Virus preparation and animal surgery
Replication-incompetent pantropic retrovirus pseudo-typed with the VSV-G glycoprotein (1 × 106
cfu/mL) carrying the enhanced green fluorescent protein (GFP) reporter gene was produced from a stably transfected packaging cell line (generous gift of Drs. Fred Gage and Theo Palmer). Embryonic day (E)15–16 rat embryos were injected with retrovirus in utero as previously described (Noctor et al., 2004
). This approach labels progenitor cells that were in contact with the ventricle at the time of the retroviral injection (Walsh and Cepko, 1988
). Briefly, the uterine horns of E15–16 pregnant rats were removed, cerebral hemispheres were transilluminated using a fiber optic light, and ≈0.5 μL of retrovirus was injected into the lateral ventricles of selected embryos. The uterine horns were lavaged with sterile saline, replaced, and the incision sutured. The lateral ventricle of E12 and E13 embryos was injected with retrovirus ex utero and the embryos cultured at room temperature for 2 hours in artificial cerebrospinal fluid (aCSF) bubbled with 95%/5% O2
containing (in mM): NaCl 125, KCl 5, NaH2
25, and glucose 20, pH 7.4, at 25°C, 310 mOsm/L. All surgical procedures were performed in accordance with the UCSF IACUC.
Organotypic slice cultures
Two hours after E12–E13 retrovirus injections, brains were removed and embedded in 4% agar. One day after E15–E16 retroviral injections embryos were sacrificed, brains removed, and embedded in 4% agar. All brains were sectioned at 375 μm on a vibratome (Leica, Deerfield, IL) in ice-chilled aerated aCSF. Slices were plated on slice culture inserts (Millicell, Millipore, Bedford, MA) in culture well plates (Corning, Corning, NY) with culture medium containing (by volume): BME 66%, Hanks 25%, FBS 5%, N-2 1%, Pen/Strep/Glutamine 1% (each from Gibco, Grand Island, NY), and d-(+)-glucose 0.66% (Sigma, St. Louis, MO). Slices remained in the culture plates for the duration of time-lapse imaging experiments and were maintained in an incubator at 37°C, 5% CO2 between confocal imaging of the labeled cells.
Confocal time-lapse microscopy
GFP-labeled progenitor cells were imaged on an inverted Olympus (Lake Success, NY) Fluoview confocal microscope. Projection images were made from Z-stacks that included all visible processes of individual GFP+ cells, and all GFP+ clonal cells on a PC running Fluoview (Olympus). We used laser power levels that allowed us to visualize all cellular processes without risking overexposure to the live cells. Transmitted light images were taken at each timepoint to track movements of the GFP+ cells in the cultured slices. Cell position was maintained relative to the ventricular surface while RG cells maintained contact with the ventricular surface. Cells that lost contact with the ventricular surface were positioned relative to the ventricular surface until they left the proliferative zones, at which point they were positioned relative to the pial surface for the duration of the time-lapse experiment. At some timepoints only dividing cells were imaged to reduce laser exposure to other cells in the clone. Between timepoints, slices were kept in a humidified incubator at 37°C, 5% CO2. Cleavage plane angle of dividing GFP+ cells was measured during cytokinesis, after experiments using Adobe Photoshop v7 (San Jose, CA). The orientation of division was recorded from 0° (horizontal) to 90° (vertical) with respect to the ventricular surface. 3-Dimensional images were made from the Z-stack and the image rotated (Olympus Fluoview) to measure cleavage for cells that divided orthogonally in the slice. Montages were assembled, time-lapse sequences arranged, and images adjusted to improve contrast/brightness using Photoshop. Line drawings were prepared using Adobe Illustrator.
Morphological analysis of VZ cells
In utero injections of lower titer retrovirus (1 × 105 cfu/mL) were performed at E15, as described above, and embryos sacrificed 1 day later. Brains were perfused transcardially with chilled phosphate-buffered saline (PBS) followed by 4% paraformaldehyde (PFA, Fisher, Pittsburgh, PA). Brains were removed, fixed overnight in PFA and vibratome-sectioned at 50, 100, or 200 μm. All eGFP-labeled cells that contacted the ventricular surface were analyzed. We determined whether VZ cells had a pial directed process, and if so whether the process was restricted to the VZ, the IZ, the CP, or the MZ.
Organotypic slice cultures were transferred to a recording chamber on an Olympus BX50WI upright microscope after time-lapse recordings and were perfused with aerated aCSF. GFP+ cells were identified under epifluorescence and recordings performed using an EPC-9 patch-clamp amplifier (Heka Electronics, Canada) controlled by an Apple computer running Pulse v8.0 (Heka). Glass recording electrodes (5–7 M°) were filled with (in mM): KCl 130, NaCl 5, CaCl2 0.4, MgCl2 1, HEPES 10, pH 7.3, EGTA 1.1. Epifluorescent images of the recorded cells were collected using Scion Image (NIH, Bethesda, MD), and arranged using Photoshop. Electrophysiological responses were measured and analyzed using Pulse, and traces arranged using Igor Pro (Wavemetrics), and Free-hand (Macromedia). We obtained some RG daughter cell and VZ cell recordings in acute slice preparations. Slices were prepared from embryonic rat neocortex that had been injected with eGFP retrovirus in utero as described above and 500 μM Alexa 594-conjugated biocytin (Molecular Probes, Eugene, OR) was added to identify recorded cells.
E12 embryos (n = 8) were immersion-perfused in PFA overnight at 4°C and frozen in OCT media. E15 (n = 3), E17 (n = 3), and E20 embryos (n = 3), and postnatal (P)7 neonates (n = 3) were perfused transcardially with chilled PBS followed by PFA. Brains were removed, fixed overnight in PFA, frozen in 2-methyl butane, and stored at 80°C until cryostat (Leica) sectioning at 10 μm. Sections were mounted on glass slides and stained with Cresyl Violet to label Nissl substance. Sections were photomicrographed and all anaphase and telophase cells were identified at the ventricular surface and in abventricular positions. The orientation of cleavage plane angle was calculated by averaging the angle of the sister chromatids in anaphase and telophase cells with respect to the ventricular surface. Results were statistically analyzed using Prism (Graphpad Software, San Diego, CA).
Coronal sections were labeled with goat polyclonal antibodies against doublecortin (DC). We tested DC antibodies that were raised in goat against the N-terminus of human doublecortin (amino acids 40–90, Santa Cruz Biotechnology, Santa Cruz, CA, #SC-8067 N-19, Lot F2204, dilution 1:100), and the C-terminus of human doublecortin (amino acids 350–402, Santa Cruz Biotechnology #SC-8066 C-18, Lot D1105, dilution 1:100), and determined that they provided comparable staining patterns. Western blotting of mouse embryo extract and 3T3-L1 doublecortin-expressing whole-cell lysate with both antibodies reveals a 40-kD band, which is consistent with the molecular weight of doublecortin protein (manufacturer’s technical information). Immunoreactivity obtained with these antibodies in our hands was completely abolished through preadsorption with peptide sequences #SC-8067P, Lot O402 and #SC-8066P, Lot E059, respectively (see ). Coronal sections were also labeled with mouse monoclonal NeuN (Chemicon, Temecula, CA, #MAB377, clone A60, Lot #0601019159, dilution 1:1000). The NeuN antibody was raised in mouse against purified cell nuclei from mouse brain and recognizes two or three bands in the 46–48-kD range and another band at 66 kD in Western blotting (manufacturer’s technical information). The NeuN antibody is reported to label most classes of neurons, and negative controls run by the manufacturer to test specificity include nonneuronal cells such as fibroblasts (manufacturer’s technical information). We find that the antibody does not label any dividing cells in the embryonic neocortex, consistent with data showing that NeuN labels only neurons. Finally, we labeled coronal sections of embryonic neocortex with rabbit polyclonal Tbr2 antibodies (1:2,000; gift of Dr. Robert Hevner; see Englund et al., 2005
). This antibody was generated in rabbit against the unique peptide sequence EYSKDTSKGMGAYYAFYTSP from mouse Tbr2. Western blotting of mouse brain homogenates shows a 73-kD band, matching the predicted molecular weight of Tbr2 (Quinn et al., 2007
). The Tbr2 antibody does not label mature neurons and shows very little colocalization with Pax6-expressing cells in the embryonic VZ (Englund et al., 2005
). Consistent with these data, we find that the Tbr2 antibody does not label RG cells ().
Fig. 6 The embryonic subventricular zone (SVZ) is a neurogenic compartment. A: Nissl-stained tissue costained with doublecortin (DC) antibodies shows the pattern of DC-immunoreactivity in a coronal section of E17 rat cortex. The cortical plate (CP) is densely (more ...)
Fig. 8 Radial glial (RG) cells do not express Tbr2. A–C: GFP-labeled RG cells (green) in the embryonic ventricular zone (VZ) do not express Tbr2 (red). RG cells in interphase (A), G-2 phase (B), and prophase (C). Only RG daughter cells that are intermediate (more ...)
Sections were rinsed in PBS 0.1 M, pH 7.4, incubated overnight in antibody buffer containing 2% serum, 0.1% Triton X, and 0.2% gelatin diluted in PBS. Sections were rinsed, incubated with biotinylated secondary antibodies (1:100, Jackson Laboratories, West Grove, PA) for 1 hour at room temperature (RT). Sections were stained with avidin-biotin complex (Vector, Burlingame, CA) for 1 hour at RT, rinsed, and placed in 0.04% DAB (Sigma) with 0.001% H2O2 for 2–5 minutes. Sections were counter-stained with Cresyl Violet before coverslipping with DPX (Sigma). In some cases fluorescent immunolabeling was produced using goat antimouse secondary antibodies (1: 100; Jackson Laboratories) or goat antirabbit secondary antibodies (Abcam, Cambridge, UK; 1:100). We tested the specificity of all secondary antibodies through omission of the primary antibody.