Embryos were removed by cesarean section from timed pregnant mice (OF1 strain; IFFA Credo, Arbresle, France). The plug date was considered E1. Fetal brains were removed under sterile conditions in iced HBSS containing 10 mM HEPES. The cerebral hemispheres were detached by a medial longitudinal section. The neopallium including the ventricular zone, the intermediate zone, and the cortical plate was isolated.
Cortical precursor cultures
Cortical cells underwent enzymatic dissociation (trypsin-EDTA 0.25%, 1:100, 3 min at 37°C). Trypsin activity was stopped by washing in Glasgow modified essential medium (GMEM; Biomedia) supplemented with 10% fetal calf serum (FCS). Cells then underwent a mechanical dissociation (up and down aspiration through a P1000 pipette) and were centrifuged for 5 min at 4°C and resuspended in GMEM and 10% FCS. Viability was estimated by means of a trypan blue exclusion assay (0.2% trypan blue was incubated for 2 min), and cells were counted under a hemocytometer. Cells were seeded at a density of 4 × 105 cells per 14-mm-diameter poly-L-lysine-laminin-coated glass coverslip. Cells were cultured in 500 μl of GMEM and 10% FCS. The medium was renewed every 4 d. Cells attached within 3 hr of plating. Cultures were treated with growth factors (25 or 50 ng/ml) 5–12 hr after plating. The proliferation rates were assayed after 2 or 3 d in vitro (DIV).
Cell viability: trypan blue exclusion assay
The proportion of dying cells was quantified in all experiments, for each experimental condition on sister cultures by incubating the cells with 0.2% trypan blue for 20 min. After a thorough wash, cultures were fixed with 2% paraformaldehyde. The cultures were regularly scanned with a 25× objective, and the proportion of blue cells with respect to the total number of cells was computed. After Hoechst staining, apoptotic cells can be easily visualized, because they show condensed chromatin (Wyllie et al., 1980
). Such cells were very rare. Rates of cell death at 2 DIV varied from ~4 to 10% in E14-E16 control cultures. Addition of NT3 or bFGF (25–50 ng/ml) in the cultures led to a nonsignificant reduction of rates of cell death, which was of the same order in both instances.
Bromodeoxyuridine incorporation protocol
Bromodeoxyuridine (BrdU; 20 μg/ml) was added to the medium for 2–3 hr. The cultures were then washed with phosphate buffer and fixed with 70% ethanol at −20°C.
Cumulative labeling. BrdU (20 μg/ml) was added to the medium, which was partially renewed every 10 hr when long exposure periods were required. Each time point was repeated on three to four sister coverslips. Cultures were washed with phosphate buffer before being fixed with 70% ethanol at −20°C.
Percentages of labeled mitotic figures. BrdU (20 μg/ml) was added to the medium for 1 hr. Cultures were rinsed twice before adding new medium. Two repeats were done at each time point. After appropriate survival periods, cultures were fixed with 70% ethanol at −20°C. Immunocytochemistry to reveal BrdU was performed as below using DAB as a chromogen. Cells were finally counterstained with Hoechst to allow the identification of mitotic figures.
Proliferating cell nuclear antigen-BrdU double immunostaining
Cultures were first incubated for 20 min in Tris-buffered saline (TBS) and 0.6% H2O2, followed by a 20 min incubation in normal goat serum (1:5). Proliferating cell nuclear antigen (PCNA) was revealed according to the following three-step procedure: mouse anti-PCNA (DAKO-PC10; Dako, Glostrup, Denmark; 1:75 in TBS) for 30 min at room temperature, biotinylated goat anti-mouse antibody (Dako; 1:400 in TBS) for 30 min at room temperature, and peroxidase-conjugated streptavidin (Dako; 1:500 in TBS). Peroxidase activity was revealed by incubating the cultures in DAB (Sigma, St. Louis, MO; 1 mg/ml in 0.05 M Tris) for 5 min and then adding 3% H2O2 for 10 min. DNA denaturation was subsequently performed by 2N HCl for 30 min at 37°C, followed by two rinses (15 min each) in borate buffer, pH 8.5. Mouse anti-BrdU (BU33; Sigma; 1:400) was incubated overnight at 4°C. Labeling was revealed by a last incubation of FITC rabbit anti-mouse antibody (Dako; 1:100) or Cy2 rabbit anti-mouse antibody (Jackson ImmunoResearch, West Grove, PA; 1:400). Cell nuclei were counterstained with Hoechst.
Cell cycle kinetics
Cell density determination. Cultures were scanned at regular intervals, and densities were estimated by computing the total number of cells per unit area. Statistical significance between control and experimental values was assessed with a Mann-Whitney U test.
Identification of the precursor pool. Precursor cells were identified by means of PCNA labeling (Dehay et al., 2001
). PCNA is an auxiliary protein of delta -DNA polymerase, which is present during all phases of the cell cycle (Bravo et al., 1981
). The estimation of the growth fraction (GF; i.e., the fraction of cycling cells) was determined by computing the proportion of PCNA-positive cells with respect to the total number of cells.
Labeling index determination. Cells in S phase at the time of the pulse were BrdU-positive. The labeling index (LI) was determined as the proportion of BrdU-positive cells (cells that were in S phase during the BrdU exposure) with respect to the precursor pool, i.e., the PCNA-positive cells.
S phase and G1 + G2/M phase lengths were derived from eight different cumulative BrdU experiments (Nowakowski et al., 1989
Durations of TS, TG1+G2/M, TC, and TG1 in different experimental conditions
G2/M duration was determined on an E15 culture using the percentage of labeled mitoses (PLM) method (Shackney, 1974
). BrdU was used as an S phase marker. Forty-eight hours after neurotrophins were added to the medium, a 1 hr BrdU exposure was given to control and bFGF- or NT3-treated sister cultures. The cultures were carefully rinsed, and fresh medium was added. Cultures were then fixed at hourly intervals (1–8 hr). After immunohistochemical labeling of BrdU-positive cells, the PLM was computed for each time point. This procedure measures the time required for cells in S phase cells to enter M phase and therefore returns the G2/M duration (Shackney, 1974
; Cavanagh et al., 1997
). All comparisons of the different populations of cells have been made between control and neurotrophin-treated cultures prepared from the same pool of cells.
For the PLM experiments (Quastler and Sherman, 1959
), statistical significance was tested by means of an F test applied to the ascending slope of the curve. For BrdU cumulative labeling, the statistical differences between slopes were tested by means of an F test combined with bootstrap analysis (implemented with Matlab software; The MathWorks, Inc., Natick, MA) that it makes possible to determine whether the intersection of the two slopes is on the x-axis.
After bFGF (25 ng/ml) or NT3 (25 ng/ml) exposure for 1–3 DIV, cells were scraped off the dish in ice-cold PBS, pelleted, and frozen in liquid nitrogen until use. All the subsequent steps were performed at 4°C. Approximately 5 × 106 cells were lysed for 30 min in 1 ml of lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% Nonidet P-40, 50 mM NaF, 1 mM sodium orthovanadate, 4 mM phenylmethylsulfonyl fluoride, 25 mg/ml leupeptin, 25 mg/ml aprotinin, 10 mg/ml trypsin inhibitor, 1 mM benzamidine, and 1 mM dithiothreitol). Cellular debris were removed by centrifugation (10,000 × g for 15 min). Protein content was quantified by the Coomassie blue protein assay, and 250 μg of protein lysate was used for immunoprecipitation of proteins of interest (Cyclin D2 and p27kip1). Cell lysates were used either directly for analyzing expression of cyclin-dependent kinase 2 (CDK2), CDK4, and cyclin A by immunoblotting, or the proteins of interest (cyclin D2 and p27kip1) were first immunoprecipitated from the lysates using specific rabbit polyclonal antibodies bound to protein A-Sepharose. Polyclonal anti-cyclin D2 (M-20) and anti-p27kip1 (M-197) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Three milligrams of purified antibody coupled to 25 μl of protein A-Sepharose were used for each milliliter of cell lysates. After incubating protein A-Sepharose-coupled antibodies with the cell lysates, immune complexes were collected before being analyzed by SDS-PAGE and immunoblotting.
Protein lysates were prepared exactly as described in Immunoprecipitation. 5 micrograms of protein lysates were loaded for each sample. Samples were analyzed on 10–12% SDS-polyacrylamide gels, followed by immunoblotting on nitrocellulose membranes in 12.5 mM Tris-HCl, 100 mM glycine, 0.05% SDS, and 20% methanol. Membranes were blocked in 20 mM Tris-HCl, pH 7.6, 137 mM NaCl, 0.1% Tween 20, and 5% dry milk for 1 hr to overnight. Membranes were then incubated with the first antibody (diluted at 2 μg/ml in 20 mM Tris-HCl, pH 7.6, 137 mM NaCl, 0.1% Tween 20, and 2% dry milk) for 1 hr, washed three times for 10 min each in 20 mM Tris-HCl, pH 7.6, 137 mM NaCl, and 0.1% Tween 20, and incubated 1 hr with HRP-conjugated second antibody (Amersham Biosciences, Arlington Heights, IL) diluted 1:10,000. HRP activity was revealed with the ECL detection kit (Amersham Biosciences). All incubations with antibodies were performed using Biocomp Navigator (Serlabo). Primary antibodies were as follows: rabbit polyclonal anti-CDK2 (M2), anti-CDK4 (C-22), and anti-cyclin A (H-432) were purchased from Santa Cruz Biotechnology; and mouse monoclonal anti-cyclin D2 (14821A) and anti-p27kip1 (13231A) were purchased from PharMingen (San Diego, CA).
Cyclin D2 and p27 immunochemistry
After PCNA (see above) or MAP2 (see below) immunochemistry, cultures were rinsed three times in TBS and then incubated for 20 min in normal goat serum and rinsed three times before proceeding to primary antibody incubation as follows: rabbit anti-p27 (1:20 in Dako diluent) from Santa Cruz Biotechnology (SC-528) or rabbit anti-D2 (1:20 in Dako diluent) from Santa Cruz Biotechnology (SC-593). Incubation was at room temperature for 1 hr. After three rinses, goat anti-rabbit Cy2 (1:200 in Dako diluent; Jackson ImmunoResearch) or goat anti-rabbit Cy3 (1:200 in Dako diluent; Jackson ImmunoResearch) was incubated for 1 hr at RT. After three rinses, cultures were counterstained with Hoechst (1 μg/ml) and mounted with 0.1% n-propylgallate (P3130; Sigma) in 0.1 M phosphate buffer and glycerol (1:1) to prevent fading on fluorescent illumination.
PCNA, GFAP, and MAP2 triple immunohistochemistry
After PCNA immunochemistry (see above), GFAP and MAP2 were revealed according to the following two-step procedure. Cultures were rinsed three times in TBS and 0.5% Triton X-100 and then incubated in a mixture of ethanol (95%) and acetic acid (5%) for 20 min. After three rinses in TBS, coverslips were incubated for 20 min in normal goat serum and rinsed three times before proceeding to primary antibody incubation as follows: rabbit anti-GFAP (1:100 in Dako diluent) from Sigma (G9269) and mouse anti-MAP2 (1:100 in Dako diluent) from Sigma (M4403) were incubated simultaneously overnight at 4°C. After three TBS rinses to reveal GFAP labeling, goat anti-rabbit Cy2 (1:400 in Dako diluent; Jackson ImmunoResearch) was incubated for 1 hr at room temperature. After three rinses, coverslips were further incubated with goat anti-rabbit Cy3 (1:400 in Dako diluent; Jackson ImmunoResearch) for 1 hr at room temperature to reveal MAP2 labeling. Coverslips were counterstained with Hoechst (1 μg/ml).
Microscopic observations and confocal analysis of the fluorescent labeling
Cultures were examined using an oil objective (25 or 63×) under UV light to detect FITC and Cy2 (L5 filter), Cy3 (N2.1 filter), and Hoechst (A filter) on a Leica (Nussloch, Germany) DMRB fluorescence microscope. PCNA labeling revealed by DAB staining was observed under white illumination. The cultures were scanned at regular spacing with a grid corresponding to a field of 0,46 mm2. One hundred fields were observed per coverslip.
Confocal analysis of p27 and cyclin D2 expression was performed with a Leica confocal system (TCS SP) using a 63× oil objective. Quantitative analysis of Cy2 and Cy3 fluorescence in PCNA-positive or MAP2-negative cells nuclei was performed using Leica software (TCS NT). Levels of fluorescent intensity were measured individually for each cell, and three categories of labeling intensity were defined: 20–65 (low-intensity labeling), 65–110 (intermediate-intensity labeling), and >110 (high-intensity labeling).
Time-lapse videomicroscopy recording of cell division
Cortical precursors from green fluorescent protein (GFP; Okabe et al., 1997
) +/− and −/− mice were seeded on poly-L-lysine- and laminin-coated glass coverslips to obtain 25% of GFP+ cells. This low proportion of GFP+ cells makes it possible to reliably monitor the behavior of individual fluorescent cells. Glass coverslips were placed in a 35 mm glass-bottom Petri dish (MatTeck Corp.) in GMEM supplemented with 10% FCS or in neurobasal medium supplemented with B27 (Invitrogen, San Diego, CA). Cultures were incubated at 37°C with 7.5% CO2 for 4–5 DIV in a Pecon incubating chamber placed on a Leica DMIRBE inverted microscope stage. Observations of the proliferative behavior of individual green fluorescent precursors were made with a 40× objective under halogen illumination. Using Metamorph software, 20 fields were scanned per coverslip per hour. Subsequent analysis of the movies allowed estimation of (1) the cell cycle length of individual cortical GFP+ cells, corresponding to the duration between two mitoses; and (2) the mode of division assessed by the proliferative behavior of the daughter cells (subsequent division or not).