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Mouse NG2+ oligodendrocyte precursors express mRNA for proteolipid protein but not its DM-20 variant: a study of laser microdissection-captured NG2+ cells - PubMed

  • ️Wed Jan 01 2003

Mouse NG2+ oligodendrocyte precursors express mRNA for proteolipid protein but not its DM-20 variant: a study of laser microdissection-captured NG2+ cells

Ping Ye et al. J Neurosci. 2003.

Abstract

Despite recent advances in our understanding of lineage of oligodendrocytes, detailed molecular characterization of this lineage in vivo is limited, primarily because of our inability to obtain a pure population of cells in situ. To define the molecular characteristics of oligodendrocyte lineage cells during development and their response to injury, we developed a strategy that uses laser capture microdissection (LCM) to isolate cells from sections and reverse transcription-PCR to determine mRNA expression. As a first step, we examined the expression of myelin-specific protein genes in NG2+ cells in cerebral cortex. We demonstrate that NG2+ cells in both developing and adult mice express NG2 mRNA but not mRNA for proteins specific for astrocytes, neurons, or microglia, indicating that a highly pure population of antigen-specific cells of the oligodendrocyte lineage can be obtained using LCM. Furthermore, we show that NG2+ cells express mRNAs for proteolipid protein (PLP), myelin basic protein, and 2',3'-cyclic nucleotide 3'-phosphodiesterase, but they dot not express DM-20 mRNA, a PLP mRNA splicing variant. Our data demonstrate that antigen-specific cells of oligodendrocyte lineage differentially express mRNA for myelin-specific proteins and their variants in vivo, partly define the gene expression in NG2+ cells, and raise questions about the cellular sites of DM-20 expression. This work also shows that LCM is a valuable tool to define and analyze gene expression in the cells of the oligodendrocyte lineage.

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Figures

Figure 1.
Figure 1.

Representative microphotographs of a cerebral cortical section stained for NG2 + precursors derived from a 4-month-old mouse before (A) and after (B) LCM, as well as that of the same captured cells on a membrane (C). Arrows in A indicate NG2 + cells. Note that these cells are absent in B.

Figure 2.
Figure 2.

Expression of NG2, GFAP, CD68, and NF-L mRNAs in cerebral cortical NG2 + cells and whole cerebral cortex. Total RNA was isolated from LCM-captured NG2 + cells (LCM) and from whole cerebral cortex (CTX) of a 4-month-old mouse, and each RNA preparation was subjected to RT-PCR. RT-PCR DNA products were fractionated on 1% agarose gel containing ethidium bromide and photographed under UV light. The mRNA products derived from RT-PCR are indicated on the top of each lane.A DNA ladder of 100 bp markers was loaded in the left lane, and the size of the 400 and 600 bp bands are indicated by arrows. Note that the bands at bottom of the figure are primers used for RT-PCR.

Figure 3.
Figure 3.

Expression of myelin protein mRNA in cerebral cortical NG2 + cells and whole cerebral cortex. A, Expression of PLP, CNP, MAG, and MBP mRNAs in 4-month-old mice. Total RNA was isolated from LCM-captured NG2 +cells(LCM) and from whole cerebral cortex (CTX) of amouse and was subjected to RT-PCR.RT-PCR DNA products were separated on 1%agarose gel. Each mRNA product derived from RT-PCR is indicated on the top of each lane. B, Expression of PLP–DM-20 and MBP mRNA during development. Total RNA was extracted from cerebral cortical NG2 + cells (LCM) and cerebral cortex (CTX) of a 5-d-old mouse (P5) and a 4-month-old mouse (A), respectively, and subjected to RT-PCR. In both panels, DNA 100 bp markers were loaded in the middle lane.The arrowheads on the left of the panel indicate the expected sizes for RT-PCR amplified DNA derived from PLP (687 bp) and DM-20 (582 bp) mRNA, respectively. The arrowheads on the right indicate the expected sizes for RT-PCR amplified DNA (479, 422, and 354 bp) derived from MBP mRNA variants, respectively.

Figure 4.
Figure 4.

Representative Southern blot hybridization analysis of RT-PCR amplified PLP–DM-20. A, RT-PCR amplified PLP–DM-20 DNA fragments derived from cerebral cortical NG2 + cells (LCM) and cerebral cortex (CTX) of a 5-d-old mouse (P5) and a 4-month-old mouse (A) were fractionated on 1% agarose gel. B, DNA fragments, fractionated on 1% agarose shown in A, were transferred onto a nylon membrane, hybridized with a 32P-labeled PLP ssDNA probe, and exposed to an x-ray film for ∼2 min. C, The blot shown in B was exposed with a film for an extended time (∼30 min). In B and C, arrowheads on the right of each indicate the expected sizes for RT-PCR amplified DNA derived from PLP (687 bp) and DM-20 (582 bp) mRNA, respectively.

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References

    1. Campagnoni AT, Roth HJ, Pretorius PJ, Campagnoni CW ( 1989) Expression of myelin basic protein genes in the developing mouse brain. In: Developmental neurobiology (Evrard P, Minkowski A, eds), pp 95–109.New York: Nestec.
    1. Chomczynski P, Sacchi N ( 1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156–159. - PubMed
    1. Dawson MR, Levine JM, Reynolds R ( 2000) NG2-Expressing cells in the central nervous system: are they oligodendroglial progenitors? J Neurosci Res 61: 471–479. - PubMed
    1. Diers-Fenger M, Kirchhoff F, Kettenmann H, Levine JM, Trotter J ( 2001) AN2/NG2 protein-expressing glial progenitor cells in the murine CNS: isolation, differentiation, and association with radial glia. Glia 34: 213–228. - PubMed
    1. Fujita N, Sato S, Kurihara T, Kuwano R, Sakimura K, Inuzuka T, Takahashi Y, Miyatake T ( 1989) cDNA cloning of mouse myelin-associated glycoprotein: a novel alternative splicing pattern. Biochem Biophys Res Commun 165: 1162–1169. - PubMed

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