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doi: 10.1242/10.1242/jcs.00103

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Nuclear organization in differentiating oligodendrocytes

¹ Program in Molecular and Cell Biology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814, USA
² National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA
³ Department of Anatomy, Physiology and Genetics, Program in Neuroscience, Uniformed Services University of the Health Sciences, Bethesda, MD 20814-4799, USA

View larger version (116K): [in a new window]   Fig. 1. PLP mRNA in situ hybridization in primary rat oligodendrocyte cultures. Phase contrast image analysis shows bipolar processes that are characteristic of progenitors (A), multiple branched processes characteristic of oligodendrocytes (C), and flat fibroblastic morphology of astrocytes (E). Brightfield images show that PLP mRNA was not detectable in cultured progenitors (B, same field as A) or astrocytes (F, same field as E). High levels of PLP mRNA (blue/black signal) accumulated in differentiated oligodendrocytes (D, same field as C). Bar, 50 µm.

View larger version (33K): [in a new window]   Fig. 2. SC35 immunostaining combined with genomic in situ hybridization. Merged images for PLP genomic in situ hybridization (green) combined with SC35 immunostaining (red) in astrocytes (A), progenitors (B), oligodendrocytes (C) and oligodendrocytes treated with -amanitin (D). IRBP genomic in situ hybridization (green) merged with SC35 immunostaining (red) in progenitors (E). Bar, 10 µm.

View larger version (17K): [in a new window]   Fig. 3. Quantitation of genomic in situ hybridization combined with SC35 immunostaining. The PLP gene and SC35 domains were scored as co-localized when there was pixel overlap in the red and green channels (see Fig. 2). SC35 domains are more frequently co-localized with the PLP gene in oligodendrocytes (oligos, n=110) compared with cells that do not transcribe detectable levels of PLP, i.e. astrocytes (astros, n=55), progenitors (OPs, n=116), and oligodendrocytes treated with -amanitin (oligos+AM, n=110) (P<0.05; Chi-square). As a control, the IRBP gene, which is not expressed in oligodendrocytes, had a frequency of PLP/SC35 co-localization similar to cells that do not express PLP (n=50). Error bars=standard error of the proportion.

View larger version (40K): [in a new window]   Fig. 4. 3D reconstruction of PLP genomic in situ hybridization merged with SC35 immunostaining in oligodendrocytes. The PLP gene (green) co-localizes with SC35 immunostaining (red) to appear as yellow at the periphery of nuclear domains enriched in SC35 in oligodendrocytes. Oligodendrocytes are shown as a 3D reconstruction imaged in the XY plane (A) and rotated 90 degrees to the XZ view (B). In progenitors shown as a 3D reconstruction, the PLP gene does not co-localize with SC35 imaged in the XY plane (C) or when rotated 90 degrees to the XZ view (D). The blue line outlines the nuclear periphery. Bar, 5 µm.

View larger version (14K): [in a new window]   Fig. 5. Quantitation of PLP and IRBP gene nuclear localization. The PLP and IRBP genes were classified as within the peripheral region of the nucleus if the measured distance between the center of the in situ hybridization signal and the edge of the nucleus was less than 1.5 µm. When the center of the in situ hybridization signal was located within the remaining nuclear volume, the localization was counted as central. In panel A, phase-contrast microscopy was used to identify the nuclear boundary. The PLP gene was more frequently associated with the peripheral region of the nucleus in oligodendrocyte progenitors (OPs, n=22) and oligodendrocytes (oligos, n=37). Analysis of individual IRBP alleles within each cell did not indicate a preferential association of IRBP alleles with the peripheral region of the nucleus (OPs, n=38; oligos, n=30). In panel B, both IRBP alleles were analyzed within each cell and were categorized as central-central (CC), central-peripheral (CP), or peripheral-peripheral (PP). In panel C, DAPI was used to identify the nuclear periphery. The PLP gene was non-randomly associated with a peripheral localization in astrocytes (astros, n=52), oligodendrocyte progenitors (OPs, n=103), or oligodendrocytes (oligos, n=101) (*P<0.05, chi-square). The PLP gene localization was not significantly different when comparing across cell types. Error bars=standard error of the proportion.

View larger version (24K): [in a new window]   Fig. 6. PLP gene nuclear localization. PLP genomic in situ hybridization (green or red) to detect the PLP gene combined with the nuclear stain DAPI (blue) to show the nuclear volume in astrocytes (A), progenitors (B) and oligodendrocytes (C). Bar, 10 µm.

View larger version (29K): [in a new window]   Fig. 7. Nuclear localization of SC35 splicing factors and Myt1 DNA-binding proteins in oligodendrocyte progenitors. Progenitor cultures were double immunostained using anti-SC35 detected with anti-mouse FITC (green) and anti-Myt1 detected with anti-rabbit Cy3 (red). 3D reconstruction of SC35/Myt1 double immunostain imaged in XY plane (A) and with the 3D image rotated 90 degrees to show XZ view (B). Bar, 5 µm.

View larger version (67K): [in a new window]   Fig. 8. PLP genomic in situ hybridization combined with Myt1 immunostaining. Merged images for PLP genomic in situ hybridization (green) combined with Myt1 immunostaining (red) in pre-oligodendrocyte progenitors (A), progenitors (B) and oligodendrocytes (C). IRBP genomic in situ hybridization (green) merged with Myt1 immunostaining (red) in progenitors (D). Bar, 10 µm.

View larger version (17K): [in a new window]   Fig. 9. Quantitation of PLP genomic in situ hybridization combined with Myt1 immunostaining. The PLP gene and Myt1 domains were scored as co-localized when there was pixel overlap in the red and green channels (see Fig. 8). Myt1 nuclear domains were found associated with the PLP gene in approximately 50% of the cells analyzed (pre-oligodendrocyte progenitors (preOP, n=50), progenitors (OPs, n=101), and oligodendrocytes (oligos, n=100). The IRBP gene, which is not expressed in oligodendrocyte progenitors, was also found associated with Myt1 nuclear domains in approximately 50% of the progenitor cells analyzed (n=50). No statistical difference was found between any of the groups using the chi-square statistical test. Error bars=standard error of the proportion.

View larger version (52K): [in a new window]   Fig. 10. Double genomic in situ hybridization for MBP and PLP genes. Genomic in situ hybridization with the X-linked PLP gene being detected with FITC-labeled PLP probe (green) and the autosomal MBP gene being simultaneously detected with a digoxigenin-labeled MBP probe (red). In progenitors (Fig. 10A, phase-contrast 10C) and in oligodendrocytes (10B, phase contrast 10D), the PLP and the MBP genes were spatially separated. Bar, 10 µm.