Tissue collection and reagents
Choroid plexus for cultures, and brains to be fixed in 4% paraformaldehyde and paraffin embedded for immunohistochemistry were dissected from out-bred CD-1 albino mice supplied by Charles River Mouse Farms (Kent, UK) and housed under Home Office regulations. The morning of detection of a vaginal plug was designated as embryonic day 0.5 (E0.5). Mice were killed by cervical dislocation according to Home Office regulations at the times indicated in the results section and embryos removed for immunohistochemical analysis or CPe culture preparation as described below. Paraffin sections from human embryos at 8 weeks of gestation obtained under ethical approval were provided by the Wellcome/MRC-funded Human Developmental Biology Resource. Unless otherwise specified, reagents used were from Sigma-Aldrich, UK.
Choroid plexus cell cultures
The fourth ventricle CP from E12.5 mice was dissected and CP cells isolated and cultured as previously described [30, 31]. Cells were grown in HEPES-buffered Dulbecco's modified essential medium (H-DMEM, Gibco-BRL, Paisley, UK) supplemented with 10% foetal calf serum (FCS) and penicillin-streptomycin (Gibco-BRL) either as monolayers on laminin (Sigma, UK) or seeded in Matrigel (Becton Dickenson, distributed by Stratech Scientific Ltd, Dudley, UK) to allow vesicle formation. In vesicle culture experiments 1–2 × 105 CPe cells were plated on to Matrigel in cloning cylinders of 8 mm diameter and fed every 2–3 days with the culture medium described above.
The TRCSFB-2 cell line was established from CPe cells isolated from a transgenic rat harboring the temperature-sensitive simian virus 40 (ts SV 40) large T-antigen gene . Cells were grown in DMEM (Gibco-BRL) supplemented with 10% FCS and penicillin-streptomycin on collagen I (Vitrogen, Cohesion Technologies, Inc. CA, USA) at 33°C . Before carrying out RNA expression studies, TRCSFB-2 cells were cultured for 24 h at 37°C.
Cell analysis and treatments
CPe behaviour was monitored by regular observation under an inverted microscope (IM45 Zeiss) and by time-lapse photography for up to 4 consecutive days using Openlab software (Improvision, UK). For Fgf2 treatment, Fgf2 (R&D Systems, Oxford, UK) at the desired concentration was added to the culture medium either at the time of plating CPe cells or later as specified in the Results. To study the effect of Fgf2 on vesicle recovery after inhibition of secretion and collapse, cultures were treated for 48 h either with a combination of 100 μM acetazolamide and 5 mM ouabain or with the vehicle alone (DMSO controls).
Proliferation analysis and immunohistochemistry
Fgf2 was detected by an anti-Fgf2 mouse monoclonal antibody (Transduction Laboratories, Lexington, UK, 1:100 dilution). Cell proliferation was detected by: anti-bromo-deoxyuridine (BrdU) rat monoclonal antibody to detect cells in S phase that had incorporated BrdU (Oxford Biotechnology, Oxford, UK, 1:100); anti-phosphorylated-histone 3 (p-H3) rabbit antibody to detect cells in M phase (Upstate Biotechnology, Buckingham, UK; 1:100 or 1:500 dilution, see below); a mouse monoclonal antibody to PCNA, an auxiliary factor for DNA polymerase delta, to detect cells in G1-S phase (clone PC10, Santa Cruz Biotechnology, Inc., CA, USA, 1:100). The secondary antibodies used, fluorescein-conjugated goat anti-rabbit (1:50 dilution), and peroxidase-conjugated goat anti-rabbit, goat anti-mouse and goat anti-rat immunoglobulins (1:100 dilution), were all from DAKO (Bucks, UK). Hoechst dye H33258 (1:500 dilution of a 1.2 mg/ml stock) was used to counter-stain nuclei.
Mitotic cells in CPe vesicles and aggregates were detected using the anti-p-H3 antibody. Cells grown in Matrigel inside cloning cylinders placed onto glass coverslips were fixed in 4% paraformaldehyde (PFA) for 10 min. Non-specific binding sites were blocked with 1% goat serum in PBS for 40 min. CPe vesicles/aggregates were incubated overnight at 4°C with the anti-p-H3 antibody, thoroughly washed and incubated with fluorescein-conjugated goat anti-rabbit for 30 min. Nuclei were counterstained for 15 min with Hoechst dye.
The anti-PCNA monoclonal antibody was used to stain primary CPe cells fixed for 10 min in 4% PFA. Bound antibody was detected using a fluorescein-conjugated goat anti-mouse antibody and nuclei counterstained with Hoechst dye. Paraffin sections were stained either with the anti-Fgf2 mouse monoclonal antibody or with the anti-p-H3 rabbit antibody (1:500 dilution) essentially as previously described [7, 26].
Tissues and cells were viewed under a Zeiss Axioplan microscope and digitally scanned using a Hamamatsu digital camera (C4742-95, Hamamatsu Photonics KK, Japan) into Openlab software (Improvision Ltd, Coventry, UK).
TRCSFB-2 cells were trypsinised and the pellet stored in 1 ml TRI-Reagent at -20°C until RNA extraction following the manufacturer's protocol. cDNA was synthesised from 1 μg of RNA using M-MLV reverse transcriptase and random hexamers according to the manufacturer's instructions (Promega, Southampton, UK). The PCR step was performed using Taq polymerase at an annealing temperature of 57°C with specific primer pairs for each gene, which were designed to span intron-exon boundaries to exclude genomic DNA contaminations. The linearity range of amplification for each set of primers was evaluated and the number of cycles selected accordingly. The primers (Genosys, Cambridge, UK) used and number of cycles and product size (in brackets) are: Gapdh (20, 491 bp) 5' TTCCAGTATGACTCCACTCACG 3', 5' GGATGCAGGGATGATGTTCT 3'; E2f5 (25, 227 bp) 5' TGTGGCTACAGCAAAGCATC 3', 5' GGCCCTGAGTGACTCTTCAG 3'; FoxJ1 (25, 205 bp) 5' TACTGCTGACCCAGGAGGAG 3', 5' GGTAGCAGGGCAGTTGATGT 3', TTR (30, 505 bp) 5' CAGATCCACAAGCTCCTGAC 3', 5' CTGCTTTGGCAAGATCCTGC 3'; p73 (30, 253 bp) 5' AGAGTGTGGTTGTGCCGTATG 3', 5' TCCCGGTAATGGTCTTCATC 3'. PCR products were visualized on a 1.5% agarose gel containing ethidium bromide under ultraviolet light and imaged by using a Gel imaging system and Alphaease 3.3 software (GRI Ltd, Braintree, UK).
Measurements and statistical analysis
The diameter of each vesicle was measured using an eyepiece graticule at ×20 magnification. Measurements were taken to within the nearest quarter graduation on the eyepiece. Each vesicle could be identified easily by recording the co-ordinates on a finely graduated x-y stage and was measured at 2-day intervals at approximately the same time of day on days 4, 6, 8, and 10. The diameters were recorded 'blind' to treatment group.
The number and size of aggregates and vesicles was measured using the Openlab 3.1.5 software. The size of aggregate and vesicles was estimated by measuring the surface area in μm2 of their focal equatorial plane. The measurements were automatically recorded by the Openlab 3.1.5 programme, stored in Microsoft Excel and later transferred to SPSS Statistics 11.5 (SPSS Inc.) for statistical analysis. Aggregates and vesicles were measured in the same fields at different days. Four fields of view were taken on each measurement day. Cell motility was assessed following time-lapse photography by measuring the distance travelled by a cell using the Openlab 3.1.5 software.
Data are expressed as means ± standard error of the mean (mean ± SEM). Parametric and non-parametric tests as appropriate were used to compare treatment groups at different time points. Regression analysis was used to quantify the effects of treatments over time when different vesicles were assessed on each day. Multilevel modelling was used to model the serial changes in individual vesicles over time . Results are presented with 95% confidence intervals.