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. 2018 Feb 20;7:e32021. doi: 10.7554/eLife.32021

Figure 4. OPC-specific Pten ablation promotes new myelination.

Figure 4.

(A) Experimental scheme for 4HT into Ptenf/f ± Pdgfra-CreER mice, and for mouse sampling for western blot analysis (B,C) and EM analyses (D –G). For western blot analysis, 4HT was injected into Ptenf/f (control) and Pdgfra-CreER; Ptenf/f mice at P14, P20 (twice), and P21 (1 mg per injection, a total of 4 mg), and the mice were sampled at P75. For EM, A single dose 4HT was injected at P13, 15, and 17 (a total of 3 mg), and the mice were sampled at P30. (B) Western blot analysis of cortical lysates for myelin proteins of control and OPC-targeted Pten cKO mice (P14 +61). (C) Quantification of the levels of PLP, MAG, MOG, MBP and CNP in the western blot (B). n = 3 mice per group. (D) Representative electron micrograph (EM) of the CC of the control and Pten cKO mice (P13 +17). Arrows indicate thickened myelin in Pten cKO mice. Scale bar, 500 nm. (E) Scatter plot of g-ratios. More than 100 myelinated axons per mouse were analyzed. (F) Average g-ratio. (G) Percentage of myelinated axons was increased in Pten cKO mice. More than 700 axons were analyzed for myelination per mouse. n = 3 mice per group for (D - G). (H, I) Fluorescence images of EGFP+ cells in the CC (G) and CTX (H) of Pdgfra-CreER; R26-mEGFP;±Ptenf/f mice. 4HT (1 mg per injection, two injections per day) was injected at P25 and P26 (a total of 4 mg), and the mice were killed 3, 10, or 20 days later. Arrowheads indicate increased EGFP+ slender processes, reminiscent of bundles of myelinated fibers in Pten cKO mice (P25 +20). Scale bar, 100 µm. (J) Immuno-EM of anti-EGFP immuno-gold particles in the CC of Pdgfra-CreER; R26-mEGFP; ±Ptenf/f mice (P20 +21). Arrows indicate EGFP+ newly formed immature myelin sheaths. Scale bar, 500 nm. (K) Percentage of EGFP+ myelinated axons increased at P20 +21. n = 3 mice per group. Data are represented as mean ±S.E.M. *p<0.05. Unpaired Student's t-test. The numerical data for the graphs are available in Figure 4—source data 1. Original western images are available in Figure 4—source data 2.

Figure 4—source data 1. Numerical data for graphs in Figure 4.
elife-32021-fig4-data1.xlsx (14.9KB, xlsx)
DOI: 10.7554/eLife.32021.016
Figure 4—source data 2. Original western blot images used for Figure 4A.
Full-length western blot images from three separate blots (A, B, and C) used in Figure 4A. The blots were sequentially re-probed as indicated. The original images were cut with dashed-line boxes. The blot D was used for the simultaneous detection PLP and β-actin using Odyssey infrared scanner (LI-COR).
elife-32021-fig4-data2.pptx (569.7KB, pptx)
DOI: 10.7554/eLife.32021.017