Figure 7: Crb3-Hippo pathway dissociates longitudinal from radial myelin growth.

(a) Western blots showing expression of AKT and active phosphorylated AKT (P-AKT) in primary rat Schwann cells differentiated in culture with dibutyryl-cyclic AMP (+db) following infection with GFP control or constitutively active YAP S127A Flag adenovirus. GFP and YAPS127A panels confirm infection in three independent cultures of each condition. Actin is used as loading control. (b) Ratio active P-AKT on AKT is calculated from the quantifications of the western blots shown in a and after normalization over actin. Three independent experiments. NS: not significant. (c) Representative western blots showing expressions of YAP and phosphorylated YAP (P-YAP) in sciatic nerves of transgenic Nrg1 mice (Mutant) and of control littermate. Actin is used as loading control. (d) Quantifications of western blots showing YAP and phosphorylated YAP levels in mutant mice (Mut, 5 mice) relative to control mice (Cont, 4 mice) normalized over actin levels. (e) Representative western blots showing expression of Crb3 in sciatic nerves of transgenic Nrg1 mice (Mutant) and of control littermate. Actin is used as loading control. (f) Quantifications of western blots showing Crb3 levels in mutant mice (Mut, 5 mice) relative to control mice (Cont, 4 mice) normalized over actin levels. Error bars show s.d. (g) Representative nuclear YAP immunostaining on sciatic nerve longitudinal sections of transgenic Nrg1 mice (Mut) and littermate control (Cont). Scale bar, 50 μm. (h) Quantification of YAP immunostaining fluorescence intensity in nuclei of mSCs as shown in e. Nuclei quantified: 112 (Control), 270 (Mutant). Three animals for each condition. (i) Quantification of internodal lengths after teasing sciatic nerves of transgenic mice (Mut, n=57 cells, three animals) and control littermate (Cont, n=84 cells, three animals). Cells were stained with FluoroMyelin, nuclei with TOPRO3 and nodes immunostained with Kv1.2 antibodies. Mice were 5 months old. a.u., arbitrary units. Statistical two-tailed Student’s t-tests: *P<0.05; **P<0.01; NS: not significant. Error bars show s.d for b, d and f and s.e.m for h and i.