Fig 2: No overt changes in myelination after GluA2 overexpression(A) Timeline for 4HT injections and mouse sampling. Sox10-CreER; Ai14 (control) and Sox10-CreER; Ai14; R26-Gria2 (+ GluA2) mice received 4HT at P9, P11, and P13 (0.2 mg per s.c. injection) and killed at P35.(B) Fluorescence images showing widespread tdTomato expression with or without EGFP-GluA2. Scale bar: 200 µm.(C) Confocal images of tdTomato and EGFP in the CTX of a Sox10-CreER; Ai14; R26-Gria2 mouse. Scale bar: 50 µm.(D) Confocal images of cortical tdTomato+ CC1+ OLs. Scale bar: 50 µm.(E) Densities of all tdTomato+ cells and tdTomato+ CC1+ OLs in the CTX (control versus + GluA2 group, total tdTomato+ cells: p = 0.246; tdTomato+CC1+ OLs: p = 0.972).(F) Percentage of CC1+ OLs among tdTomato+ cells. n = 5 (Sox10-CreER; Ai14, control) and 4 (Sox10-CreER; Ai14; R26-Gria2, + GluA2) mice for (E and F).(G) Fluorescence and confocal images of MBP in R26-Gria2 (control) and Sox10-CreER; R26-Gria2 (+ GluA2) mice. Upper panels: boxes correspond to confocal images in lower panels (optical thickness = 7 µm). Scale bars: 200 µm (up) and 50 µm (bottom).(H and I) Western blot analysis of myelin proteins from cortical lysates of R26-Gria2 and Sox10-CreER; R26-Gria2 mice (H) and its quantification (I). Control versus + GluA2 group, MBP: p = 0.877; MAG: p = 0.337; MOG: p = 0.611; CNP: p = 0.636. n = 3 mice per group.(J) Representative electron micrographs of callosal axons. Scale bar: 2 µm.(K) Scatterplots of g-ratios. More than 150 myelinated axons per mouse were analyzed.(L and M) Average g-ratio (L, control versus + GluA2 group, p = 0.880) and percentage of myelinated axons (M, control versus + GluA2 group. p = 0.12). For quantification of the percentage of myelinated axons, 700–900 axons were analyzed per mouse. n = 3 mice per group.Data are represented as means ± SEMs. Unpaired Student’s t test for (E), (F), (I), (L), and (M). See also Figure S1.

Fig 3: OPC-specific Tsc1 ablation impairs oligodendrocyte development in the brain.(A) Schematic diagram of the Akt-mTOR signaling pathway. The TSC1/2 complex and PTEN (orange circles) negatively regulate mTOR activity, whereas other molecules in green circles positively regulate it. (B) Experimental scheme for 4HT administration into Pdgfra-CreER; R26-EYFP; ±Tsc1f/f mice and mouse sampling. Three 4HT injections (1 mg per injection) were given between P20 and P21 (a total of 3 mg of 4HT). (C) Confocal images of phosphorylated S6 ribosomal protein (pS6) and EYFP+ cells in the CC at P20 +21. Arrowheads and asterisks indicate EYFP+ pS6+ cells and EYFP+ pS6- cells, respectively. Scale bar, 50 µm. (D) Quantification of the percentage of pS6+ cells among EYFP+ cells in the CC. n = 4 mice per group. (E) Fluorescence (left) and confocal microscopic (right) images of EYFP+ cells in the control and Tsc1 cKO mice (P20 +21). The confocal images of EYFP+ cells were taken from the CTX, and show their maturation stages. Arrowheads indicate EYFP+CC1+ OLs. Scale bars, 500 µm (left) and 50 µm (right). (F) Quantification of EYFP+ cells in the CC and CTX. (G - I) The numbers of EYFP+CC1+ OLs (G), EYFP+CC1-NG2- pre-OLs (H), and EYFP+NG2+ OPCs (I). (J) Percentages of OPC, pre-OL and OL among EYFP-labeled cells at P20 +21. n = 6 (control) or 3 (Tsc1 cKO) mice for (F - J). (K) Cell proliferation analysis with Ki67-expressing patterns. Confocal images of EYFP+NG2+Ki67+ OPCs in the CTX of a Tsc1 cKO mouse. Scale bar, 50 µm. (L) The percentage of Ki67+ cells among EYFP+ OPCs. n = 4 mice per group. (M) The number of total OPCs. (N) Western blot analysis of cortical lysates (P20 +21) for myelin proteins MOG, MBP, and CNP. Tsc1f/f (control) and 4HT-administered Pdgfra-CreER; Tsc1f/f mice (Tsc1 cKO) were used. (O) Quantification of levels of myelin proteins. n = 3 mice per group for (N, O). Data are represented as mean ±S.E.M. *p<0.05; **p<0.01; ***p<0.001. Unpaired Student's t-test. The numerical data for the graphs are available in Figure 1—source data 1. Original western images are available in Figure 1—source data 2.10.7554/eLife.32021.005Figure 1—source data 1.Numerical data for graphs in Figure 1.10.7554/eLife.32021.006Figure 1—source data 2.Original western blot images used for Figure 1K.Full-length western blot images from two separate blots (A and B). The blots were sequentially re-probed with the indicated Abs. The original images were cut with dashed-line boxes.