCAGTGCCCTTTTGTC-3′).

Histology, immunofluorescence, and measurements.
Eyeballs were fixed in 4% paraformaldehyde for 1 h at 4 °C, embedded in OCT (TissueTek 4583, Sakura, http://www.sakuraeu.com), frozen on dry ice, and cut into 12-μm sections on Superfrost plus slides (VWR, http://www.vwr.com). For S-phase analysis, BrdU (100 μg/g of body weight) was injected intraperitoneally 2 h prior to sacrifice. BrdU+ cells were detected using a biotin-conjugated sheep polyclonal antibody (1:500, Maine Biotechnology Services, http://www.mainebiotechnology.com). All other antibodies are described in Table S1. For E2f3, Mki67, and Rb staining, antigen retrieval was performed by boiling sections in citric acid solution for 15 min according to Ino [50], except on frozen sections. TUNEL was performed as described [13]. Briefly, sections were incubated for 1 h at 37 °C with 75 μl of mixture solution consisting of 0.5 μl of terminal deoxynucleotide transferase, 1 μl of biotin-16-dUTP, 7.5 μl of CoCl2, 15 μl of 5× terminal deoxynucleotide transferase buffer, and 51 μl of distilled water. After three washes in 4× SSC buffer, sections were incubated with Alexa 488– or Alexa 568−streptavidin (1:1,000; Molecular Probes, http://probes.invitrogen.com) for 1 h at room temperature. Primary antibodies or labelled cells were visualized using donkey anti-mouse Alexa 488 or Alexa 568, donkey anti-rabbit Alexa 488 or Alexa 568, donkey anti-goat Alexa 488 or Alexa 568, and streptavidin Alexa 488 or Alexa 568 (1:1,000; Molecular Probes). Nuclei were counter-stained with 4,6-diamidino-2-phenyindole (DAPI; Sigma, http://www.sigmaaldrich.com). Labelled cells were visualized using a Zeiss (http://www.zeiss.com) Axioplan-2 microscope with Plan Neofluar objectives and images captured with a Zeiss AxionCam camera. For double-labelled samples, confocal images were obtained with a Zeiss LSM 5.0 laser scanning microscope.
The retina was separated into three bins by dividing the ventricular edge of the retina into equal parts and extending a line to the vitreal edge [2]. Bin 1 contains only cells that expressed Cre as progenitors; bin 3 is at central retina and contains cells derived from progenitors that did not express Cre. For cell counts or thickness measurement we used a region 0–100 μm peripheral to the boundary separating bins 1 and 2. Measurements were performed on an Axioplan-2 microscope using Axiovison software. Quantification of S-phase, M-phase, and apoptotic cells was performed on horizontal sections that included the optic nerve. Quantification of differentiated cell types was performed using horizontal sections at equal distances from the optic nerve. A minimum of three sections per eye and three eyes from different litters were counted.

RNA extraction, reverse transcription, and PCR.
Total RNA was isolated from dissected peripheral retina using TRIzol reagent (Invitrogen, http://www.invitrogen.com) followed by digestion with RNase-free DNase (DNA-free, Ambion, http://www.ambion.com) to remove DNA contamination. First-strand cDNA was synthesized from 0.2–0.5 μg of total RNA using the SuperScript II first-strand synthesis system (Invitrogen). PCR primers are listed in Table S2. Real-time quantitative PCR was performed using an Applied Biosystems (http://appliedbiosystems.com) PRISM 7900HT. Tests were run in duplicate on three separate biological samples with SYBR Green PCR Master Mix (Applied Biosystems) exactly as we described previously [71]. Briefly, master stocks were prepared such that each 10-μl reaction contained 5 μl of SYBR Green PCR Master Mix, 0.1 μl of each forward and reverse primer (stock 50 μM), 0.8 μl of blue H2O (0.73% Blue Food Colour; McCormick, http://www.mccormick.com), 2 μl of diluted cDNA template, and 2 μl of yellow H2O (0.73% Yellow Food Colour). PCR consisted of 40 cycles of denaturation at 95 °C for 15 s and annealing and extension at 55 °C for 30 s. An additional cycle (95 °C, 15 s, 60 °C) generated a dissociation curve to confirm a single product. The cycle quantity required to reach a threshold in the linear range was determined and compared to a standard curve for each primer set generated by five 3-fold dilutions of genomic DNA or cDNA samples of known concentration. Values obtained for test RNAs were normalized to Hprt1 mRNA levels.

Western blots.
Mouse retinas were homogenized by passing them through a 30-gauge BD 9 http://www.bd.com) needle 5–10 times in 1× PBS solution. Nuclear and cytoplasmic proteins were extracted using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Product# 78833, Pierce Biotechnology, http://www.piercenet.com). Proteins were separated by 10% SDS-PAGE and transferred to nitrocellulose. After blocking overnight at 4 °C in 5% skim milk, membranes were incubated in the primary antibody for 2 h at room temperature. After three 10-min washes in TPBS (100 mM Na2HPO4, 100 mM NaH2PO4, 0.5 N NaCl, 0.1% Tween-20), membranes were incubated for 30 min at room temperature in the secondary horseradish peroxidase-conjugated antibody (Jackson ImmunoResearch Laboratories, http://www.jacksonimmuno.com). Blots were developed using the ECL-Plus chemiluminescent detection system (Amersham Pharmacia Biotech, http://www.pharmacia.ca), according to the manufacturer's instructions.
The following primary antibodies were used: E2f-1 (SC-193), E2f-3 (SC-878), Cdkn1a (p21, SC-471), Cdkn1b (p27, SC-528), Pou4f2 (Brn3b, SC-6062), and Tfdp1 (Dp1, SC-610) from Santa Cruz Biotechnology (http://www.scbt.com), pRB (554136) from BD Science-Pharmingen (http://www.bdbiosciences.com), and Slc18a3 (VAChT, G448A) from Promega (http://www.promega.com).

Electroretinography.
ERGs were recorded from dark-adapted mice as described [72]. Briefly, mice were dark-adapted overnight and anaesthetized by subcutaneous injection of ketamine (66.7 mg/kg body weight) and xylazine (11.7 mg/kg body weight). The pupils were dilated and single-flash ERG recordings were obtained under dark-adapted (scotopic) and light-adapted (photopic) conditions. Light adaptation was accomplished with a background illumination of 30 candela (cd) per square meter starting 10 min before recording. Single white-flash stimulation ranged from 10−4 to 25 cd·s/m2, divided into ten steps of 0.5 and 1 log cd·s/m2. Ten responses were averaged with an inter-stimulus interval of either 5 s (for 10−4, 10−3, 10−2, 3 × 10−2, 10−1, and 3 × 10−1 cd·s/m2) or 17 s (for 1, 3, 10, and 25 cd·s/m2). Band-pass filter cut-off frequencies were 0.1 and 3,000 Hz.

Statistics.
Different genotypes were evaluated using analysis of variance (ANOVA) followed by the Tukey honestly significant difference (HSD) test or Fisher test (XLSTAT program, http://www.xlstat.com).