Tissues were then sectioned either at 60C80?m using a vibratome (Microm) or at 15 to 25?m using a cryostat (Leica CM1950) after cryoprotection in 20% sucrose (Sigma) in PBS and freezing in OCT media on the surface of dry ice. 2.4. that deletion, while not affecting OPC production, impairs generation of a previously unknown Olig2\expressing pMN\derived cell subtype that, in contrast to OPCs, does not upregulate Sox10, PDGFR or Olig1. Instead, these cells activate expression of AP identity genes, including and and, of note, retain Olig2 expression as they populate the spinal parenchyma at embryonic stages but also as they differentiate into mature astrocytes at postnatal stages. Thus, our study, by revealing the existence of Olig2\expressing APs that segregate early from pMN cells under the influence of Sulf2, supports the existence of a common source of APs and OPCs in the ventral spinal cord and highlights divergent regulatory mechanism for the development of pMN\derived OPCs and APs. mice were generated by crossing heterozygous knock\in line (Dessaud et al., 2007) with mice carrying one floxed allele and the R26R\tomato reporter (mice were crossed and and littermate embryos were selected by genotyping. GFAP\GFP and Aldh1L1\GFP transgenic mice were genotyped as previously reported (Gong et al., Maprotiline hydrochloride 2003; Heintz, 2004; Nolte et al., 2001). Parental mice were generated by crossing heterozygous mutant mouse to an transgenic mouse. Then, mice were crossed and and littermate embryos were selected by genotyping. All mice were maintained on a C57BL/6 background. 2.2. Thymidine analogue labeling For proliferation assays, the timed pups (P0) were injected intraperitoneally with 5\ethylyl\2\deoxyuridine (EdU, Invitrogen) at 50?g/g body weight once a day from postnatal Day 0 to postnatal Day 2. The animals were then sacrificed 2 hr after the last injection. EdU incorporation was detected using the AlexaFluor\647 Click\iT Imaging Kit (Invitrogen). 2.3. Tissue processing Spinal cords at the brachial level were isolated from E12.5 to E18.5 mouse embryos and Maprotiline hydrochloride fixed in 4% paraformaldehyde (PFA, Sigma) in phosphate\buffered saline (PBS) overnight at 4 C. Adult mice were perfused intracardially with 4% PFA (Sigma) in PBS. Adult spinal cords were dissected and RPS6KA5 postfixed in 4% PFA in PBS overnight at 4 C. Tissues were then sectioned either at 60C80?m using a vibratome (Microm) or at 15 to 25?m using a cryostat (Leica CM1950) after cryoprotection in 20% sucrose (Sigma) in PBS and freezing in OCT media on the surface of dry ice. 2.4. In situ RNA hybridization and immunofluorescent staining Simple or double in situ hybridization and immunofluorescent staining were performed on transverse sections as previously reported (Touahri et al., 2012; Vento et al., 2012). Digoxigenin\ or Fluorescein\labeled antisens RNA probes for and were synthesized using DIG\ or Fluorescein\labelling kit (Roche) according to the manufacturer’s instructions and revealed using either NBT/BCIP (Roche), INT/BCIP (Roche) or Alexa\fluor 488 Tyramide (Molecular Probes). Antibodies used in this study were as follows: goat anti\AldoC (Santa Cruz Biotechnology), rabbit and mouse anti\GFAP (DAKO), rabbit anti\NFIA (Active Motif), rabbit and goat anti\Olig2 (Millipore and R&D Systems), goat anti\Olig1 (R&D Systems), goat anti\Sox10 (Santa Cruz Biotechnology), rat anti\PDGFR (BD Pharmingen), rabbit anti\Zeb1 (Novus), mouse anti\Nkx6.1 (Hybridoma Bank), mouse anti\Cx43 (BD Biosciences) and rabbit anti\Cx30 (Thermo Fisher Scientific). Alexa Fluor\594\ or Alexa Fluor\488 or Alexa Fluor\647\conjugated secondary antibodies (Thermo Fisher Scientific) were used. 2.5. Imaging, cell counting, and statistical analysis Confocal images were acquired from tissue sections using Leica SP5 or SP8 confocal microscopes and were always represented as single optical plane sections. In some instances, Z\stacks were acquired at high magnification (63 objective) and images along the Z\axis were obtained using the Reslice feature of ImageJ software along a straight or broken Maprotiline hydrochloride line drawn in the X/Y plane. Images of simple or double bright\field ISHs were collected with Nikon digital camera DXM1200C on a Nikon Eclipse 80i microscope. Double ISH stainings and double ISH\immunofluorescence staining were imaged Maprotiline hydrochloride on Leica SP5 confocal microscope according to the method of Maprotiline hydrochloride Trinh et al. (2007) which allows acquiring in the same optical plane a high resolution confocal image of NBT/BCIP stain in the near infrared range together with the immunofluorescence or.