In situ hybridization was performed on frozen tissue with digoxigenin (DIG)-labelled
riboprobes. 5-7μl RNA-antisense probe (corresponding to 0.5-1.0μg RNA) were diluted in 150μl hybridization buffer and denaturated for 5 minutes at 70°C, applied onto the microscope slides carrying the sections of interest and sealed with a clean coverslip. Slides were incubated overnight at 65°C in a sealed box with Whatman paper, soaked with 1x SSC in 50% formamide, in a hybridization oven. Slides were thereafter washed for 10 minutes in pre-warmed (65°C) washing solution at 65°C and the coverslip was removed. Further 2-3 washes at 65°C for 30min were done, followed by 2 washes in MABT for 30min at RT. Sections were blocked in 2% blocking-solution for 1h at RT. Anti-digoxigenin Fab fragments coupled to alkaline phosphatase were diluted 1:2500 in blocking-solution and 150μl of this antibody-solution were applied per slide. Sections were covered with parafilm. The antibody incubation was performed in a humid chamber overnight at RT. Slides were washed 4-5 times in MABT for 20 minutes at RT, and rinsed twice in Alkaline-phosphatase (AP) staining buffer for 10 minutes at RT. 150μl NBT/BCIP-containing staining solution was added per slide and covered with parafilm. Slides were incubated 12 to 24h (occasionally up to 3 days) at RT. When the staining was strong enough, the reaction was stopped by rinsing the slides in AP staining buffer and shortly in water. Slides were dried for several hours at RT and mounted in AquaPoly/Mount.
The plasmids used for in situ hybridization are listed in the materials part.
5.6 Cell culture
5.6.1 Cortex dissection and dissociation
Embryos were removed by caesarean section from time-pregnant mice anesthetized by diethylether (Sigma). The meninges were removed, the telencephalic hemispheres separated, the hippocampus and the olfactory bulbs were removed. Cortices were dissected from rat embryos at E15, E19, or from mice hGFAP-eGFP at E14, E18 and AP2γ conditional mouse embryos at E12, E13, E14, E17, as gestational lengths vary between rats and mice and rats 1 day older were considered equivalent to mice 1 day younger. Cortices were dissected in ice cold Hanks balanced salt solution (HBSS, GIBCO) and then incubated in trypsin-EDTA
(GIBCO) for 15 minutes at 37°C. After mechanical trituration and several washes in medium containing 10% fetal calf serum (FCS, GIBCO), 5x105 rat and mouse cortex cells were plated on poly-D-lysine- coated glass coverslips in 24 well plates (NUNC) in 500µl DMEM/10%FCS (GIBCO) for 1 day, 3 days or 7 days.
5.6.2 Clonal analysis and overexpression in vitro
About 2 hours after plating the rat cortex cells, cells sorted from hGFAP-eGFP mouse cortices were added at a density of 200 cells per well (24 well plate). Cortices from WT and AP2γ conditional mouse embryos at E14 were plated directly on PDL coated glass coverslips. The following day, 500µl of chemically defined medium (SATO) was added and further medium changes were performed as described previously (Götz et al., 1998). Comparison of cell counts after sort and stainings of surviving cells after 2 hours or 7 days revealed an in vitro survival of about 80%, further improved in comparison to previous data [(69±15% in (Malatesta et al., 2000)]. Immunostaining for M2M6 labelling of the sorted mouse cells revealed single cells 2 hours after plating, confirming the low proportion of duplets in our sorted population. To label the cells that continue to divide in vitro, the DNA base analogue 5-bromo-2’-deoxyuridine (BrdU, Sigma) was added at a final concentration of 10µM. After 3 days or 1 week in vitro, all cell-types (neurons, astrocytes, oligodendrocytes) were detected by immunostaining in comparable numbers to those determined in previous retroviral or time-lapse cell lineage experiments (Malatesta et al., 2000; Qian et al., 1998; Williams and Price, 1995).
For overexpression experiments, retroviruses were added 2 hours after plating WT and AP2γ
conditional mouse cortices (E14). Further medium changes were performed as described above.
5.6.3 Neurosphere cultures
Embryonic cortical cells were isolated, counted and plated at a density of 5000 cells per well in a 24 well plate without Polylysine or similar coating.
Cells proliferate into spheres that were maintained 7 days in DMEM/F12 media at 37°C with no medium change, counted, redissociated to single cells, counted and plated again at the same density. After seven more days in culture, the number of neurospheres was counted again.
5.6.4 Dual-luciferase reporter assay
Mouse neuroblastoma (Neuro-2A) and Human embryonic kidney (Hek) cells were plated on DMEM/10%FCS/PS media (1x105 cells/24 well) and transfected with expression plasmids (3µg), firefly (Photinus pyralis) luciferase reporter (1.5µg) under the Tbr2, Math3 or AP2γ
promoter and the pRL-TK plasmid encoding Renilla (sea pansy-Renilla reniformis) luciferase (0.1µg, Promega). Total amount of plasmids was kept constant (4.6µg). After 24h, medium was changed and on the next day cell extracts were prepared and assayed for luciferase activity. Relative light units were normalized to Renilla luciferase activity and results were expressed as the ratio of firefly to Renilla luciferase activity (Fluc/Rluc, Fig. 39). Luciferase activity was measured with the Dual-Luciferase Reporter Assay System (Promega) using a luminometer (Berthold Centro LB 960). Values represent the mean±SE of three experiments. The level of significance was p <0.05.
5.7 Fluorescence activated cell sorting (FACS) and cell cycle analysis
Dissociated cells from the cortices of E14 or E18 hGFAP-eGFP heterozygous mice were re- suspended in DMEM and GFP- and/or prominin-positive cells were isolated using a FACSAria (BD) set at purity mode and the appropriate sort rate (below 1000 cells per second for high purity and recovery of cells). Gating parameters were determined by side and forward scatter to eliminate debris, dead and aggregated cells and by green (530nm) and red (575nm) fluorescence to separate positive from negative cells (determined by the use of negative control cells from GFP-negative littermates). Dissociated cells were stained using propidium iodide (PI) to reveal dead cells (less than 4%) as PI-positive by analysis with the FACSAria. Less than 1% of non-fluorescent cells were included in the sort gate. Live staining for prominin (anti-prominin PE) was combined with GFP-fluorescent protein to sort the double-labelled cells and compensation was determined using cells with only one staining. Sorted cells were routinely re-sorted to determine their purity (95-98%). After sorting, the cells were centrifuged for 30 minutes at 1000rpm and re-suspended in 100µl of lysis buffer (RLT from QIAGEN with β-mercaptoethanol) for RNA isolation. Alternatively, cells were plated on either embryonic rat cortex feeder layer or in some experiments without feeder layer on poly-D-lysine-coated plates (2000 cells plated per well of a 24 well plate and fixed after 2hours). Cells were stained for cell-type specific antibodies and analyzed for their GFP-fluorescence revealing about 5% non-fluorescent cells. These are maybe contaminants of non-fluorescent cells or GFP+ cells that lost the GFP protein due to membrane damage during dissociation and plating.
Cortical cells from WT and AP2γ-/-, similarly to hGFAPeGFP cells used for sorting with prominin, were isolated as described before and dissociated by disruption of cation-mediated cell adhesion, to preserve the cell surface molecules. Therefore isolated cortices were placed in dissociation buffer (Ca2+/Mg2+-free HBSS, 1mM EDTA), washed several times in this buffer and incubated for 15 minutes at 37°C. The cells were dissociated mechanically with a fire-polished Pasteur pipette, and washed twice in DMEM/10% FCS to remove traces of EDTA. For permeabilization, the pelleted cells were resuspended in 200µL of DMEM/10%FCS/NaN3. β-III-tubulin antibody was added and the solution was incubated for 15 minutes on ice in the dark. To remove the primary antibody, the cells were washed twice in permeabilization solution and finally resuspended in a 200µL DMEM/10%FCS. For cell cycle analysis, dissociated cells from E14 WT and AP2γ conditional knock-out embryos were ressuspended in 70% (v/v) ethanol overnight at -20°C. The fixative was washed away with PBS and cells were ressuspended in PBS containing 1% FCS. The DNA content of cells was stained with propidium iodide (PI, final concentration 1mg/ml) for 5 minutes and used for FACS analysis.