ARTICLE |
Correspondence to: M. Said Ghandour, UMR 7004 ULP/CNRS, Faculté de Médecine, 11 rue Humann, 67085 Strasbourg, France. E-mail: ghandour@neurochem.u-strasbg.fr
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Summary |
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Oligodendrocytes, the myelin-forming cells in the central nervous system, were visualized with excellent resolution at the light microscopic level using in situ hybridization (ISH). Digoxigenin (Dig)-tagged probes were synthesized and efficiently labeled by PCR. Specific probes to myelin genes were made by RT from brain total RNAs, followed by PCR with designed specific primers in the presence of Dig-11-dUTP. Probes specific to proteolipid protein (PLP), PLP and its isoform DM20 (PLP/DM20), and myelin oligodendrocyte glycoprotein (MOG) were synthesized and labeled. ISH was then applied on vibratomed tissue sections from mouse brains. Despite a low expression of MOG-specific and PLP-specific mRNAs in adult and newborn mouse brains, an oligodendrocyte population was detected. The specificity of Dig-labeled probes was confirmed with the double labeling of carbonic anhydrase II (CA II) and glial fibrillary acidic protein (GFAP) immunocytochemistry and ISH. This versatile and easy method for synthesis and labeling of specific probes to oligodendrocytes can be also applied to detect many other mRNAs in the nervous system and in other tissues. (J Histochem Cytochem 51:913919, 2003)
Key Words: PLP, MOG, nonradioactive PCR labeling, high-resolution in situ, hybridization, oligodendrocyte, carbonic anhydrase II, GFAP, myelin
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Introduction |
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A VARIETY of published methods have been extensively used for in situ hybridization (ISH) to detect gene expression in the nervous system (
The present study describes a simple and rapid method for the synthesis and labeling by the polymerase chain reaction (PCR) of desired probes for ISH. Proteolipid protein of the myelin and myelin oligodendrocyte glycoprotein (MOG) cDNA probes were synthesized and labeled with digoxigenin by PCR. These probes were used for ISH on vibratomed sections to visualize the myelin-forming cells in the central nervous system, the oligodendrocytes.
PLP is highly expressed in neural tissue, whereas MOG is considered a minor constituent of myelin (for reviews see
The detection of the targeted mRNAs was compared to the localization obtained with other ISH techniques described in the literature. A double-labeling technique using Dig and tetramethyl rhodamine isothiocyanate (TRITC)-labeled probes for ISH in conjunction with immunohistochemistry (IHC) of GFAP, an astrocyte marker, and carbonic anhydrase II (CA II), an oligodendrocyte marker (
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Materials and Methods |
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RNA Isolation
Total RNA was isolated from C57BL/6 mouse brains using TRIzol reagent according to manufacturer instructions (GIBCO BRL; Cergy Pantoise, France). To eliminate any eventual contamination with genomic DNA, isolated RNAs were then treated with DNase I, RNase free (Amersham Pharmacia Biotech; Orsay, France). In addition, before reverse transcription (RT), and the mRNA, we checked for genomic contamination by amplifying the RNA in a PCR reaction with primers that detect genomic PLP/DM20 (
Probe Preparation by RT-PCR Dig or TRITC Labeling
cDNAs were prepared from total mouse brain RNA using a Qiagen Omniscript kit (Qiagen; Courtaboeuf, France). Briefly, total RNA (12 µg/reaction) in the presence of Oligo dT15 primers (Promega; Charbonnieres, France), and Omniscript Reverse Transcriptase was incubated at 37C for 1 hr. Three µl of cDNA from the RT reaction was then subjected to PCR using Ready-to-Go PCR beads (Amersham Pharmacia Biotech) in the presence of digoxigenin-11-dUTP or TRITC-5-dUTP (Roche; Meylan, France) and 25 ng of each specific primer to PLP, PLP/DM20, or MOG. PLP/DM20, (
Tissue Preparation
Mice at postnatal days 1, 20, and 60 and one 1-year-old mouse were anesthetized with 9% chloral hydrate and the brains were fixed by intracardiac perfusion with a fresh solution of 4% paraformaldehyde (Electron Microscopy Sciences; Fort Washington, PA) in 0.1 M phosphate buffer, pH 7.4, containing 0.9% NaCl (PBS). Brains and spinal cords were removed and then postfixed in the same fixative for 24 hr at 4C. Fifty- and 100-µm sagittal sections were cut with a vibratome (Technical Products International; St Louis, MO). Sections were then kept at 4C overnight in PBS or 2 x SSC buffer.
Dot-blot Probes
Dig-labeled probes were tested by the dot-blot method using a nitrocellulose membrane (Millipore; Saint Quentin, France). The denatured probes were serially diluted and spotted onto a nitrocellulose membrane. Spotted probes were then incubated with alkaline phosphatase (AP)-tagged anti-Dig antibody and detected as described below.
In Situ Hybridization
Sections were washed in 0.1 M Tris-buffered saline, pH 7.5 (TBS) twice for 10 min at room temperature (RT), treated with 0.5% Triton X-100 (SigmaAldrich; Saint Quentin Fallavier, France) for 15 min at RT, and then briefly washed in the same buffer. Sections were treated with proteinase K (GIBCO BRL) 10 (µg/ml in 0.1 M Tris buffer, pH 8.0, 0.05 M EDTA for 10 min at 37C, immersed in 0.1 M triethanolamine (TEA) for 10 min, in 0.25% acetic anhydride in 0.1 M TEA for 15 min, and then washed in TBS. One µl of 1:2 diluted labeled RT-PCR product was added to 100 µl prehybridization solution (50% formamide, 2 x SSC, 5% dextran sulfate, 1 x Denhardt's, and 0.1 mg/ml salmon testis DNA), boiled for 10 min, and snap-cooled on ice.
Sections were incubated in the above hybridization solution overnight at 40C. Next day the sections were briefly washed in 4 x SSC, followed by two sequential washes of 5 min in 2 x SSC, 1 x SSC, and 0.5 x SSC. After three washes of 10 min in 0.1 M Tris buffer, tissue nonspecific binding was blocked by incubation of sections in 3% DNA blocking solution (Roche) for 1 hour at RT. Sections were rinsed in TBS and incubated with AP-labeled anti-DIG antibodies (anti-digoxigeninAP, Fab fragments; Roche) at 1:200 dilution in 1 x Denhardt's in TBS at RT for 1 hr. Sections were then washed in TBS three times for 10 min and finally with 0.1 M Tris-HCl, 0.1 M NaCl, 0.05 M MgCl2, pH 9.5 buffer (TBS 9.5). The color was developed in TBS 9.5 in the presence of nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) (Roche) for 30 min to 1 hr.
Sections were rinsed several times in TBS and then mounted on glass slides with AquaPoly/Mount (Polysciences; Warrington, PA). Controls for ISH consisted of sections incubated in prehybridization solution without addition of cDNA probes. Other control experiments were performed using sense Dig-labeled probes.
Double Labeling for PLP/DM20 ISH and Immunolocalization of CA II and GFAP
One-month-old mice were used. Tissue sections labeled for ISH with AP for PLP/DM20 or MOG were then treated for IHC. After several washes in TBS, pH 7.5, sections were incubated with 0.3% Triton X-100 for 15 min, then incubated with 1 x DNA blocking solution (Roche) for 30 min at RT and then with a rabbit polyclonal antibody to GFAP at 1:200 dilution (Dako; Glostrup, Denmark). Peroxidase-labeled secondary antibody to rabbit IgG at 1:200 dilution was used and peroxidase activity was detected using 3,3' diaminobenzidine4HCl (DAB) and hydrogen peroxidase. All substances if not otherwise stated were purchased from Sigma. A similar protocol was used for the double ISH with PLP/DM20TRITC probes and CA II immunofluorescence. CAII antibodies bound to tissue sections were revealed with fluorescein (FITC)-tagged anti-rabbit IgG.
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Results |
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Probe Synthesis and Labeling
cDNA probes to a major myelin protein in the CNS, PLP and its isoform DM20, were synthesized by RT-PCR from 20-day-old brain total RNA (
The PCR products were separated on a 1.5% agarose gel after electrophoresis. The bands revealed with ethidium bromide correspond to the predicted sizes of the amplified fragments. The theoretically estimated sizes of the probes were 730, 604/499, and 113 bp for MOG, PLP/DM20, and PLP, respectively (Fig 1A). RT-PCR products for PLP/DM20 and MOG from 1-, 20-, and 130-day-old mice total brain RNAs (Fig 1B) showed high expression at postnatal day 20, whereas the expression was much lower in 1- and 130-day-old mice. To label the cDNA probes, Dig-11-dUTP was introduced to the reaction mixture before PCR cycling. The efficiency of cDNA labeling was checked using a series of dilutions of PCR products on dot-blots (not shown). The probes were then used without purification after the PCR step. However, a purification step of PCR products did not further improve the ISH technique used in this study.
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In Situ Hybridization
We used the probes Dig-labeled by RT-PCR for ISH. To determine the specificity of the method, we used three probes to assess PLP/DM20, PLP, and MOG mRNAs in oligodendrocytes. Oligodendrocytes were stained in brain and spinal cord throughout different stages of mouse postnatal development. Of the three probes, PLP/DM20 gave the most intense staining in brain (Fig 2A2D). Higher magnification of the corpus callosum at day 20 showed typical perinuclear cytoplasmic staining. Other authors (
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The proteolipid protein gene produces two alternatively spliced proteins: DM20 and PLP. Our primers specific for PLP only were chosen mainly from the latter half of exon 3, which is missing in the splice form that produces mRNA for DM20. ISH using this probe labeled oligodendrocytes in brain and spinal cord from day 1 (Fig 2E2G), day 20 (Fig 2H), and adult mice (Fig 2I). Higher magnification (Fig 2G and Fig 2I) showed the same pattern of staining as with PLP/DM20 probes, which was predicted. Control experiments using hybridization buffer without cDNA (Fig 2J) or with labeled sense probes (not shown) did not show any significant staining in brain sections.
PLP and DM20 are highly expressed in oligodendrocytes, accounting for approximately 40% of total protein. To determine the sensitivity of our method, we assessed expression of MOG mRNA. MOG is a minor component of myelin, representing 0.050.1% of total myelin protein (
Combined IHC and ISH
We also assessed the possibility of using this method in combination with IHC. We used the PLP/DM20 probe for ISH and for IHC we used a classical marker for astrocytes, GFAP. The hybridization of the Dig-cDNA labeled probes and the detection of AP activity with BCIP and NBT were restricted to oligodendrocytes, while the typical astrocyte morphology was revealed with GFAP using immunoperoxidase IHC. No overlapping was observed between the two cell populations (Fig 4A and Fig 4B). Furthermore, we combined ISH of PLP/DM20 using cDNA probes labeled with dUTPTRITC and the immunofluorescence of CA II, a marker for oligodendrocytes (
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Discussion |
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PCR represents the most common and widespread method for direct amplification of specific sequences of nucleic acid target molecules. The most sensitive method uses single-stranded cRNA riboprobes. Although these probes give little background, their synthesis requires elaborate techniques. The use of nonradioactive probes synthesized by PCR provides a significantly more rapid procedure in comparison with the preparation of probes using plasmid vectors. The use of crude Dig-labeled probes by PCR is less time-consuming because plasmid amplification, cutting, separation, and cleaning of DNA are excluded and concentration of probe by amplification and labeling takes place in one step in the PCR reaction. Dig-labeled DNA probes using RT-PCR have been used to assess mRNA in bone tissue (
Another advantage of the PCR-generated Dig-labeled probes is that the size and sequence can be easily selected. In addition, several sequences from the same mRNA can be easily generated and compared. We found that the size of the Dig probes between 400 and 700 bp gives the most intense staining for ISH. However, low sizes of 100 bp, which are hard to construct for RNARNA ISH, can be successfully labeled by PCR. We demonstrated this by using primers of 113 bp to detect the expression of PLP-specific mRNA. However, fewer cells were stained with the PLP-specific probe compared with PLP/DM20 probes. This observation is in accordance with previous studies that detected expression of DM20 before PLP in brain as early as E9.5 (
In addition, the sensitivity of the ISH technique used in this study was proved by detection of low-expression MOG transcripts in oligodendrocytes. Published data (
We also combined the ISH method with IHC to detect two types of cells, oligodendrocytes and astrocytes, in the same tissue section and to visualize the same cell population detected for the mRNA content and for antibodies to the cell markers. The combined ISH and immunohistofluorescence technique used in the present study provides evidence for the specificity of the cDNA probes and the antibodies alike. The present report shows that in situ hybridization with Dig-labeled cDNA by RT-PCR provides a simple and sensitive method to detect myelin mRNAs.
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Footnotes |
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1 Authors contributed equally to this study.
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Acknowledgments |
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Supported by Association Française contre les Myopathies (AFM) to MSG and NINDS 38236 to RPS. We wish to thank Prof D. Grucker for support and encouragement and Ms D. Bessert and E. Scherrer for technical assistance.
Received for publication August 12, 2002; accepted February 12, 2003.
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