Article Figures & Data
Figures
- Figure 1.
mTOR expression is reduced in the mTOR cKO. A, GFP reporter expression induced by active Cre recombinase in white matter at PND7, PND14, and 8 weeks. B, Images of individual recombined (GFP+, green), Olig2+ (blue) cells (i,ii), and nonrecombined (Tomato Red+) Olig2+ (blue) cells (i) at PND7 in the corpus callosum of the mTOR cKO. C, PCR products amplified from PND14 spinal cord genomic DNA showing the floxed-mTOR (fl-mTOR) and the recombined-mTOR (recom-mTOR) alleles. D–F, Representative Western blots and quantification of mTOR, Phospho:total S6 ribosomal protein, and Phospho:total Akt expression in PND14 spinal cord. Blue bars represent PND14 mTOR cKO. G–K, Representative Western blots and quantification of mTOR, P-S6, total S6, P-Akt, and total Akt expression in PND25 spinal cord. Green bars represent PND25 mTOR cKO. Quantifications are expressed as percentage control, with the exception of the ratio of Phospho:total S6 and Phhospho:total Akt at PND14. Scale bars: A, 100 μm; B, 50 μm. All statistical comparisons were conducted with a Student's t test. *p < 0.05.
- Figure 2.
The extent of myelination is significantly reduced in mTOR cKO spinal cord. A, C, Representative electron micrographs from the spinal cord of control or mTOR cKO animals at PND14 (A) and 8 weeks (C). Scale bars: left, 2 μm; right, 0.5 μm. B, D, Scatter plots of G-ratios of representative animals at PND14 (B) and 8 weeks (D). G-ratio measurements were performed on ≥200 axons/animal; n = 3/group. p ≤ 0.01 at both time points.
- Figure 3.
Lack of mTOR results in a deficit in the initiation of myelination. Quantification of the percentage of axons that were myelinated in relation to axon diameter at PND14 (A) and 8 weeks (B). Differences in myelination were compared using two-way ANOVA and Tukey's post hoc test. *Significant differences in percentage myelinated axons at a given diameter (p ≤ 0.02).
- Figure 4.
Myelin protein and mRNA expression in the spinal cord of mTOR cKO. mRNA (A, C, E, G) and protein (B, D, F, H) expression was examined for MBP (A, B), PLP/DM20 (C, D), MOG (E, F), and MAG (G, H) in isolated spinal cord at the onset of myelination (PND14) and in the adult (8 weeks). Representative blots at each time point are shown for protein expression. mRNA analyses were performed on n = 6/group from at least two independent litters. Protein analyses were performed on n = 9/group from at least two independent litters. All quantifications are expressed as percentage control ± SE. *p ≤ 0.05.
- Figure 5.
Differentiation is impaired in the mTOR cKO spinal cord during development. A, Representative images of PDGFR∝ (yellow)/Olig2 (blue; nuclear) or CC1 (red)/Olig2 (blue; nuclear) immunofluorescence to identify progenitors and mature oligodendrocytes in the spinal cord at PND14. Scale bar, 100 μm. B–D, Quantification of average number Olig2+ cells (B), PDGFR∝+/Olig2+ cells (C), and CC1+/Olig2+ cells per section (D). *p ≤ 0.05.
- Figure 6.
Differentiation is impaired in the mTOR cKO adult spinal cord. A, Representative images of CC1 (red)/DAPI (blue; nuclear) immunofluorescence to identify mature oligodendrocytes in the spinal cord at 8 weeks. Scale bar, 100 μm. B, Quantification of the number of CC1+/DAPI+ cells in the white matter. *p < 0.01.
- Figure 7.
Myelin protein and mRNA expression in the cerebellum of mTOR cKO. Myelin protein (B, D, F, H) and mRNA (A, C, E, G) expression was determined for MBP (A, B), PLP/DM20 (C, D), and MOG (E, F) in isolated cerebellum of control and mTOR cKO mice at PND14, PND25, and 8 weeks. Representative blots at each time point are shown for protein expression. mRNA analyses were performed on n = 6/group from at least two independent litters. Protein analyses were performed on n = 9/group from at least two independent litters. All quantifications are expressed as percentage control ± SE. *p ≤ 0.05.
- Figure 8.
Myelin protein and mRNA expression in the cortex of mTOR cKO. mRNA (A, C, E, G) and protein (B, D, F, H) expression was determined for MBP (A, B), PLP/DM20 (C, D), MOG (E, F), and MAG (G, H) in isolated cortex of control and mTOR cKO mice at PND14, PND25, and 8 weeks. Representative blots at each time point are shown for protein expression. mRNA analyses were performed on n = 6/group from at least two independent litters. Protein analyses were performed on n = 9/group from at least two independent litters. All quantifications are expressed as percentage control ± SE. *p ≤ 0.05.
- Figure 9.
Differentiation and myelination in the brain of the mTOR cKO. A, Electron micrograph images: at PND14 (Scale bar, 100 nm) and at 8 weeks (Scale bar, 1 μm). B, Representative images of PDGFR∝ (yellow)/Olig2 (blue; nuclear) and CC1 (red)/Olig2 (blue; nuclear) immunofluorescence to identify early and late progenitors in the corpus callosum at PND14. Scale bar, 10 μm. C–E, Quantification of total Olig2+ cells (C), percentage PDGFR∝+/Olig2+ cells (D), and percentage CC1+/Olig2+ cells (E). *p ≤ 0.05.
