Figure 1. Nf1 inactivation during early cerebellar development disrupts neuronal lamination and causes BG abnormalities.

(A) Sagittal sections from control and Nf1^hGFAPCKO cerebella at 2 months of age were stained with H&E and imaged at three increasing magnifications. Each cerebellar folium is numbered by Roman numerals. Arrows in (A, B) highlight folia V/VI where mutants exhibited extra foliation and arrows in (B′′) point to the cells clustered in the ML and on the pial surface of the Nf1^hGFAPCKO cerebellum. The total number of cells in the ML and IGL were quantified in (A′) and (A′′). (B) Cerebellar sections were stained for Calbindin (CalB) /NeuN. Arrows point to mispositioned CalB⁺ Purkinje cells. (C) Sections from adult Nf1^hGFAPCKO cerebellum were stained with β-gal/CalB and Nf1. Purkinje cells (arrows) were β-gal⁻ and expressed Nf1. (D) Cerebellar sections were stained for NeuN and GABARα6. The insets are the high magnification images of the boxed areas highlighting the co-localizing cells. The number of NeuN⁺ cells in the ML and IGL were quantified in (D′) and the percentage of NeuN⁺ cells in the IGL among total NeuN⁺ cells (ML + IGL) is shown. The percentage of GABARα6⁺NeuN⁺/NeuN⁺ cells in the ML was quantified in (D′′). (E) The cell bodies (arrows) and processes of BG in control and mutant cerebella were labeled by BLBP staining. The total number of BLBP⁺ BG cells was quantified in (E′). (F) Sections were stained for CalB/BLBP. Boxed areas compare the Purkinje cell alignment in the less affected areas (lower box) and severely affected areas (upper box) in the Nf1^hGFAPCKO cerebellum. Note the correlation between increased number/misalignment of BG and the severity of the disruption of Purkinje cell layer. (G) Sections were stained for CalB/GFAP. Boxed areas highlight the severely disrupted BG alignment and Purkinje cell patterning. All the quantification data are presented as mean ± SEM. DAPI labels the nuclei. Scale bars: 50 μm.

DOI: http://dx.doi.org/10.7554/eLife.05151.004

Figure 1—figure supplement 1. Purkinje cell defects in the Nf1^hGFAPCKO cerebellum are non-cell-autonomous.

(A) The developmental lineages of the major cerebellar cell types are illustrated. Cell types expressing distinct Cre recombinases (hGFAP, L7, Math1) are highlighted by red dashed boxes. Of note, these Cre drivers except for L7-cre also target cells in the other regions of the brain. (A′) The laminar structure, the alignment and marker expression of Bergmann glia, Purkinje cells and granule cells in adult cerebellum are illustrated. (Ba-d) X-gal staining was performed on cerebellar sections from P0.5 (A, B) and P60 (C, D) double-transgenic hGFAP-cre+; R26^LacZR mice to determine the pattern of Cre-mediated recombination. Arrows point to Purkinje cells, which were not stained. Inset in (D) shows a positive control for the X-gal staining in the Purkinje cells that were targeted by Synapsin I-cre (Zhu et al., 2001). PCZ, Purkinje cell zone; PCL, Purkinje cell layer. EGL, external granule cell layer (B′) DNA extracts of the whole cerebellum and tail from hGFAP-cre+;Nf1^flox/+ mice were analyzed by PCR analysis, which distinguishes Nf1 wildtype (+), flox (x1) and deleted (Δ) alleles. Note that in the cerebellum the flox allele was completely recombined and became deleted allele. (C) Adult Nf1^hGFAPCKO mice exhibited a unique ‘handstand’ behavior. (D–F) Cerebellar sections from adult control and Purkinje-cell-specific Nf1^L7CKO mice were analyzed. (D) The specificity of L7-cre-mediated recombination in Purkinje cells was confirmed by CalB and β-gal staining. (E, F) CalB⁺ Purkinje cells, BLBP⁺ BG cells and NeuN⁺ granule cells were compared between control and Nf1^L7CKO cerebella and no difference was identified. DAPI labels the nuclei. Scale bars: 50 μm.

DOI: http://dx.doi.org/10.7554/eLife.05151.005