The used pregnant mice (C57B L/6J) were purchased from The Jackson Lab. This work was performed according to the German Ethical Guidelines for Laboratory Animals recommendations. The protocol was sanctioned by the ethics committee of the City of Freiburg, (authorizations: G11/56 and X-16/10F), and the Institutional Animal Care and Use Committee of the Freiburg University.
Different embryonic ages of wild-type mice (C57B L/6J) have been used, starting from E12, E14 and E18, to elucidate the role of SHH accessory receptors (Boc and Gas1) and SHH downstream components (Ptch1 receptors, Gli1, Gli2 and Gli3 transcription factors) in the development (differentiation and survival) of the mDA neurons. Pregnant C57BL/6J mice were sacrificed by cervical dislocation. Embryos were obtained by the cesarean section, rinsed with 1xPBS (pH7.4), fixed overnight at 4oC in 0.1M PBS containing 4%PFA. After fixation, embryos were rinsed 3x PBS for 30 minutes (min). and then cryoprotected in 15% sucrose in PBS overnight at 4oC. Later, embryos were immersed in 30% sucrose in PBS and kept at 4oC overnight until the embryos sank. Following overnight immersion in sucrose embryos were coated with Tissue Tek (OCT compound), frozen in liquid nitrogen and finally stored at –20°C until further processing. Sagittal and coronal serial sections at 10μm thickness were sectioned by Cryostat, mounted on super-frost slides, and air dried for 40 min before performing in-situ hybridization or immunohistochemical staining.
Mouse anti-TH (MAB318, Millipore) and rabbit anti-TH (AB152) (Merck-Millipore, Darmstadt, Germany) were used for immunoperoxidase light microscopy. Goat anti-Gas1 (AF2644, R&D Systems) and anti-Boc (AF2385, R&D Systems) used for immunofluorescent (IF) staining. Goat anti-Ptch1 (G-19; sc-6149, Santa Cruz Biotechnologies, Heidelberg, Germany), rabbit anti-Smoothened (ab38686, Abcam, Cambridge, UK) for IF staining to MN9D cell line. Secondary antibodies were goat anti-mouse or goat anti-rabbit IgG coupled to horseradish peroxidase, donkey anti-mouse, donkey anti-goat or donkey anti-rabbit IgG Alexa 568 (Dianova, Germany). DAPI- Fluoromount G (Biozol, Germany) was used for counterstaining of nuclei and Aquatex (Merck-Millipore, Germany) for mounting.
Frozen sections were dried at room temperature (RT) for 30 min then washed once for 10 min in 1x PBS. Antigen retrieval was performed in 0.1 M citrate buffer solution (pH6) for 3 min using a microwave at (600 Watt). Sections were washed 3x 5 min each with 1x PBS, incubated in 30% hydrogen peroxide (H2O2) in methanol (75ml methanol+750µl H2O2) for 30 min to block the endogenous peroxidase activity. Afterwards, the cryosections were washed for 5 min with 1x PBS, incubated with blocking solution [10% normal donkey serum (NDS)+ 0.2% Triton-X100/PBS] for 2h at RT, incubated with rabbit anti-TH diluted at 1:500 in blocking solution overnight at 4◦C. On the second day sections were rinsed in PBS 3x 5 min each, incubated with secondary antibody conjugated to biotin (diluted in 1.5 % NDS + 0.2% Triton-X100/PBS) for 2h at RT, followed by incubation with avidin and biotinylated horseradish-peroxidase macromolecular complex (ABC kit) for 45 min. Finally, the antigen-antibody reaction was visualized using the DAB peroxidase substrate (15-20 min), rinsed with distilled water, mounted with Aqua-Tex and analyzed with a bright field (Axioplan2, Zeiss) microscope.
The sections were treated as mentioned above without H2O2 treatment. The cerebellum was used as a positive control for Gas and Boc. They were incubated with the primary antibodies (anti-Gas1 or anti-Boc at dilutions 1:200 and 1:100, respectively) overnight at 4°C. On the second day, the sections were washed in PBT (3x 10 min each), incubated with Alex Fluor® secondary antibodies for 2h at RT in a dark place. Finally, the sections were washed 3x 5 min each in 1xPBS, mounted with Flouromount-G mixed with 4'-6-diamidino-2-phenylindole (DAPI) diluted 1:1000 in PBS for counterstaining the nuclei. Then the reaction was visualized and imaged under the fluorescence microscope (Axioplan2, Zeiss).
In situ hybridization (ISH)
This technique was performed, as described by , to detect specific ribonucleic acid sequence using labelled mRNA probe with a molecular marker, such as Digoxigenin (DIG; non-radioactive antibody) to investigate the expression of a gene of interest at the transcriptional level. ISH was performed on frozen sections using DIG labelled mRNA probes (Roche, Mannheim, Germany) for the SHH signalling components such as, Ptch1 receptor, Gli1, Gli2 and Gli3 transcriptional factors, neurofilaments (NF) and TH. The sense probes were used as negative controls. The neurofilament marker was used as a positive control for all ISH experiments. In addition, cerebellum was used as a positive control for Gli1, 2, 3. ISH was performed in RNase free conditions and all used solutions were prepared using DEPC water and under sterile conditions. The DIG-labelled RNA probes were diluted with Tris-EDTA buffer (1:10), afterwards, the diluted DIG labelled RNA probes were added to 150 µl of hybridization buffer and applied on the freshly dried frozen sections. The buffer box containing the sections were placed in humidified chamber and incubated overnight at 68°C. On the second day, slides were washed in washing buffer at 70°C for 10 min. Post hybridization washes were performed twice for 30 min at 70°C in washing buffer, immersed in 1x MABT (maleic acid buffer with tween) at RT for 30 min twice. Afterwards, the sections were blocked with blocking reagent (1.2 ml lamb serum: 4.8ml MABT) for 2h, followed by incubation with sheep anti-DIG-AP (anti- DIG conjugated to alkaline phosphatase (AP) in blocking reagent (1:1500) at RT overnight in a humid chamber. On the third day, sections were washed with 1x MABT for 20 min at RT, covered with 400 µl MABT twice for 20 min /each, covered with 400 µl three times with filtered AP buffer for 10 min. To visualize the DIG- labelled mRNA probes; Nitro-blue tetrazolium chloride in combination with 5-bromo-4-chloro-3’-indolyphosphate p-toluidine (NBT/BCIP, Roche) was used as a substrate (colouring solution) overnight at RT. On the fourth day, the sections were washed in 1x PBS, mounted with aquatex and examined with a bright field (Axioplan2, Zeiss) microscope.
In vitro cell/tissue culture
MN9D is a dopaminergic cell line, which was established by hybridoma fusion of cells from E14 murine mesencephalic cells with neuroblastoma cell line N18TG2 . We aimed to investigate whether the SHH signalling downstream components are expressed in this dopaminergic cell line. Cells were plated on poly-D-lysine (PDL) coated 12-mm2 glass coverslips in 24-well plates at a density of 5x104 cells/ coverslips. Cells were cultured in Dulbecco’s Modified Eagle Medium/F12 (DMEM/F12 1:1), supplemented with 5% fetal bovine serum (FBS) and 1% penicillin/streptomycin/ neomycin (to prevent bacterial contamination) and maintained at 37°C, 5% CO2, and 95% humidified air atmosphere. The cells were passaged when they reached approximately 80- 90% confluency. Cells were treated with 1nM SHH for 48h. Subsequently, after 2 days in-vitro (DIV2) controls and SHH treated cells were either fixed for IF or processed for RNA extraction and reverse transcription (RT-PCR). Coverslips of foetal mouse mesencephalic neural progenitor cells (NPCs) were kindly provided by Prof. Bjoern Spittau.
The coverslips of the SHH-treated MN9D cells and the vehicle control were fixed with 4% PFA for 20 min at RT and washed 3x 5 min in PBS. Afterwards, 1% SDS was used to permeabilize the cell membrane, then blocked in 1% bovine serum albumin (BSA) in PBS for 30 min at RT. MN9D cells were incubated with primary antibodies; rabbit anti-Smo (1:100; Abcam, ab38686), goat anti-Patch1 (1:100; Santa Cruz Biotechnology, SC6149), mouse anti-TH (1:1000; Merck-Millipore, Darmstadt, Germany, MAB318) diluted in PBS overnight at 4°C. The primary cultured mesencephalic dopaminergic neurons were incubated with primary antibodies; mouse anti BIII-tubulin-c (1:100; Developmental Studies Hybridoma Bank; DSHB Hybridoma Product E7) and rabbit anti-Smo (1:100; Abcam, ab38686) diluted in PBS overnight at 4°C. On the following day, cover slips were washed with PBS 3x 5 min each, incubated with the corresponding secondary antibodies coupled to Alexa Fluor 594 or Alexa Fluor 488 for 2h at RT. The coverslips were washed in PBS 3x 5 min, mounted with mixture of Flouromount-G and DAPI for counterstaining of the nuclei.
RT-PCR (reverse transcriptase-polymerase chain reaction)
Total RNA was isolated from SHH-treated MN9D cells and control, reverse transcribed for Synthesis of cDNAand processed for PCR, as described earlier .
PCR (Polymerase chain reaction)
The primers used for Gli1, Gli2, SHH, and ptch1 were supplied from the Gene-bank. Conventional PCR was performed on an Eppendorf Mastercycler (Wesseling, Germany). For detection of the active transcribed genes from the cDNAs, the following protocol was used; denaturation of the DNA for 5 min at 95°C, the optimum number of cycles -depending on primer pairs- of PCR amplification were performed at following conditions: denaturation for 30 sec at 95°C, primer annealing for 45 sec, and elongation for 45 sec at 72°C. Final extension for 10 min at 72°C was terminated by rapid cooling at 4°C. PCR products were run in agarose gel electrophoresis, and the bands of the reaction products were visualized using a UV transilluminator and TS view software.
To elucidate whether the genes of interest were up- or down-regulated, the reciprocal of each calculated value was estimated. The size and intensity of the bands of interest was measured using the ImageJ software. The optical density of each amplified band was calculated and numerically expressed as the relative density in comparison to the optical density of the background. The ratio of the determined intensity of the bands of interest was divided by the ratio of the intensity of the bands from “housekeeping” gene, GAPDH. The control was set to 1 and the different conditions were normalized to the control.
Data were presented as the mean ± SEM and were statistically analyzed using unpaired Student’s t-test, to compare treated group with the untreated control. Differences were considered statistically significant at (***P ≤ 0.001, **P ≤ 0.01 and *P ≤ 0.05). p>0.05 was considered not statistically significant. GraphPad Prism 6 (GraphPad Software Inc.) was the used software for statistical analysis.