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First published online November 28, 2005
doi: 10.1242/10.1242/dev.02162

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Inactivation of NF1 in CNS causes increased glial progenitor proliferation and optic glioma formation

¹ Center for Developmental Biology and Kent Waldrep Foundation Center for Basic Research on Nerve Growth and Regeneration, University of Texas Southwestern Medical Center, Dallas, TX 75390-9133, USA
² Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9133, USA
³ Department of Neurology, Washington University School of Medicine, St Louis, MO 63110, USA
⁴ Division of Molecular Medicine and Genetics, Departments of Internal Medicine and Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI 48109, USA

View larger version (104K): [in a new window]   Fig. 1. Comparison of the number of BrdU-positive cells in adult and developing control and mutant brains. (A) The number of BrdU-positive cells in the cerebral cortex (Ctx), thalamus (Th) and cerebellum (Cb) of adult mutant brains (n=6) was not significantly different from those in adult control brains (n=6). The data plotted are mean±s.e.m. Cerebral cortex, P=0.42; thalamus, P=0.46; cerebellum, P=0.57. (B) The number of BrdU-positive cells in developing mutant brains (n=5) was significantly increased relative to those in controls (n=3). Cerebral cortex, P=0.001; thalamus, P=0.007. **P<0.01. (C) Sections from the P8 control cerebral cortex (a,c) and thalamus (e,g), and the P8 mutant cortex (b,d) and thalamus (f,h) were stained with an anti-BrdU antibody. In contrast to the control P8 brains, which have low proliferation, the mutant brains contain significantly more BrdU-positive cells. Scale bars: 100 µm.

View larger version (103K): [in a new window]   Fig. 2. Proliferating cells in P8 control and mutant brains express progenitor cell markers, not a mature astrocyte marker. Sections from P8 control and Nf1hGFAPKO mutant brains were subjected to double-labeling immunofluorescence with anti-Ki67 (red) and anti-nestin (green) (A,B), with anti-BrdU (red) and anti-BLBP (green) (C,D), and with anti-BrdU (red) and anti-GFAP (green) (E,F). BrdU staining in the external granule cells in the P8 cerebella serves an internal positive control for this study (E,F). (G,H) Higher magnification of sections in E,F, showing that only a small number of BrdU-positive cells in the mutant brain but not in the control brain expressed GFAP (arrows in H). (A-D) Sections from the thalamus. (E-F) Sections from the brainstem. IV, the fourth ventricle. Scale bars: 100 µm.

View larger version (112K): [in a new window]   Fig. 3. hGFAP-cre is expressed in glial progenitor cells during postnatal development. (A) Sections from the thalamus of P8 control (a,c,e) and mutant (CKO) brains (b,d,f) were subjected to double-labeling immunofluorescence with anti-Cre (red) and anti-GFAP (green) (a-d), and anti-Cre (red) and anti-BLBP (green) (e,f). (B) Sections from the cerebellar white matter of P8 control (a,c,e) and mutant brains (b,d,f) were subjected to double-labeling immunofluorescence with anti-Cre/anti-GFAP (a-d) and anti-Cre/anti-BLBP (e,f). The mutant cerebellar white matter has significantly more GFAP-positive cells than control (marked by broken lines in a-d). Most of the Cre-positive/GFAP-negative glial progenitor cells are distributed in the periphery of the cerebellar white matter (outside the broken lines). Arrows indicate glial progenitor cells expressing Cre and BLBP, but not GFAP. Genotypes: control mice, Nf1flox/+;hGFAP-cre+; mutant mice, Nf1flox/-;hGFAP-cre+ or Nf1flox/flox;hGFAP-cre+. Scale bars: 100 µm.

View larger version (106K): [in a new window]   Fig. 4. Nf1-/- glial progenitor cells undergo astrocytic differentiation. (A) Sections from P8 control (a,c,e) and Nf1hGFAPKO mutant (b,d,f) brains were subjected to double-labeling immunofluorescence with anti-Cre (red) and anti-GFAP (green). (a,b) Sections from the control and mutant cerebral cortex (CC); (c-f) low (c,d) and high (e,f) magnification of sections from control and mutant hippocampal dentate gyrus (DG). Most of the GFAP-positive cells also express the Cre transgene. (B) Sections from P8 control (a,c,e) and Nf1hGFAPKO mutant (b,d,f) brains were subjected to double-labeling immunofluorescence with anti-nestin (red) and anti-GFAP (green). (a,b) Sections from the corpus callosum; (c,d) sections from the brainstem; (e,f) sections from the dentate gyrus. Most of the GFAP-positive astrocytes in both control and mutant P8 brains do not express nestin, except in the dentate gyrus (arrows in e,f), where a small number of cells express both GFAP and nestin. There are low levels of nestin expression in the endothelial cells. Genotypes: control mice, Nf1flox/+;hGFAP-cre+; mutant mice, Nf1flox/-;hGFAP-cre+ or Nf1flox/flox;hGFAP-cre+. Scale bars: 100 µm.

View larger version (88K): [in a new window]   Fig. 5. A subset of Nf1-/- astrocytes in the adult brain express nestin. Sections from adult control (A) and mutant (B) dentate gyrus (DG), as well as control (C) and mutant (D) cerebral cortex were subjected to double-labeling fluorescence with anti-nestin (red) and anti-GFAP (green). LV, lateral ventricle. Reactive astrocytes expressing both GFAP and nestin were observed only in the mutant brain (arrows in B,D). Immunohistochemical analysis with anti-P-erk of the control (E) and mutant (F) dentate gyrus. A population of nestin-positive reactive astrocytes (arrows in H, see inset for high magnification) was identified only in a subset of aged mutant brains, but not in control or mutant mice at young age (G). Scale bars: 100 µm.

View larger version (76K): [in a new window]   Fig. 6. Cre-mediated recombination leads to enlarged optic nerves. X-gal staining of the proximal optic nerve immediately adjacent to the retina (A) and distal optic nerve near the chiasm (asterisk) (B). Optic nerves were dissected from 2-month-old mice with the genotype of hGFAP-cre+; Rosa26-lacZ/+ (n=3). (C,D) Adjacent sections to A,B were subjected to double-labeling immunofluorescence with anti-lacZ (red) and anti-GFAP (green). Most of the lacZ-positive cells are GFAP-expressing astrocytes. (E) hGFAP-cre-mediated recombination in the optic nerve was revealed by PCR analysis. Upper panel: a PCR assay identifying the floxed NF1 allele (x1) and recombined floxed allele () indicated that a significant number of the floxed NF1 alleles in the optic nerve (ON), cerebral cortex (Ctx) and hippocampus (Hp) of the Nf1flox/+;hGFAP-cre+ mice transformed into the recombined alleles. Bottom panel: a PCR assay that identifies the wild type (+) and the floxed (x1) NF1 allele confirmed the genotype of the tissues analyzed. (F) A representative of control (left) and mutant (right) eyes with the optic nerves and chiasm (arrowheads). High-magnification of view of control (G) and mutant (H) optic nerves with chiasms (asterisk). The mutant nerve has a conspicuous enlargement (indicated by broken lines and arrowheads). Scale bars: 100 µm in A-D; 1 mm in F-H.

View larger version (76K): [in a new window]   Fig. 7. Nf1hGFAPKO optic nerves are hyperplastic. Sections from control (A,C) and mutant (B,D) distal optic nerves and chiasms (asterisk) were stained with Hematoxylin and Eosin. Arrows in A,B indicate the enlarged area of the nerves shown in C,D, respectively. Adjacent sections from control (E,G) and mutant (F,H) optic nerves were stained with anti-nestin (red) and anti-GFAP (green). Mutant optic nerves are enlarged (B), hyperplastic (D) and disorganized (F,H). Sections from control (I) and mutant (J) proximal optic nerves were stained with Hematoxylin and Eosin. The most pronounced hypercellularity was observed in the proximal optic nerves (J). Scale bars: 100 µm.

View larger version (83K): [in a new window]   Fig. 8. Nf1hGFAPKO mice develop optic pathway gliomas. Two independent optic gliomas (B,C) are shown with Hematoxylin and Eosin staining and compared with the control (A). Adjacent sections from control (D) and mutant nerves with gliomas (E,F) were stained with anti-nestin (red) and anti-GFAP (green). Unlike the well-organized normal astrocytes that express both nestin and GFAP in control (D), most of the cells in optic gliomas express only GFAP (E,F, arrowheads). Scale bars: 100 µm.

View larger version (118K): [in a new window]   Fig. 9. Nf1hGFAPKO hyperplastic nerves have increased proliferation and express glial progenitor cell markers. (A) The quantification of BrdU-positive cells in the adult control (CTR) and mutant (CKO) distal (DN) and proximal (PN) nerves. The data plotted are mean±s.e.m. (distal nerve, CTR, n=4, CKO, n=5, **P<0.001; proximal nerve, CTR, n=3, CKO, n=6, **P<0.001). Sections from control (B,D) and mutant (C,E) distal (B,C) and proximal (D,E) optic nerves were stained with anti-BrdU (red) and anti-GFAP (green). Sections from control (F) and mutant (G) nerves were stained with anti-Ki67 (red) and anti-nestin (green). Arrows in B and C indicate proliferating cells that do not express GFAP. Arrows in F and G indicate nestin-positive proliferating cells (inset in G provides higher magnification). Sections from control proximal (H,J), mutant proximal (I,K), control distal (L) and mutant distal (M) optic nerves were stained with anti-BLBP. Arrow in J indicates a BLBP-positive cells with normal nuclei in the normal nerve; arrows in K,M indicate BLBP-positive cells with atypical nuclei in the mutant nerve. Sections from control (N) and mutant (O) optic nerves were stained with anti-Pax2. Scale bars: 100 µm.

View larger version (125K): [in a new window]   Fig. 10. Glial cells in mutant optic nerves have elevated levels of activated MAPK. Sections of the proximal optic nerves from control mice (A,C) and mutant mice (B,D) were stained with anti-P-erk. The arrow in D indicates the tumor cells with activated MAPK. Sections from control (E) and mutant (F) optic nerves were subjected to triple labeling with anti-P-erk (red), anti-GFAP (green) and DAPI (blue). DAPI was used to label the nuclei. In contrast to the control nerves where little or no cells had activated MAPK (E), mutant nerves contained numerous P-erk positive cells, some of which co-expressed GFAP (F, arrows) and some of which failed to express GFAP (F, arrowheads). Scale bars: 100 µm.

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