Olig2 Ablation in Immature Oligodendrocytes Does Not Enhance CNS Myelination and Remyelination

Deletion of Olig2 in immature OL at the neonatal stages inhibits OL maturation. A, Control (Ctrl; Plp1-CreERT) and iKO-Plp mice carrying the tdTomato reporter were given TAM daily from P5 to P9 and harvested at indicated stages. B, Representative grayscale images of cortical sections from Ctrl and iKO-Plp mice at P14 stained for MBP. Scale bars, 100 µm. C, Representative images of tdTomato (red) and staining for Olig2 (blue) and CC1 (green) cells in the cortex of Ctrl and iKO-Plp mice at P14. Arrows indicate the tdTomato⁺ cells. Scale bars, 30 µm. D, Percentages of tdTomato reporter⁺ cells in cortex that express Olig2 (left) and CC1 (right) at P14. E, Representative images of tdTomato (red) and staining for Olig2 (blue) and CC1 (green) cells in the corpus callosum of Ctrl and iKO-Plp mice at P14. Arrows indicate the tdTomato⁺ cells. Scale bars, 30 µm. F, Percentages of tdTomato reporter⁺ cells in corpus callosum that express Olig2 (left) and CC1 (right) at P14. G, Representative images of tdTomato (red) and staining for cytoplasmic Olig1 (Olig1, green) in the corpus callosum from Ctrl and iKO-Plp mice at P14. Scale bars, 20 µm. H, Left, Percentages of tdTomato⁺ cells in the corpus callosum with cytoplasmic staining for Olig1. Right, Total number of tdTomato⁺ cells and cytoplasmic Olig1⁺ cells in the corpus callosum per mm² at P14. I, Representative images of GST-π immunostaining in the cortex from Ctrl and iKO-Plp mice at P14. Scale bars, 60 µm. J, Fold change of the GST-π⁺ cell number in the iKO-Plp mice relative to Ctrl mice at P14. K, Percentages of CC1⁺ cells among tdTomato⁺ cells in the cortices of Ctrl and iKO-Plp mice at indicated stages. n = 5 animals per genotype. Data are mean ± SEM. ***p < 0.001; **p < 0.01; unpaired two-tailed Student's t test.

Mobp- and Mog-Cre expression in postmitotic immature and mature OLs in the developing brain and spinal cord. A, Representative images of fluorescent tdTomato expression (red) in postmitotic OLs in the developing corpus callosum and spinal cord in Mobp-iCre;Rosa-tdTomato mice at P14 stained for CC1 (green) and PDGFRα (purple). Scale bars, 50 µm. B, Enlarged inset of image in A. Arrows indicate CC1⁺ postmitotic OLs positive for tdTomato. Arrowhead indicates PDGFRa⁺ OPC that does not express tdTomato. Scale bar, 20 µm. C, Representative images of fluorescent tdTomato expression (red) in postmitotic OLs in the developing corpus callosum and spinal cord in Mog-iCre;Rosa-tdTomato mice at P14 stained for CC1 (green) and PDGFRα (purple). Scale bars, 50 µm. D, Enlarged inset of the spinal cord in C. Arrows indicate CC1⁺ postmitotic OL cells positive for tdTomato. Arrowhead indicates PDGFRa⁺ OPC that does not express tdTomato. Scale bar, 20 µm.

Olig2 ablation causes maturation arrest and myelination deficits. A, Representative images of the cerebral white matter of control (Olig2^fl/fl) and cKO-Mobp mice at P7 stained for MBP. Scale bars, 100 µm. B, Representative images of P7 spinal cords from control and cKO-Mobp mice immunostained for Olig2 (red) and MBP (green). Scale bars, 20 µm. C, MBP density in the brain and spinal cord of cKO-Mobp mice relative to control mice at P7. D, Representative images of the cerebral white matter of control (Olig2^fl/fl) and cKO-Mog mice at P7 stained for MBP. Scale bars, 100 µm. E, Representative images of P7 spinal cords from control and cKO-Mog mice immunostained for Olig2 (red) and MBP (green). Scale bars, 100 µm. F, MBP density in the brain and spinal cord of cKO-Mog mice relative to control mice at P7. G, Representative images of P14 corpus callosum immunostained for CC1 (green) and Olig2 (red). Dotted lines indicate the subcortical white matter tracts. Scale bars, 20 µm. H, Fold change in CC1⁺ cell number (left) and percentage of CC1⁺ cells among Olig2⁺ cells in the cKO-Mog mice compared with control mice. I, Representative images of P14 cortex from control and cKO-Mog mice immunostained for Olig1 (red) and Olig2 (green). Scale bars, 20 µm. J, Frequency of cytoplasmic Olig1⁺ cells in the cortex of cKO-Mog mice relative to control mice. n = 4 animals per genotype. Data are mean ± SEM. ***p < 0.001; **p < 0.01; *p < 0.05; unpaired two-tailed Student's t test.

Olig2 deletion in mature OLs does not cause demyelination. A, Ctrl (Plp1-CreERT) and iKO-Plp adult mice (8-week-old) carrying the tdTomato reporter were administered with TAM daily for 7 d and killed at 1 (1 M) or 11 months (11 M) after TAM. B, C, Representative images of tdTomato (red), Olig2 (green), and staining for SOX10 (B) and GST-π (C) in the cortex from Ctrl and iKO-Plp mice at 1 month after TAM injection. Arrows indicate the tdTomato⁺ cells. Scale bars, 20 µm. D, Percentages of tdTomato⁺ cells in the cortex that express Olig2 (left), SOX10 (middle), and GST-π (right) in Ctrl and iKO-Plp mice. n = 3 animals per genotype. E, Representative images (left) of tdTomato (red), Olig2 (green), and staining for cleaved caspase 3 (CC3, purple) and quantifications (right, n = 3 animals per genotype) in the cortex from Ctrl and iKO-Plp mice at 1 month after TAM injection. Scale bars, 20 µm. F, Representative images of Ctrl and iKO-Plp mice cortex stained for MBP or Olig2 at 11 months after TAM injection. Scale bars, 50 µm. G, Quantifications for the area of MBP⁺ cells (left) and Olig2⁺ cells (right) in the cortex of iKO-Plp mice relative to controls. Scale bars, 20 µm. n = 3 animals per genotype. H, Representative EM images showing optic nerve and spinal cord in the Ctrl and iKO-Plp brains at 11 months after TAM injection. Scale bars, 2 µm. I, Quantification of myelin g-ratio in the optic nerve (left) and spinal white matter (right) from Ctrl and iKO-Plp mice. More than 150 axons were counted from 3 animals per group. n = 3 animals per genotype. Data are mean ± SEM. **p < 0.01; *p < 0.05; unpaired two-tailed Student's t test.

Olig2 activates the transcriptional program required for OL maturation from iOL. A, Visualization of Olig2 and H3k27ac occupancy on representative genes involved in transcriptional regulation in immature OLs and maturing OLs. B, Visualization of Olig2 and H3k27ac occupancies on representative myelin gene loci in immature OLs and mature OLs. C, Left, Representative images of Ctrl (Plp1-CreERT) and iKO-Plp brains at P14 stained for ASPA. Scale bars, 50 µm. Right, Fold change in ASPA⁺ cell frequency. n = 5 animals per genotype. D, Representative images of coronal sections from Ctrl and iKO-Plp cortices at P14 immunostained for Sox10. Scale bars, 50 µm. E, Representative images of the cortex of tdTomato (red) and staining for Sox10 (green) in Ctrl and iKO-Plp mice at P14. Arrows indicate the tdTomato⁺ cells. Scale bars, 50 µm. F, Left, Fold change in Sox10⁺ cell frequency (left) and the percentage of tdTomato⁺ cells that express SOX10 (right) in the cortex of cKO-Plp mice relative to Ctrl mice. n = 5 animals per genotype. Data are mean ± SEM. ***p < 0.001; **p < 0.01; unpaired two-tailed Student's t test.

Olig2 is required for CNS remyelination. A, TAM treatment scheme for LPC-induced demyelination in the spinal white matter of Ctrl (Plp1-CreERT) and iKO-Plp mice carrying the tdTomato reporter. B, C, Representative images of spinal LPC lesions (dashed lines) of Ctrl and iKO-Plp mice at dpi 3 stained for MBP (green) and DAPI (blue). C, Relative lesion size and MBP⁺ areas. Dashed lines indicate the DAPI dense lesion sites. Scale bars, 20 µm. D, Representative images of spinal LPC lesions (dashed lines) of Ctrl and iKO-Plp mice at dpi 21 stained for Olig2 (green) and CC1 (blue). Scale bars, 20 µm. E, Fold change in Olig2⁺ (left) and CC1⁺ cell (right) frequencies in the cKO-Plp mice relative to Ctrl mice at dpi 14 and 21. F, Left, Representative images of spinal LPC lesions (dashed lines) of Ctrl and iKO-Plp mice at dpi 21 stained for MBP (green) and with DAPI to mark nuclei (blue). Scale bars, 20 µm. Right, Relative intensity of MBP staining in spinal LPC lesions of Ctrl and iKO-Plp mice at dpi 14 and 21. G, ISH for Plp1 mRNA in spinal LPC lesions (dashed lines) of Ctrl and iKO-Plp mice at dpi 14 and 21. Scale bars, 50 µm. H, Quantification of Plp1⁺ OLs in spinal LPC lesions of Ctrl and iKO-Plp at dpi 14 and 21. I, Left, Representative images of cross semithin sections of LPC lesions (dashed lines) stained with toluidine blue (scale bars, 20 µm) and transmission electron microscopy images (scale bars, 2 µm) from Ctrl and iKO-Plp mice. Right, Percentages of axons that are myelinated in spinal LPC lesions of Ctrl and iKO-Plp mice at dpi 14. n = 5 animals per genotype. Data are mean ± SEM. ***p < 0.001; **p < 0.01; unpaired two-tailed Student's t test.