All breeding and animal experiments were approved by the McMaster University Animal Research Ethics Board and were carried out in accordance with guidelines of the National Institutes of Health and the Canadian Council on Animal Care. Six to eight week old male mSOD mice were mated with eight to ten week old female B6SJL mice purchased from Jackson Laboratories. We checked daily for a vaginal plug, and considered embryonic age as E 0.5 days when one was first seen. At E 13.5 days the dams were euthanized and embryonic spinal cords dissected. Embryo tails were genotyped for the mSOD transgene using the PCR protocol outlined on the Jackson Laboratories website.
Primary cell culture
Primary mixed cultures enriched for motor neurons were undertaken as previously described [14, 15]. Embryonic spinal cords were carefully dissected under microscopy and were processed individually. The isolated spinal cords with meninges removed were cut into small pieces and dissociated in 1% trypsin (Sigma) for 15 minutes. After trypsinization, an equal volume of trypsin inhibitor (Sigma) was added and the mixture was lightly triturated until a single cell suspension was achieved. The cell suspension was then transferred to neurobasal medium containing 1% glutamax (Invitrogen) and centrifuged at 400 g for 5 minutes without brake. The supernatant was discarded and the cell pellet resuspended in complete neurobasal medium containing 1% glutamax, 3% horse serum, 1× B-27 supplement (all from Invitrogen), 5 ng/ml ciliary neurotrophic factor (CNTF) and 5 ng/ml brain-derived neurotrophic factor (BDNF) (Leinco). 2.5 or 5 × 104 cells per well were plated onto poly-D-lysine (Sigma) coated 8-well chamber slides (Labtek) and grown in a 37°C incubator in 5% CO2 environment. Unless otherwise specified, half of the culture volume was replaced with fresh medium every the third day.
Shh and cyclopamine administration
At day 2 in culture (d2), either recombinant human Shh protein (Leinco) or cyclopamine (Sigma) was added to complete neurobasal medium. Cells grown in complete neurobasal medium served as controls. The recombinant Shh protein encompassed amino acids Cys24-Gly197 of the N-terminal portion with Ile-Ile substituted for Cys24, and was resuspended in distilled H2O without any carrier protein. Stock solutions of cyclopamine were diluted in pure dimethyl sulfoxide (DMSO). Even though the final amount of DMSO added to the cultures was minuscule, we initially carried DMSO controls. There was no effect of the DMSO, and normal controls (without Shh or cyclopamine) were subsequently carried as above.
Motor neuron survival assay
To study the effect of Shh or cyclopamine on motor neuron survival, 2.5 × 104 cells per well from 2 embryos of WT mice were plated. At d2, Shh (250 ng/ml) or cyclopamine (12.5 μM) was added to cell culture medium. The medium was replaced every 2 days containing Shh or cyclopamine until d11. At d11, the cell cultures were processed for immunofluorescent staining with the neurofilament heavy antibody (SMI32), a marker of motor neurons. The number of SMI32+ cells per well was counted under widefield deconvolution microscopy (Leica DMI 6000 B, Germany) at 20× magnification. 7–8 fields were counted and counts averaged.
Assessment of motor neurons and motor neuron ciliation
In this experiment, 2 embryos from WT mice and 2 from mSOD mice were used. With each embryo, 5 × 104 cells per well were plated onto poly-D-lysine coated 8-well chamber slides. At d2 of culture, the cells were treated with Shh (0, 250, 500 ng/ml) for 10 days. Half of the culture volume was replaced every third day. At d11, the cell medium were removed, fixed with 4% paraformaldehyde (PFA), and processed for immunofluorescent staining to determine the percentage of motor neurons and motor neuron ciliation. Adenylyl cyclase type 3 (ACIII) was used as a marker of primary cilia, while SMI32 was used to label motor neurons. Motor neuron numbers were estimated by counting SMI32+ cells, and motor neuron percentages calculated relative to DAPI stained nuclei. Motor neuron ciliation was assessed as the percentage of SMI32+ cells staining with ACIII.
BrdU (bromodeoxyuridine) labeling for cell proliferation, cell survival, and cell differentiation
Cell proliferation was determined by BrdU (Sigma) incorporation. 5 × 104 cells per well were plated onto poly-D-lysine coated 8-well chamber slides. At d2 of culture, the cells were treated with Shh (250 ng/ml for G93A SOD mice or 500 ng/ml for WT mice) or cyclopamine (5 μM for G93A SOD mice or 10 μM WT mice) for 10 days. Half of the culture volume was replaced every third day. At d11, 5 μM BrdU was added to the medium. For cell proliferation assays, cells were incubated with BrdU for 24 hours, after which the cell medium was removed, fixed with 4% PFA, and processed for immunofluorescent staining to determine the percentage or number of BrdU labeled cells. For cell survival and cell differentiation, the cells were incubated with BrdU for 72 hours and then BrdU was removed. The cells were continued in medium containing Shh (500 ng/ml) till d26. The cells were then fixed with 4% PFA prior to immunofluorescent staining for BrdU, oligodendrocyte transcription factor 2 (Olig2), homeobox gene Hb9 (Hb9), and SMI32.
Immunofluorescent staining for primary cilia, motor neurons, and BrdU labeling cells
At the indicated time points, cells were fixed with 4% PFA at room temperature for 10 minutes, after which cells were washed twice with PBS (phosphate buffer solution). For detection of BrdU labeled cells, cells were pre-treated with deionized formamide for 2 h at 65°C, 2 N HCl for 30 min at 37°C, and 3% normal goat serum (Vector Laboratories) for 30 min at room temperature. For all other staining, cells were permeabilized with methanol for 10 minutes at −20°C. Fixed permeabilized cells were blocked with 5% goat serum (Invitrogen)/0.3% Triton X-100 (Sigma) in PBS for 1 hour at room temperature. All cells were then incubated with primary antibodies overnight at 4°C. Primary antibodies include rabbit anti-ACIII (1:1000, Santa Cruz Biotechnology), rat anti-BrdU (1:200, Serotec, Martinsried, Germany), mouse anti-nestin monoclonal antibody (1:200, Millipore), mouse anti-SMI32 (1:1000, Abcam), mouse anti-Olig2 polyclonal antibody (1:200, Millipore), rabbit anti-SMI32 (1:200, Abcam), rabbit anti-mouse Gli3 (1:200, Abcam), chicken anti-mouse glial fibrillary acidic protein (GFAP) (1:200, Millipore). The next day, cells were rinsed in PBS and then incubated with secondary antibodies for 4 h at 4°C. Secondary antibodies include Alexa Fluor 488 goat anti-rat antibody (1:500), Alexa Fluor 568 goat anti-mouse highly cross-adsorbed antibody (1:500), and Alexa Fluor 647 goat anti-chicken antibody (1:500) (all from Molecular Probes, Carlsbad, USA). Then cells were rinsed several times and mounted on slides and coverslipped with ProLong anti-fade with DAPI (Molecular Probes, Carlsbad, USA) and examined by widefield deconvolution microscopy (Leica DMI 6000 B).
Cilial and cell counting
100 SMI32+ cells per treatment, an equal number from each embryo, were randomly selected to count ciliated motor neurons at 63× magnification. Only cells separate enough to be easily distinguished one from another were selected. For BrdU labeled proliferating cells, 8 to 11 random fields were captured during one session with constant camera settings (Hamamatsu Orca ER-A) at 20× magnification. For BrdU labeled surviving and differentiating cells, 15 random fields containing BrdU labeled cells were imaged at 63× magnification. (Higher magnification was used for the chronic studies since some of the BrdU label was diluted due to cell divisions, and was easier seen at higher power). Images were converted to TIFF files by using image analysis software (Image J). Single, double or triple labeling cells were counted by using LSM5 image browser (Carl Zeiss).
A one-way analysis of variance (ANOVA) (Statistica, version 6.0, StatSoft, Tulsa, OK) was used to determine significant differences. When there was significant difference, Newman-Keuls significant difference test was used post-hoc. Unless otherwise noted, all data are presented as means ± standard error of the mean (SEM). Significant differences were defined as p ≤ 0.05, two tailed.