Animals, Tissue Processing and Immunohistochemistry
Olig1-Cre heterozygous mice were crossed with Dicer1lox/lox
mice to generate Olig1Cre+/-
mice, which were then bred with Dicer1lox/lox
mice to produce Dicer1CKO offsprings (Olig1Cre+/-
mice developed and behaved the same as WT. Dicer1lox/lox
mice were purchased from the Jackson laboratory (strain name, B6.Cg-Dicer1tm1Bdh
/J) (Harfe et al., 2005
). All animal use and studies were approved by the Institutional Animal Care and Use Committee of the University of Texas Southwestern Medical Center at Dallas, USA and Sichuan University at Chengdu, P.R. China.
For immunohistochemistry, mouse brains or spinal cord were short-fixed 1 hr in 4% paraformaldehyde and processed for vibratome-section. For BrdU pulse labeling, animals were injected intraperitoneally with 100 mg BrdU/kg body weight 3 hrs prior to sacrifice. We used antibodies to Olig2 (gift of C. Stiles, Harvard Medical School), Sox6 (gift of M. Wegner), PDGFRα (BD Bioscience, 558774), CC1 (Oncogene Research, OP80), MBP (Covance, SMI-94R), BrdU (BD Bioscience, 555627), GFAP (Sigma, G3893) and Hes5 (Santa Cruz, sc-13859). Monoclonal antibody to RIP were obtained from the Developmental Studies Hybridoma Bank at the University of Iowa under the auspices of the National Institute of Child Health and Human Development. The protocols of miRNA in situ hybridization and immunostaining following miRNA in situ hybridization are provided in detail in supplemental methods.
For electron microscopy, spinal cord and optic nerves were dissected and fixed in 2% glutaraldehyde and 4% paraformaldehyde in 0.1M cacodylate buffer (pH7.2) for 24 hrs and processed as previously described (Xin et al., 2005
RNA Extraction and qRT-PCR
Total RNAs were purified from tissues or cell cultures using TRizol reagent according to the manufacturer's instructions (Invitrogen). RNA was transcribed to cDNA with First-Strand cDNA Synthesis Kit (GE Healthcare). Quantitative real-time PCR was performed using the ABI Prism 7700 Sequence Detector System (Perkin-Elmer Applied Biosystems) and the relative gene expression was normalized to internal control such as Gapdh or Rpl13a. Primer sequences for SybrGreen probes of target genes are as follows: mouse Sox6: “tcagagcaatcaccacaccagaca” and “aaggttgaatgtcagggcaaaggc”; mouse Hes5: “tacctgaagcacagcaaagccttc” and “taaagcagcttcatctgcgtgtcg”; mouse Dicer1: “accagcgcttagaattcctgggag” and “gctcagagtccattccttgc”; mouse GAPDH: “gtgtgaaccacgagaaata” and “gttgtcatggatgacctt”; zebrafish mbp: “atcttcaacctgggagaaagccga” and “tgcttcccgtccatttcactctct”; zebrafish ef1a: “tcaagcctggtatggttgtgacct” and “acggatgtccttga cagacacgtt”; zebrafish rpl13a: “tcctccgcaagagaatgaacacca” and “tcaaacaccttcagc ctgtccaga”. miRNA-specific primers were purchased from Applied Biosystems, Inc. and miRNA qRT-PCR was performed according to manufacturer's instruction.
miRNA Microarray, Northern blot and Western blot analyses
miRNAs microarray assays were performed by using a service provider (LC Sciences). The assay started from 10 μg of total RNA samples on Dual sample chips with Rodentia miRNA Array based on Sanger miRBase Release 13.0.
For miRNA Northern blot analysis, RNA extracted from spinal cord was electrophoresed on a 20% polyacrylamide (7.6 M urea) gel in 1× TBE. Ten micrograms of RNA was denatured for 5 min at 70°C in a buffer containing 50% formamide and 10 mM EDTA (pH 8.0) before loading. After electrophoresis, RNA was then transferred onto a Zeta probe membrane (Biorad) in 0.5× TBE buffer at 80 V for 1 h. Hybridization was performed at 37°C according to a standard protocol. 32P-labeled Star-Fire oligonucleotide probes (IDT) against mature miR-219, miR-338-5p, and U6 were purified on Sephadex G-25 microspin columns (BioRad) and used in the hybridization. For Western blot analysis, protein lysates were resolved by SDS-PAGE and blotted using standard procedures. Antibodies used were as follows: Sox6 (Santa Cruz) and GAPDH (Abcam). Signals were revealed by using chemiluminescence with the ECL kit (Pierce) according to the manufacturer's instruction.
Culture of Rodent Oligodendroglial Precursor Cells and Rat Hippocampus-Derived Adult Neural Progenitor Cells
For mouse primary OPC-enriched cell cultures, cortical precursors were isolated from E15.5 as described previously (Chen et al., 2007
). Oligodendrocyte precursors were enriched in serum-free oligodendrocyte growth medium supplemented with bFGF and PDGF-AA. For miRNA tranfection study, primary OPCs were seeded in 24-well plates in oligodendrocyte growth medium and transfected with 50 nM of miRNA mimics (Pre-miRNAs, Ambion) using Lipofectamine 2000 (Invitrogen). A Cy3 labeled scrambled miRNA (Pre-miR™ miRNA Precursor Negative Control, Ambion Inc.) transfection was included as negative control and the transfection efficiency is >50% in OPC culture. Four days post-transfection, cultures were harvested for immunocytochemistry and qRT-PCR assay. In knockdown study, OPC-enriched cultures were transfected with 25 nM miRCURY LNA™ miRNA knockdown probes (Exiqon, Vedbaek, Denmark. mmu-miR-219, 139113-04; mmu-miR-338-5p, 139531-04; mmu-miR-338-3p, 139532-04) using Lipofectamine 2000, and control LNA knockdown probe was included as negative control (Exiqon, Product # 199002-04). The control knockdown probe has similar probe length as LNA knockdown probes, and it has no homology to any known miRNA or mRNA sequences in mouse, rat or human. Cells were then cultured in oligodendrocyte differentiation medium without mitogens and four days after transfection, cultures were harvested for analysis.
Isolation and culture of rat OPCs followed the protocol as previously described (Chen et al., 2007
). The rat hippocampus-derived adult neural progenitor cells (HCN) were originally isolated from adult female Fisher 344 rats. They give rise to homogeneous populations of oligodendrocytes on IGF-1 stimulation (Hsieh et al., 2004
). The progenitor cells expressed Olig2 when cultured in N2-FGF media (DMEM:F12 with N2 supplement and bFGF). HCN progenitor cells were grown in N2-FGF media, transfected with miRNAs or plasmids using Amaxa electroporator according to manufacturer's protocol and assayed for immunocytochemistry and qRT-PCR analysis.
miRNA Expression Vectors and Luciferase Reporter Assays
miR-219 locus on chromosome 2 and mir-338 locus on chromosome 11 with their ~200 bp flanking sequences were PCR amplified from mouse genomic DNA and inserted into pCMV6 vector or pCIG vector (Megason and McMahon, 2002
). For generating miRNA mutant constructs, the seeding sequence for miR-219 was changed from “GATTG” to “CTAAC”, seeding sequences for miR-338-5p and miR-338-3p were changed from “ACAAT” to “TGTTA” and from “CAGCA” to “GTCGT”, respectively.
Segments carrying putative miR-219 and miR-338 binding sites in 3′UTR of Sox6, Hes5 and Zfp238 were cloned into pMIR-REPORT vector (Ambion, Inc). For mutagenesis of 3′UTR for Sox6, the predicted binding site in pMir-reporter-Sox6UTR-I for miR-219 was changed from “ACAAT” to “GTCGA”, the two predicted binding sites in pMir-reporter-Sox6UTR-II for miR-338-3p was changed from “ATGCTG” to “GTCGAC” and from “ATGCTG” to “GTCGAC”, respectively by using QuikChange Multi Site-Directed Mutagenesis Kit (Stratagene, San Diego, CA).
Luciferase reporter constructs were cotransfected with vectors expressing miRNAs, their reverse sequences (219rev, 338rev) or mutant miRNAs (219Mu, 338Mu) into COS7 cells by Lipofectamine 2000. The pRSV-renilla luciferase plasmid was included as a control for transfection efficiencies. Luciferase activity was assayed 48 hours after transfection using the dual-luciferase reporter assay system (Promega). At least three transfection assays were performed to obtain statistically significant data. Statistic analyses were performed as previously described (Ye et al., 2009
In ovo and in utero Electroporation
Chicken eggs were incubated at 38 °C. Approximately 1 ul (3 μg/μl) of pCIG expression vectors carrying mir-219 or miR-338 was injected into a chicken embryo neural tube at stage HH 13–15 (E2.5) with the aid of Picospritzer III (Parker Hannifin). In situ hybridization or immunohistochemistry were performed as perviously described (Ye et al., 2009
For in utero electroporation, DNA solution (~2 μl) in PBS containing 0.01% fast green was injected into the lateral ventricle of the embryos at e14.5. After injection, electroporation (five 50 ms square pulses of 40 V with 990 ms intervals) was carried out. Plasmid DNAs (3 μg/μl) used for electroporation were pCMV-miR219 and pCMV-miR338 and control pRFP. Embryos were harvested 72 hrs after electroporation and processed for immunohistology. At least five embryos with expression of each transgene were analyzed and characterized.
Zebrafish Morpholino Injections
Approximately 1nl of morpholinos at 0.12 mM concentration for miR-219 and miR-338 (Gene Tools, LLC) was injected individually into Tg(olig2:egfp) zebrafish embryos at the one-cell stage. The morpholino sequences used were miR219: 5′ acagatgtccaggcacaattctt gg-3′ and miR338: 5′- caacaaaatcactgatgctggagtg-3′. Injected embryos at 3 dpf were paraformaldehyde-fixed, cryosectioned and immunostained using an anti-Sox10 antibody or processed for in situ RNA hybridization using probe for Mbp.