Extracellular matrix-induced myelin sheet formation by oligodendrocytes requires Scribble.

<p>A,B; To assess differentiation of OPCs derived from neonatal rat pups into oligodendrocytes, cells were immunolabelled with antibodies against O4 (red) and myelin basic protein (MBP, green). C; Scribble knockdown did not change the proportion of O4-positive cells that express MBP on either poly-D-lysine (PDL, grey bars) or poly-D-lysine-Matrigel (PDL-MG, black bars) substrates. D; Culturing oligodendrocytes on PDL-MG increased the proportion of cells exuding myelin-like membrane sheets (arrows in A). While Scribble knockdown in oligodendrocytes cultured on PDL did not affect the proportion of cells with membrane sheets, the percentage of cells with membrane sheets (arrows in B) was significantly decreased on PDL-MG. Percent of O4-positive oligodendrocytes with myelin membrane sheets: non-targeting PDL = 13.6% ± 0.6%, non-targeting PDL-MG = 23.5% ± 0.8%, Scribble PDL-MG = 13.3% ± 0.9%. Numerical results are presented as mean ± standard error of the mean (SEM). ANOVA with Tukey's multiple comparison test was used, <i>n</i> = 6 coverslips per condition, with four fields analysed per coverslip. *** <i>p</i> < 0.005. Scale bar = 50 μm.</p

Scribble regulates myelin thickness and ERK activation in oligodendrocytes.

<p>A,B,D,E; At P40, decreased myelination of small-diameter axons in both optic nerve (B) and spinal cord (E) of Scribble cKO mice persists relative to wild-type littermates (A,D). This is quantified for optic nerve in C (0.3–0.4μm: WT = 68.4% ± 6.4%, cKO = 36.2% ± 10.1% myelinated, <i>p</i> = 0.0002; 0.4–0.5μm: WT = 87.8% ± 6.0%, cKO = 68.2% ± 4.1% myelinated, <i>p</i> = 0.0034) and for spinal cord in F (0.3–0.4 μm: WT = 34.0% ± 4.4%, cKO = 9.3% ± 3.7% myelinated, <i>p</i> = 0.039; 0.4–0.5μm: WT = 47.5% ± 4.5%, cKO = 22.3% ± 2.5% myelinated, <i>p</i> = 0.033) Numerical results here and below are presented as mean ± SEM. Two-way ANOVA with Bonferroni's multiple comparisons test was used. <i>n</i> = 3 animals per genotype. At least 200 axons were analysed per animal, per region. G–I; Axons that are myelinated in cKOs have thicker myelin sheaths than do axons of similar diameters in wild-type littermates, as decreased mean g-ratios were observed in both optic nerve and spinal cord (G; optic nerve: WT = 0.755 ± 0.004, cKO = 0.704 ± 0.016, <i>p</i> = 0.012; spinal cord: WT = 0.760 ± 0.010, cKO = 0.702 ± 0.008; <i>p</i> = 0.018; Student's <i>t</i> test was used to compare means from each animal. At least 100 g-ratios were analysed per animal, per region.). The best fit lines obtained by linear regression differed significantly between cKO and wild-type datasets in both optic nerve (H; WT: slope = 0.063 ± 0.012, intercept = 0.720 ± 0.007, cKO: slope = 0.075 ± 0.015, intercept = 0.666 ± 0.010; <i>p</i> = 0.548 [slope], <i>p</i> < 0.0001 [intercept]) and spinal cord (I; WT: slope = 0.027 ± 0.004, intercept = 0.728 ± 0.006, cKO: slope = 0.012 ± 0.005, intercept = 0.688 ± 0.007; <i>p</i> = 0.022 [slope]). J–L; Increased abundance of phosphorylated ERK1/2 relative to total ERK1/2 was observed in western blots of lysates of optic nerve and spinal cord from Scribble conditional mutants (J). The ratio of phospho-ERK/ERK densitometry is provided in arbitrary units for optic nerve (K): p44ERK/ERK1: WT: 0.306 ± 0.064, cKO: 0.666 ± 0.027, <i>p</i> = 0.0066; p42ERK/ERK2: WT: 1.511 ± 0.025 cKO: 5.391 ± 0.924, <i>p</i> = 0.014; and spinal cord (L): p44ERK/ERK1: WT: 0.127 ± 0.046 cKO: 0.335 ± 0.051, <i>p</i> = 0.038; p42ERK/ERK2: WT: 0.997 ± 0.069, cKO: 5.218 ± 1.278, <i>p</i> = 0.030, Student's <i>t</i> test was used. A,B: Scale bar = 1 um. E,F: Scale bar = 1.5 um. * <i>p</i> < 0.05, ** <i>p</i> < 0.01, *** <i>p</i> < 0.005 versus Scrib WT.</p

Scribble cKO oligodendrocytes produce shorter myelin internodes.

<p>Myelin internodal lengths were determined in teased fibre preparations from P16 Scribble WT (A) and cKO (B) mice by measuring the distance between adjacent Caspr-positive paranodes (green). Internodal length was decreased in Scribble cKO large-diameter spinal cord axons relative to those observed in wild-type littermates (C; Scrib WT: 422.2 ± 16.0 μm, Scrib cKO: 345.8 ± 22.9 μm, <i>p</i> = 0.0262). Numerical results are presented as mean ± SEM. Student's <i>t</i> test was used; <i>n</i> = 3 animals per condition, at least 30 internodes per animal. * <i>p</i> < 0.05. Scale bar: 50 μm.</p

Decreased remyelination initiation is observed in Scribble cKO mice following focal demyelination in corpus callosum.

<p>A–F; Focal LPC lesions were induced in corpora callosa of 16-wk-old wild-type and Scribble cKO mice, and myelination was assessed at 21 d following focal demyelination caused by injection with lysophosphatidylcholine (LPC). In the contralateral (unlesioned) hemisphere (CL), no difference was seen in the proportion of axons in the corpus callosum that were myelinated in Scribble cKO compared to wild-type animals (A–C). Fewer remyelinated axons were observed in corpus callosum in the lesion area in Scribble cKO mice (E) relative to controls (D) as quantified in F; 0.4–0.5 μm: WT = 80.0% ± 9.8%, cKO = 41.3% ± 8.9% myelinated; 0.5–0.6 μm: WT = 79.4% ± 6.3%, cKO = 41.5% ± 9.5% myelinated; 0.6–0.7 μm: WT = 91.6% ± 3.0%, cKO = 44.3% ± 8.7% myelinated; >0.7 μm: WT = 92.7% ± 1.9%, cKO = 47.9% ± 2.5% myelinated. Numerical results here and below are presented as mean ± SEM. Two-way ANOVA with Bonferroni's multiple comparisons test was used. <i>n</i> = 3 mice per genotype. At least 200 axons were analysed per animal, per region. G–I; Myelin sheaths in the unlesioned hemispheres of Scribble cKO corpora callosa were thicker than those observed in wild-type animals, with both mean g-ratio (G; WT: 0.761 ± 0.010, cKO: 0.675 ± 0.011) and the intercept of the best-fit line (H; WT: slope = 0.240 ± 0.029, intercept = 0.634 ± 0.017, cKO: slope = 0.266 ± 0.022, intercept = 0.538 ± 0.012) being significantly decreased. Following remyelination, mean g-ratios increased significantly as compared to WT contralateral (unlesioned) hemisphere (indicating thinner myelin) and did not differ significantly between wild-type and Scribble cKO remyelinated axons (G), although the Y-intercepts of the best-fit lines obtained by linear regression differed slightly but significantly between cKO and wild-type datasets (I; WT: slope = 0.162 ± 0.011, intercept = 0.692 ± 0.007, cKO: slope = 0.158 ± 0.017, intercept = 0.708 ± 0.012), indicating slightly thinner myelin in Scribble cKO animals. Two-way ANOVA with Bonferroni's multiple comparisons test was used. <i>n</i> = 3 mice per genotype. At least 20 g-ratios were analysed per animal, per region. J–M; The density of Olig2+CC1- OPCs (L) and CC1 oligodendrocytes (M) did not differ between lesioned Scribble cKO corpora callosa (K) and those of wild-type animals (J). Student's <i>t</i> test. <i>n</i> = 3 mice per genotype. * <i>p</i> < 0.05, ** <i>p</i> < 0.01, *** <i>p</i> < 0.001 versus Scrib WT CL. Scale bars: A,B,D,E: 2 μm. J,K: 50 μm.</p

Scribble is required for normal myelin initiation in vivo.

<p>A–C; Fewer myelinated axons were observed in P14 optic nerve cross-sections from Scribble conditional knock-out (B) relative to wild-type controls (A). This was observed in axons of all diameters (C; 0.5–0.6 μm: WT = 45.1% ± 6.2%, cKO = 10.3% ± 3.0% myelinated; 0.6–0.7 μm: WT = 76.9% ± 9.9%, cKO = 29.0% ± 4.1% myelinated; ≥0.7 μm: WT = 96.1% ± 3.8%, cKO = 77.7% ± 3.2% myelinated). D–F; Fewer myelinated axons were also observed in P14 spinal cord cross-sections from Scrib cKO (E) relative to wild-type controls (D) for all but the largest-diameter axons (F; 0.4–0.5 μm: WT = 49.7% ± 10.6%, cKO = 19.7% ± 2.3% myelinated; 0.5–0.6 μm: WT = 83.7% ± 11.5%, cKO = 48.7% ± 3.1% myelinated; 0.6–0.7 μm: WT = 94.7% ± 5.3%, cKO = 63.1% ± 10.7% myelinated; 0.8–0.9 μm: WT = 100% ± 0%, cKO = 72.2% ± 2.8% myelinated). Numerical results are presented as mean ± SEM. Two-way ANOVA with Bonferroni's multiple comparisons test was used. <i>n</i> = 3 mice per genotype. At least 200 axons analysed per region, per mouse. * <i>p</i> < 0.05, ** <i>p</i> < 0.01, *** <i>p</i> < 0.005 versus Scrib WT. Scale bars in A,E = 2μm.</p

GFP induction and mKate2 expression is uniform in most organs of <i>CAGs-rtTA3</i> and <i>CAGs-RIK</i> mice.

<p>Immunofluorescence stains for GFP and mKate2 in the small intestine and pancreas of ‘no rtTA’, <i>R26-rtTA</i>, <i>CAGs-rtTA3</i> and <i>CAGs-RIK</i> mice following 1 week of doxycycline treatment. All rtTA strains show strong GFP induction in small intestine (<b>A</b>), but only <i>CAGs-rtTA3</i> and <i>CAGs-RIK</i> show robust and uniform GFP expression (and mKate2 for <i>RIK</i>) in the pancreatic acinar tissue (<b>B</b>).</p

Oligodendroglial differentiation is unaffected in Scribble cKO mice.

<p>A–D; The number of OPCs positive for Olig2 (red) and not labelled by antibody CC1 (green), and Olig2 and CC1 double-positive oligodendrocytes present in the optic nerves of wild-type (A) and Scribble CKO (B) did not significantly differ (C,D). E–H; Similarly, the number of OPCs and oligodendrocytes in wild-type (E) and Scribble cKO (F) ventral spinal cord white matter did not differ significantly (G,H). Numerical results are presented as mean ± SEM. Student's <i>t</i> test was used, <i>n</i> = 4 animals per genotype, at least three fields were analysed per section in at least two non-adjacent sections per animal. Scale bar: 50 μm.</p

Scribble is required for normal myelin initiation in vitro.

<p>A,B; Transfected OPCs were seeded on dorsal root ganglion (DRG) neuron cultures and maintained for 3 d, then immunolabelled with antibodies against MBP (red), Caspr (white), and NFH (blue). MBP-positive oligodendrocytes labelled with siGLO (green) were scored using the categories shown in C as having “contacting” (A, square arrowhead), “extending” (A, curved arrowhead), or “wrapping” morphology (A, arrow). D; Decreased contact and wrapping by MBP-expressing oligodendrocytes was observed in Scribble siRNA-transfected oligodendrocytes (shown in B) relative to those that were mock-transfected or transfected with non-targeting siRNAs (shown in A). Contacting: non-targeting = 55.2% ± 4.6%, Scribble = 85.7% ± 0.4%; extending: non-targeting = 27.4% ± 2.7%, Scribble = 12.6% ± 1.2%; wrapping: non-targeting: 17.4% ± 4.8%, Scribble = 1.7% ± 0.9%. Numerical results are presented as mean ± SEM. ANOVA with Tukey's multiple comparison test was used; <i>n</i> = 6 coverslips per condition, five fields were analysed per coverslip. * <i>p</i> < 0.05 versus Mock. ** <i>p</i> < 0.01 versus Mock, *** <i>p</i> < 0.005 versus Mock. Scale bar in A = 100 μm.</p

Mendelian transmission of Rosa26-targted CAGs-rtTA3 transgenes.

<p>*Numbers represent: observed (expected) from heterzygote x wild-type crosses.</p

Adenoviral Cre induces mosaic activation of rtTA and GFP induction in <i>CAGs-LSL-rtTA3</i> and <i>CAGs-LSL-RIK</i> animals.

<p><b>A</b>. Immunofluorescent stains for GFP and mKate2 in liver sections of <i>TG-Ren.713;CAGs-LSL-rtTA3</i> and <i>TG-Ren.713;CAGs-LSL-RIK</i> mice 1 week following intravenous injection of Adenoviral Cre (5×10⁸ PFU) or PBS (<i>CAGs-LSL-RIK</i> only – left panel) and dox treatment. Double transgenic mice exposed to AdenoCre show mosaic expression of GFP (<i>CAGs-LSL-rtTA3</i>) or GFP and mKate2 (<i>CAGs-LSL-RIK</i>). No GFP of mKate2 expression was observed in animals not exposed to Cre. <b>B</b>. Immunofluorescent stains for GFP and mKate2 in lung sections of triple transgenic mice (<i>CAGs-LSL-rtTA3 or RIK;TG-Ren.713;LSL-Kras^G12D</i>). Kras^G12D-induced lung adenomas show strong expression of GFP and mKate2. Lowe panel: higher magnification of the lesion. White arrows indicate rare cells that show mKate2, but not GFP expression.</p