Animals
Experiments were performed with male OF1 strain mice pups from adult females purchased from Charles River (L’Arbresle, France). Samples of tissue has been collected according the directive 2010/63/EU of the European Parliament.
Facs Analysis
After removing the cerebellum and olfactory bulbs, the brains were dissociated into single cell suspensions using the Neural Tissue Dissociation Kit containing papain (Miltenyi, Germany) (Post-natal day < 10) or Adult Brain dissociation kit (Miltenyi Biotec, Germany) (Post-natal day 10 to 21) and the gentleMACS Octo Dissociator with Heaters (Miltenyi Biotec, Germany). Cells were filtrated on cell strainers (70µm, Miltenyi Biotec, Germany).
After counting and resuspension at 10.106 cells/ml in FACs buffer (Dulbeccos’s Phosphate Buffer Saline (Gibco Life Technologies, UK), 2mM EDTA (Sigma Aldrich, USA), 0,5% Bovine Serum Albumin (Miltenyi Biotec GmbH, Germany), cells were incubated with Fc blocker (BD Biosciences, France), then fluorophore-conjugated antibodies against mouse O4 and PDGFRα (both Miltenyi Biotec) or their corresponding control isotypes (Miltenyi Biotec), and viability marker (FVS 780, BD Biosciences) at concentrations recommended by the manufacturers or calculated after titration. Cells were washed with FACs buffer, resuspended in PBS and FACs analysis was done within 24hrs.
After doublets exclusion based on morphological parameters, we analyzed the percentages of PDGFRα+, O4+, and PDGFRα+/O4 + cells.
O4 + and/or PDGFRα cell sorting
Brain cell suspensions were obtained as described above (see FACS analysis). O4 + positive cells were first isolated using anti-O4 MicroBeads (Miltenyi Biotec, Germany) and negative fractions were then incubated with anti-PDGFRα MicroBeads (Miltenyi Biotec, Germany) to isolated PDGFRα-positive cells according to manufacturer instructions and as previously described (Bokobza et al., 2021; Boccazzi et al., 2021).
Primary PDGFRα + OPC culture
Brains single cell suspensions from post-natal day 5 pups were prepared using Neural Tissue Dissociation Kit containing papain (Miltenyi, Germany). PDGFRα-positive cells were isolated using the anti-PDGFRα MicroBeads (Miltenyi Biotec, Germany). PDGFRα-positive cells were cultured on poly-D-lysine-coated 24-well plates (30,000 cells/well) in proliferation medium consisting of Neurobasal (Gibco, France) supplemented with 2% B27 (Gibco, France), 1% penicillin/streptomycin, 20 ng/ml PDGF-AA, and 40 ng/ml bFGF (Sigma-Aldrich, France). After 3 days in proliferation medium, cells were switched to differentiation medium consisting of DMEM/High Glucose (Gibco, France) supplemented with 1% N2, 2% B27, 1% penicillin/streptomycin, 0.01% BSA, 10 nM d-biotin, 5ug/ml N-acetyl-cysteine and 40 ng/ml T3 (Sigma-Aldrich, France). Cells were treated with WNT Agonist II, (SKL2001, Calbiochem, USA), at different concentrations (3 or 10 µM) or with vehicle and fixed after 24, 48 and 72 hours for immunocytochemistry (ICC) analysis (see below).
Oli-neu Cell Line Culture
The oligodendroglial precursor cell line Oli-neu was cultured in the proliferative medium DMEM Glutamax high glucose (Gibco, France) supplemented with 1% horse serum, 1% of N2 supplement (Gibco, France), 10 µg/ml insulin, 0.35 µg/ml 3′,5-Triiodo-L-thyronine, 0.4 µg/ml L-Thyroxine, and 1% penicillin/streptomycin (Sigma-Aldrich, France). When cells reached 60% of confluence, 1⎧M of PD174265 (EGFR tyrosine kinase inhibitor, ChemCruz, USA) was added to the medium to differentiate cells. Proliferative cells were maintained in proliferative medium containing 0.1% of DMSO.
Pharmacological Modulation Of Wnt Pathway In Oli-neu Cell Line
Vehicle (DMSO 0.1%) or pharmacological treatments with WNT agonist II (Calbiochem, USA), CT99021 (Selleck Chemicals, USA) and XAV939 (Sigma-Aldrich, France) were added with PD174265 at the beginning of differentiation. Vehicle corresponded to medium containing 0,1% of DMSO.
Plasmid Constructs And Transfection Assays In Oli-neu Cell Line
The human GPR17 promoter luciferase construct was previously described [19]. The mouse Gpr17 promoter was cloned and subsequently modified in the PGL4.17 vector (Promega, France) upstream to the firefly luciferase reporter gene using the NEBuilder technology (New England Biolabs, France). The mouse 1.1kb promoter is the antisense mm10 sequence Chr18:31,949,371 − 31,950,469, corresponding to the − 2460/-1362 region relative to the transcription start site, the A of the ATG codon being considered at position + 1 (mm10 Chr18:31,948,009). The sequence was inserted between the HindIII and XhoI sites. Constructs comprising different mutated promoters (targeted mutations) were generated using Q5 Site-directed mutagenesis kit (New England Biolabs, France) according to manufacturer’s protocol. Two targeted mutations were generated: WRE1 (TCTTTG to CCCTCG at position − 2416/-2411) and WRE2 (CAAAGA to CCAGGG at position − 1850/-1845). To overexpress GPR17, a plasmid encompassing the coding sequence of the mouse Gpr17 gene under the control of the CMV promoter, referred as pcDNA3.1-GPR17 [20], was used. pcDNA3.1-EGFP (Promega, France) plasmid was used as a control.
For transient transfection assays of luciferase plasmids and siRNA, human GPR17 or mouse Gpr17 promoter luciferase fusion constructs (100 ng/well) were mixed with pRL-SV (10 ng/well in 96-well plates). For modulation of Id2 expression, a mix of siRNA containing anti-Id2-siRNA (Qiagen, SI010727 -43, -50, -57, -64) and Scramble siRNA (Qiagen, Ctrl siRNA 102781) was used to obtain 0, 5, 10, 25 or 50 nM of anti-Id2-siRNA. Scramble siRNA was used as negative control siRNA to reach 50 nM of total siRNA in each condition. The siRNA mixes were added to luciferase plasmids or used alone for RT-qPCR analysis. Plasmid DNA and/or siRNA were mixed with MACSFectin reagent (Miltenyi Biotec, Germany) in OptiMEM (Gibco, France) for 20 min according to manufacturer’s protocol (0,5 µl MACSFectin and 50 µl OptiMEM per well for luciferase experiments in 96 well plates, 4 µl MACSFectin and 200 µl OptiMEM per well for RT-qPCR experiments in 12 well plates). Transfection mix was added to Oli-neu suspension at 1.5x105 cells/ml just before plating in poly-L-lysine coated 96-well plates (luciferase experiments, 50 µl transfection mix added to 100 µl cell suspension) or 12 well plates (RT-qPCR, 200 µl transfection mix added to 1 ml cell suspension). After 48h in proliferation medium, transfected cells were switched to differentiating medium containing 1 µM PD174265 and simultaneously exposed to 3 µM of WNT agonist II or DMSO (0.1%) during 24h. Firefly and Renilla luciferase activities were evaluated using DualGlo Luciferase Assay System (E2920, Promega, France) according to manufacturer’s protocol in a microplate luminometer (Berthold, Germany).
For transient transfection assays of the expression vectors, pcDNA3.1-GPR17, named GPR17 plasmid [20], and pEGFP.N1, named CTRL plasmid, were used. Both Gpr17 and egfp coding sequences inserted in these plasmids were under the control of CMV promoter. Cells were plated on poly-L-lysine coated glass coverslips in 24 well plates at the density of 32,000 Oli-neu cells per well. On the next day, cells were transfected with 1 µg plasmid using 2 µl of MACSFectin (Miltenyi, Germany) per well according to manufacturer’s protocol. Briefly, plasmid and MACSFectin were mixed in 100 µl of Opti-MEM, incubated 20 min and added to the cell culture medium. 4 to 5h after transfection, the medium was changed with proliferation medium. After 48h in proliferation medium, transfected cells were switched to differentiating medium containing 1 µM PD174265 and simultaneously exposed to 3 µM of WNT agonist II or DMSO (0.1%) during 48h. At the end of the treatment, cells were fixed with 4% paraformaldehyde during 20 min for subsequent immunostaining.
Rt-qpcr
Total RNA was extracted with the NucleoSpin® RNA plus XS (Macherey-Nagel, Germany) according to the manufacturer’s instructions. RNA concentration was assessed by spectrophotometry with the NanodropTM apparatus (ThermoFisher Scientific). Total RNA (100–500 ng) was subjected to reverse transcription using the iScriptTM cDNA synthesis kit (Bio-Rad). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed in triplicate for each sample using SYBR® Green Supermix (Bio-Rad) for 40 cycles with a 2-step program (5 s of denaturation at 95° C and 10 s of annealing at 60° C). Amplification specificity was assessed with a melting curve analysis. Primers were designed using Primer3 plus software (sequences are provided in Supplementary Table 1). Specific mRNA levels were calculated after normalization of the results for each sample with those for Rpl13a mRNA (reference gene). The data are presented as relative mRNA units with respect to control group (expressed as fold change over control value).
Immunocytochemistry
Cells were fixed in 4% paraformaldehyde and subjected to immunocytochemistry (ICC) as previously described [21]. Mouse anti-CNPase (1/250; Millipore MAB326), mouse anti-NG2 (1:200; Abcam, UK), rabbit anti-GPR17 (1:100, custom antibody produced by PRIMM, Milan, Italy or, for Oli-neu ICC, CAYMAN #10136) and/or mouse anti-MBP (1:200, Chemicon MAB 382, Japan) were incubated overnight (4°C) and the secondary antibodies goat anti-rabbit, goat anti-rat or goat anti-mouse antibodies conjugated to AlexaFluor®488 or AlexaFluor®555 were incubated 1h at room temperature (1:600, Life Technologies, USA). Nuclei were counterstained with DAPI (Sigma-Aldrich, France). Cells were finally analyzed by a fluorescent microscope (Nikon Eclipse Ti-E).
The total number of PDGFRα + cells in each coverslip was evaluated by counting DAPI + nuclei, and the number of GPR17+, MBP+, or NG2 + cells with the ImageJ (NIH) software. Number of cells was determined in 20–25 optical fields chosen according to a pre-established scheme to represent the entire slide under a ×20 magnification using Nikon Eclipse Ti-E microscope. For CNPase-IR, images were acquired using Nikon Eclipse Ti‐E microscope. The number of DAPI + nuclei and the mean density of CNPase-IR were determined using an appropriate macro design with the ImageJ (NIH) software. 29–31 optical fields per group repeated on 2 independent experiments were analyzed.
Statistical analysis
Data were expressed as mean value with standard error of the mean (SEM). Using GraphPad Prism Software, data were tested for normality using the Kolmogorov–Smirnov normality test. Multiple comparisons in the same data set were analyzed by One way analysis of variance (ANOVA) with the Dunnett’s or Bonferroni post-hoc test or Two way ANOVA with the Bonferroni post hoc test. Single comparisons were made using Student’s t test.