Figure 4. Neuron-specific Nf1 inactivation leads to cell-autonomous defects in the glutamatergic interneuron lineages.

(A) X-gal staining on P21 control and Nf1^Math1CKO cerebella labeled cells that underwent Math1-cre-mediated recombination. Arrows point to the abnormally accumulated cells in the ML of the mutant cerebellum. (B, B′) Cerebellar sections were stained for NeuN/β-gal and Cal/β-gal, and GFAP/β-gal and BLBP/β-gal. The total number of NeuN⁺/β-gal⁺ and NeuN⁺/β-gal⁻ cell per high magnification field was quantified in (B′′). (C–D) Control and Nf1^Math1CKO mice were analyzed 32 hr and 48 hr after BrdU-pulse at P8. Cerebellar sections were stained for BrdU/MAP2 and imaged at folium V. High magnification images comparing the nuclear morphology of BrdU⁺ cells are shown in (C′) and (D′). (E, E′) Cerebella sections from P8 control and Nf1^Math1CKO mice were stained for Tbr2/MAP2 (E) and Tbr2/NeuN (E′) and imaged at folium V. The inset in (E) shows an example of Tbr2⁺MAP2⁺ cells in the mutant ML. (F, G) 24 hr and 48 hr after BrdU-pulse, cerebellar sections from P8 control and Nf1^Math1CKO mice were stained for Tbr2/BrdU. The distribution of Tbr2⁺BrdU⁺ cells (arrows) in the EGL-ML-IGL was quantified 24 hr and 48 hr after BrdU-pulse in (F′) and (G′), respectively. (H) Golgi staining was performed on P8 cerebellar sections. Cells with UBC morphology are highlighted by red arrows and shown in high magnification (lower panels). Dashed red lines delineate the ML. PC, Purkinje cell; GC, granule cell; bt, the brush tip of UBCs. (I) Cerebellar sections from adult control and Nf1^Math1CKO cerebella were stained for Tbr2/NeuN and imaged at folium V. (I′) The number of Tbr2⁺NeuN⁺ (arrows) and Tbr2⁺NeuN⁻ cells (arrowheads) in the ML of folium V per high magnification image was quantified and compared with Nf1^hGFAPCKO mice. The percentage of Tbr2⁺NeuN⁺ cells among total NeuN⁺ cells was also quantified. (J) A proposed model summarizes the role of Nf1 in preventing the ectopic differentiation of GCPs into UBCs. As a comparison, the timeline of normal UBC genesis is shown in (J′). 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.05151.010

Figure 4—figure supplement 1. Neuron-specific Nf1 inactivation does not impact on the proliferation or migration of GCPs.

(A) Cerebellar sections from adult control and Nf1^Math1CKO cerebella were stained by H&E. Lower panels are the high magnification view of folia V/VI in upper panels. (A′) The total number of cells in the ML of folia V/VI was quantified and compared with Nf1^hGFAPCKO mice. (B) P8 cerebellar sections were stained for BLBP/NeuN and imaged at folium V. Dashed lines delineate the ML. (C) Sections of P8 control and Nf1^Math1CKO cerebella were analyzed and stained for BrdU/Ki67 24 hr after BrdU pulse. (C′) The mitotic index of EGL precursors was quantified as the percentage of Ki67⁺ cells of total DAPI⁺ cells in the EGL. (C′′) The cell cycle exit index (fraction of cells exiting cell cycle) was quantified as the ratio of BrdU⁺/Ki67⁻ cells (cells exiting cell cycle) to BrdU⁺/Ki67⁺ cells (cells remaining or reentering cell cycle) in the EGL. (D–E) P8 control and Nf1^Math1CKO mice were analyzed 48 hr after BrdU pulse. (D) Cerebellar sections were stained for BrdU/MAP2. The number of MAP2⁺BrdU⁺ cell in the ML was quantified in (D′). (E) Sections were stained for BrdU/Ki67. The number of BrdU⁺/Ki67⁻ cells (newly differentiated cells) in the inner EGL/ML and IGL was quantified in (E′). oEGL, outer EGL; iEGL, inner EGL. (F) Cerebellar sections from P8 control and Nf1^Math1CKO mice were stained for p27 and the number of p27⁺ cells in the inner EGL was quantified in (F′). (G) GCPs were isolated from control and Nf1^Math1CKO cerebella at P8 and cultured in differentiation media for 2 days. Cells were then stained for MAP2 and quantified for the percentage of cells expressing MAP2 among total DAPI⁺ cells (G′). (H) Purified P6 granule cells from control and Nf1^Math1CKO cerebella were cultured with wildtype cerebellar glia at a ratio of 1:2. Cells were then stained for Tuj1 and GFAP or subjected to Transwell migration assay. The number of granule cells that migrated through the membrane was quantified, normalized by the membrane area (per mm²) and compared (H′). 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.05151.011