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First published online August 18, 2003
doi: 10.1242/10.1242/dev.00675

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Oligodendrocyte ablation impairs cerebellum development

Carole Mathis^*, Ludovic Collin and Emiliana Borrelli

Institut de Génétique et de Biologie Moléculaire et Cellulaire, INSERM/CNRS/ULP, BP 10142, 67404 Illkirch Cedex, C.U. de Strasbourg, France

View larger version (80K): [in a new window]   Fig. 1. Ablation of oligodendrocytes alters cerebellum development. (A-D) HSV1-TK expression is restricted to MBP-positive oligodendrocytes (arrowheads). Cerebellar sections from a 2-day-old (P2) untreated MBP-TK mouse were double stained with anti-HSV1-TK (red) and MBP (green) antibodies. Nuclei were stained with DAPI (blue). (D) Merging of the different stains shows nuclear localization of TK in MBP-positive cells. Sections were analyzed by confocal microscopy. (E,F) Immunofluorescence analysis of TK-positive oligodendrocytes in cerebellar folia IX of P6 untreated (E) versus treated (F) MBP-TK mice, using the anti-HSV1-TK antibody. (G-J) Cresyl Violet staining of paraffin wax-embedded cerebella sagittal sections from wild-type (G,I) and MBP-TK (H,J) mice treated from day 1 to 20. The cerebellum of the treated wild-type animal presents a normal architecture both at high (G) and low (I) magnification. The cerebellum of the treated MBP-TK mice is smaller with an abnormal foliation (J; star) and a highly disorganized cortex (H). The fissura intercruralis in both wild-type and MBP-TK mice is indicated by a star (I,J). Arrows (G,H) show Purkinje cell bodies. ML, molecular layer; IGL, internal granular layer; WM, white matter tract. (K,L) Cerebellar sagittal sections immunostained with an anti-MBP antibody from 3-week-old wild-type (K) and MBP-TK (L) mice treated from day 1 to 21. Note rare MBP-positive fibers in treated MBP-TK mice (arrow; L). Scale bars: 5 µm (A-D), 80µm (E-F), 30 µm (G-H), 300 µm (I-J), 100 µm (K-L).

View larger version (141K): [in a new window]   Fig. 2. Disorganization of the Bergmann glial network in young treated MBP-TK mice. (A-D) Immunofluorescence analysis of cerebellar sections from treated 6-day-old (A,B) or 21-day-old (C,D) wild-type (A,C) and MBP-TK (B,D) mice. Astrocytes were identified using specific antibodies directed against brain lipid binding protein (BLBP; A,B) and glial fibrillary acidic protein (GFAP; C,D). In the wild-type, the BLBP-positive soma of Bergmann glial cells (A; arrowheads) are located at the level of the Purkinje cell layer and their radial fibers extend across the ML toward the pial surface (C). In MBP-TK, Bergmann glial cells are dispersed throughout the folia (B) and their radial scaffold is strongly perturbed (D). Scale bars: 75 µm (A,B); 12 µm (C,D).

View larger version (136K): [in a new window]   Fig. 3. Alteration of the Purkinje cell monolayer and dendritic formation in treated MBP-TK mice. (A,B) In situ hybridization using a zebrin-II antisense probe. (C-F) Immunofluorescence using an anti-calbindin antibody to visualize soma and dendritic extensions of Purkinje neurons. (A,B) Cerebellar coronal, and (C-F) sagittal sections of 3-week-old wild-type (A,C,E) and MBP-TK treated (B,D,F) (1-20d) mice. In wild type, Purkinje cell soma are aligned in a monolayer (A) and their dendritic tree extends throughout the ML (C). In MBP-TK cerebella, Purkinje cells form a multicellular layer (B) and the formation of their dendritic arborization is clearly altered (D). (E) Purkinje axons in wild type converge in the white matter tract to form bundles of myelinated fibers. (F) In MBP-TK mice, Purkinje axons are defasciculated. Scale bars: 75 µm (A,B); 25 µm (C-F).

View larger version (70K): [in a new window]   Fig. 4. The postnatal development of granule cells is altered in treated MBP-TK mice. Granule cells were analyzed in sagittal cerebellar cryosections from (A,C) wild-type and (B,D) MBP-TK 6-day-old treated mice. (A,B) In situ hybridization using a PAX6 antisense riboprobe to label granule cells in the EGL, and (C,D) immunofluorescence using an anti-phosphorylated histone 3 (PH3) antibody to reveal proliferating cells in this layer. Arrowheads delimit the thickness of the EGL in wild-type (A,C) and MBP-TK (B,D) cerebella. The arrowhead external to the cerebellum in D also indicates the reduced thickness of the EGL in the region in which the fissura intercruralis should have formed. Squares (A,B) indicate the regions that have been analyzed at higher magnification (C-F). (E,F) TUNEL fluorescent labeling of apoptotic nuclei in the EGL of treated 6-day-old (E) wild-type and (F) MBP-TK mice. Nuclei were counterstained with DAPI. (G,H) Dark-field micrographs of in situ hybridization experiments using a RU49 antisense riboprobe in cerebellar sections from 3-week-old (G) wild-type and (H) MBP-TK treated (1-20d) animals. (I-L) Double immunostaining using anti-PAX6 (I) and anti-HSV1-TK (J) revealed, as expected, a complete absence of TK-positive granule cell precursors. Nuclei were counterstained with DAPI. Scale bars: 100 µm (A,B), 35 µm (C,D), 35 µm (E,F), 100 µm (G,H), 15 µm (I-L).

View larger version (87K): [in a new window]   Fig. 5. Interneuron migration to the cerebellar cortex is impaired in treated MBP-TK mice. Cerebellar sections from 3-(A,B) and 6-day-old (C-F) wild-type (A,C,E) and MBP-TK (B,D,F) treated mice were analyzed using a PAX2 antisense riboprobe (A-D). (E,F) Serial sections from the same 6-day-old treated mice were immunostained with an anti-PAX2 antibody (E, wild type; F, MBP-TK). In the wild type, migrating PAX2-positive cells are found in the white matter tract (arrowheads; A,C,E). In MBP-TK cerebella, a strong reduction in migrating PAX2-positive cells (arrowhead in F) is observed (B,D,F). (G,H) In situ hybridization using a GAD67 riboprobe. Insets show higher magnification of the ML of each section. (I,J) Immunofluorescence using an anti-parvalbumin antibody on cerebellar sections of 21-day-old wild-type and MBP-TK (1-20d) treated mice. (G) In wild-type mice, the GAD67 probe labels Purkinje cell bodies and interneurons localized in the ML and in the IGL. (H) In MBP-TK mice, only the Purkinje multicellular layer and interneurons in the IGL can still be detected. Higher magnification of the ML (insets in G,H) shows the presence and absence of GAD-positive interneurons in wild-type and MBP-TK mice, respectively. (I) Basket and stellate cells (arrowheads) visualized by anti-parvalbumin immunostaining within the ML of the wild-type cerebella. (J) Interneurons are absent in the ML of the MBP-TK cerebella. Arrowhead in J indicates parvalbumin-positive interneurons. Scale bars: 170 µm (A-D); 70 µm (E,F); 200 µm (G,H); 50 µm (I,J).

View larger version (115K): [in a new window]   Fig. 6. The cerebellar phenotype of MBP-TK mice is dependent on oligodendrocyte ablation timing. Cerebella of 3-week-old wild-type (A,E) and MBP-TK (B-D,F-H) mice treated with three different FIAU injection protocols (1-20d: A,B,E,F; 1-6d: C,G; 6-20d: D,H) were used for double immunostaining with anti-calbindin (green) and anti-parvalbumin (red) antibodies (A-D), and histological analysis of Cresyl Violet stained sections (E-H). Parvalbumin-positive interneurons are indicated by arrowheads (A-D). ML, molecular layer; IGL, internal granular layer; WM, white matter. Scale bars: 25 µm (A-D); 50 µm (E-H).

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