Figure 5. Glia-independent and -dependent UBC abnormalities in the Nf1^hGFAPCKO cerebellum.

(A) The overall expression pattern of Tbr2 in the adult control and Nf1^hGFAPCKO cerebella was compared. High magnification images were merged to provide a high-resolution view. (A′) Tbr2 staining in folium V/VI was compared. Arrows point to Tbr2⁺ cells in the ML. The total number of Tbr2⁺ cell in the ML of each folium was quantified in (A′′). (B) Sections were stained for Tbr2/NeuN and imaged at folium V. The number of Tbr2⁺NeuN⁺ cells and Tbr2⁺NeuN⁻ cells per high magnification field was quantified in (B′). The percentage of Tbr2⁺NeuN⁺ cells among Tbr2⁺ cells is illustrated. (C) Tbr2 staining in folium X was compared. The total number of Tbr2⁺ cells in the ML, IGL and WM of folia X was quantified in (C′). (D) Sections were stained for Tbr2/NeuN and imaged at folium X. The number of Tbr2⁺NeuN⁺ cells and Tbr2⁺NeuN⁻ cells per high magnification field was quantified in (D′) and (D′′). The percentage of Tbr2⁺NeuN⁺ cells is illustrated. (E) Sections were stained for GFAP and BLBP and imaged at folium X. The total number of GFAP⁺ cells in the WM was quantified in (E′). (F) Cerebellar sections from control and Nf1^Math1CKO were stained for Tbr2/NeuN and compared. (G–G′′) Control and Nf1^NcreERCKO mice were TM-induced at E17.5 and analyzed at P21. Tbr2/β-gal staining was imaged at folium X (G) and high magnification views of the white matter (G′) and ventral folium X (G′′) were compared. Dashed lines mark the border between the WM and IGL in folium X. Of note, ectopic Tbr2⁺ cells in the WM of Nf1^NcreERCKO were exclusively β-gal-negative (Nf1-wildtype) (G′). In contrast, the increase of Tbr2⁺ cells in the mutant ML was mostly contributed by Tbr2⁺β-gal⁺ cells (Nf1-deficient) (arrows, G′′). Arrows label co-localizing cells and arrowheads label non-co-localizing cells. 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.05151.012

Figure 5—figure supplement 1. The ectopic accumulation of Tbr2⁺ UBC in the ML of Nf1^hGFAPCKO cerebella occurs during postnatal development.

(A) Cerebellar sections were stained for Tbr2/CR and imaged at folium V. The number of Tbr2⁺CR⁺ cells and Tbr2⁺CR⁻ cells per high magnification field was quantified in (A′). The percentage of Tbr2⁺CR⁺ cells is illustrated. (B) Sections of P8 control and Nf1^hGFAPCKO cerebella were stained for Tbr2/NeuN and imaged at folium V. The number of Tbr2⁺ cells in the ML of folium V was quantified in (B′). (C, C′) Control and Nf1^hGFAPCKO mice were pulsed with BrdU at P8 and sacrificed at P18. Cerebellar sections were stained for Tbr2/BrdU (C) and NeuN/BrdU (C′) to label newly generated Tbr2⁺BrdU⁺ UBCs and NeuN⁺BrdU⁺ granular cells (arrows) (D) Adjacent sections from the same Nf1^hGFAPCKO adult cerebellum were stained for GABAα6/NeuN and Tbr2/NeuN. * labels a blood vessel structure found in both sections, which was used as a reference to match these two sections. Boxed areas highlight a group of cells that express a significant level of GABAα6 but are mostly Tbr2⁻. (E) Sections of adult control and Nf1^hGFAPCKO cerebella were stained for NeuN/GABA and the number of GABA⁺ GABAergic neurons in the ML were quantified in (E′). 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.05151.013

Figure 5—figure supplement 2. A subpopulation of UBCs in the Nf1^hGFAPCKO cerebella fail to migrate into the IGL and are ectopically present in the WM during postnatal development.

(A) The low magnification view of Figure 5D is shown to compare the overall distribution of Tbr2⁺ cells in folia IX and X. (B) Sections of adult control and Nf1^hGFAPCKO cerebella were stained for Tbr2/CR and imaged at folium X. Dashed lines mark the border of white matter in folium X. The number of Tbr2⁺CR⁺ cells (arrows) and Tbr2⁺CR⁻ cells (arrowheads) per high magnification field were quantified in (B′, B′′). The percentage of double-labeled Tbr2⁺CR^high cells among total Tbr2⁺ cells was illustrated. (C, C′) The distribution of Tbr2⁺ cells in E17.5 (C) and P0.5 (C′) control and Nf1^hGFAPCKO cerebella was compared. Boxed areas in (C′, upper panels) are shown at higher magnification in lower panels to indicate the absence of Tbr2⁺ cells in the EGL of both control and mutant cerebella. (D) Sections of P8 control and Nf1^hGFAPCKO cerebella were stained for Tbr2/NeuN and imaged at folia IX/X. The number of Tbr2⁺ cells in the WM of folium X was quantified in (D′). 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.05151.014