Figure 7. MEKi treatment rescues glia-dependent cerebellar defects in the Nf1^hGFAPCKO cerebellum.

(A) Cerebellar sections of vehicle- and MEKi-treated (5 mg/kg) control and Nf1^hGFAPCKO mice were stained for GFAP/NeuN and CalB/BLBP and imaged at folium V. 9 out 14 MEKi-treated mutant mice (G-responder) exhibited a significant improvement, although the remaining 5 (P-responder) still had large number of cells in the EGL/ML and displayed severe laminar disruption. The number of NeuN⁺ cells in the ML was quantified and compared in (A′). For MEKi-treated mutants, green and yellow color represents G-responders and P-responders, respectively. G-responders and P-responders were separated into two groups and compared in (A′′). (C, D) Cerebellar sections were stained for Tbr2/NeuN and imaged at folia V and X. (B, B′, C′, D′) The correlation between the number of NeuN⁺ cells in the ML, and the number of BLBP⁺ BG cells (B), mispositioned Purkinje cells (B′), ectopic Tbr2⁺ cells in the WM (C′) and ML (D′) was plotted and compared. (E) Sections of P8 control and Nf1^hGFAPCKO cerebella were stained for GFAP and p-Erk. Arrows highlight the GFAP⁺p-Erk⁺ cells in the ML, IGL and WM of the Nf1^hGFAPCKO cerebellum. (F) Three Nf1^hGFAPCKO mice treated with MEK inhibitor at 20 mg/kg from P0.5–P21 were analyzed at P60. GFAP/NeuN and CalB/BLBP staining show consistent rescue of both neuronal and glial defects. Individual data points are presented, as well as mean ± SEM for each group. DAPI labels the nuclei. Scale bars: 50 μm.

DOI: http://dx.doi.org/10.7554/eLife.05151.017

Figure 7—figure supplement 1. Ras/Erk signaling is differentially activated in neuronal and glial precursors of the developing Nf1^hGFAPCKO cerebellum.

(A, B) Sections of P8 control and Nf1^hGFAPCKO cerebella were stained for GFAP and p-Erk. Arrows highlight the GFAP⁺p-Erk⁺ cells in the ML, IGL and WM of the Nf1^hGFAPCKO cerebellum. (B) is the high magnification view of (A). The different p-Erk level in the neuronal vs glial precursors Nf1^hGFAPCKO cerebella is more evident in the images with lower exposure in (B). Boxed images are also shown in Figure 7E. DAPI labels the nuclei. Scale bars: 50 μm.

DOI: http://dx.doi.org/10.7554/eLife.05151.018

Figure 7—figure supplement 2. High-dose MEKi-treatment produces more consistent phenotypic rescue.

(A) Control and Nf1^hGFAPCKO mice were treated with vehicle or MEKi (20 mg/kg) from P0.5–P21 and analyzed at P60. H&E staining of the overall cerebellar structure and folium V are presented. Low magnification view of NeuN staining, as well as high magnification merged images of NeuN/GFAP, CalB/BLBP/DAPI are compared. Arrows point to misaligned Purkinje cells in the ML or IGL. Boxed images are also shown in Figure 7F. The number of NeuN⁺ cells in the ML, the total number of BLBP⁺ cells and the number of mispositioned Purkinje cells are quantified in (B), (C) and (D), respectively. DAPI labels the nuclei. All the quantification data are presented as mean ± SEM. Scale bars: 50 μm.

DOI: http://dx.doi.org/10.7554/eLife.05151.019