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First published online 27 July 2004
doi: 10.1242/dev.01299

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Compromised generation of GABAergic interneurons in the brains of Vax1^-/- mice

Dulbecco Telethon Institute at CNR-ITB, via fratelli Cervi, 93 20090 Segrate (MI), Italy

View larger version (126K): [in a new window]   Fig. 1. Vax1 expression in the ventral telencephalon. (A-D,F) In situ hybridization revealing Vax1 expression in telencephalic coronal sections in E12.5 wild-type mice. H is a magnification of F. (A-D) Different rostrocaudal levels of the same brain (A is the most rostral and D is the most caudal). Vax1 is strongly expressed in the SA, LGE, MGE and post-optic area. There is sharp downregulation of Vax1 expression at the corticostriatal boundary (arrowhead in A). (E) Immunohystochemical localization of the cell cycle marker Ki67 on an adjacent section to that shown in F. G is a magnification of E. (E-H) In the ganglionic eminences (F) and in the septum (H), Vax1 expression overlaps with Ki67 (E,G) with an inverse gradient. III, third ventricle; AEP, anterior entopeduncular area; CGE, caudal ganglionic eminence; LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence; POA, pre-optic area; HT, hypothalamus; SA, septal area. Scale bars: 500 µm in A-D; 200 µm in E,F; 100 µm in G,H.

View larger version (133K): [in a new window]   Fig. 2. Ventral telencephalic defects in Vax1 mutant brains. Coronal sections from wild-type (A,C,E,G,I,K,M,O) and Vax1 knockout (B,D,F,H,J,L,N,P) E13.5 embryos. The expression of several pallial and subpallial markers has been analyzed by RNA in situ hybridization (C-L,O,P) or by immunohistochemistry (M,N). Nissl analysis of comparable sections from wild-type and mutant telencephalon (A,B) reveals that in the absence of Vax1 the interganglionic sulcus is missing (black arrows). This defect is accompanied by the modification of the expression pattern of Gli1 (C,D), which shows a diffuse labeling pattern in the territory surrounding the mutant interganglionic sulcus. (E,F) In wild-type brains, the homeobox gene Nkx 2.1 is expressed in the proliferative zone of the MGE (E); in Vax1-/- embryos, its expression is expanded (F). (G,H) Ebf1 is a marker of the mantle zone of the LGE (G). In the mutant brain, its expression is strongly reduced but correctly restricted to this region (H). (I-J) The MGE mantle marker Lhx8 shows a significant reduction in mutants (J) compared with wild types (I). (K-N) The boundary between ventral pallium and subpallium (broken black line) is maintained in the absence of Vax1: expression domains of the pallial markers Ngn2 (K,L) and Pax6 (M,N) are unaltered. (O,P) The subpallial marker Gsh2 does not show a dorsal shift. LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence. Scale bars: 500 µm.

View larger version (132K): [in a new window]   Fig. 3. Abnormal differentiation in Vax1-/- brains. Proliferating cells at E13.5 (A,B) and E16.5 (C,D) were revealed with anti-Ki67 antibody. Ki67 shows strong labeling in the VZ and SVZ of the wild-type LGE and MGE (A,C). Labeling in the Vax1 mutant is significantly expanded in the MGE and presumptive septal area (D). The expansion of the VZ in the mutant MGE is confirmed by the complementary Tuj1 staining at E15.5 (E,F). There is strong expansion of the Tuj1-negative domain in the mutant brain (F). (G,H) Birthdating analysis. Brains were injected with BrdU at E13.5 and analyzed at P0. Cells labeled with BrdU at E13.5 are dispersed in the wild-type P0 brain (G, arrowheads); by contrast, BrdU-positive cells linger at the border between proliferating and differentiated regions in the mutant MGE (H, arrowheads). Acb, nucleus accumbens; Acb*, presumptive nucleus accumbens; LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence; SA, septal area; SA*, presumptive septal area; VZ, ventricular zone. Scale bars: 500 µm in A-F; 100 µm in G,H.

View larger version (102K): [in a new window]   Fig. 4 Vax1 mutant MGE shows reduced levels of Dlx proteins and loss of Gsh1 expression. Coronal sections from control (A,C,E,G) and Vax1 mutant (B,D,F,H) telencephalons at E13.5 analyzed by immunohistochemistry (A-F) and in situ RNA hybridization (G,H). Dlx proteins are expressed in the VZ and SVZ of wild-type SA, LGE and MGE (A,C) in a gradient similar to that of Vax1 mRNA. In Vax1-/- ganglionic eminences, Dlx expression is reduced in the MGE and presents a shallower graded pattern in the LGE (B,D). Dlx-positive cells are reduced in the cortex of mutant mice (arrowheads in E and F). In the MGE of mutant brains, Gsh1 expression is almost completely lost (compare G with H). LGE, lateral ganglionic eminence; LV, lateral ventricle; MGE, medial ganglionic eminence; SA, septal area; SA*, presumptive aplastic septal area; SVZ, subventricular zone; VZ, ventricular zone. Scale bars: 500 µm in A,B,G,H; 100 µm in C-F.

View larger version (81K): [in a new window]   Fig. 6. Tangential migration from MGE and POA. (A-D) Cell tracing by DiI injection. Labeled cells from E13.5 MGE after 48 hours in culture can migrate to the cortex. (A,C) Bright-field images of a wild-type (A) and a mutant (C) slices with DiI crystals placed in the MGE. (B,D) Fluorescent images of the region boxed in A and C, respectively; white arrowheads show migrating cells in the cortex of wild-type and mutant embryos. (E-H) Immunohistochemistry for calbindin on coronal sections at E13.5 wild-type (E,F) and Vax1-/- embryos (G,H). (F,H) High-magnification images of the region boxed in E and G, respectively; black arrows indicate labeled cells in a tangential orientation. Clear leading processes are present. Arrows in G indicate an aberrant accumulation of calbindin-positive cells in the area of the MGE and POA. CX, cortex; LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence; POA, preoptic area. Scale bars: 100 µm.

View larger version (34K): [in a new window]   Fig. 5. Decreased number of GABAergic interneurons in the cortex of Vax1 mutant mice. GABA-positive cells were counted in sagittal sections from wild-type (A,C) and mutant (B,D) P0 mice. (A,B) DAPI staining of two comparable sections from wild-type (A) and mutant (B) brains. (C,D) Immunohistochemistry with FITC-labeled anti-GABA antibody on the same sections; images are high magnifications of the regions indicated with asterisks in A and B, respectively. (E) Summary of cell counts (see text for details). Graphical representation of the results, indicating the amount of GABAergic cell decrease in Vax1-knockout brains (wild type is 100%). Scale bars: 500 µm in A,B; 50 µm in C,D.

View larger version (105K): [in a new window]   Fig. 7. Tangential migration from the septum is severely impaired in Vax1-/- embryos. (A,B) Nissl staining of coronal sections from wild-type and mutant brains at E16.5. There is complete loss of the septum in Vax1-/- telencephalon. (C,F) (Top) The experimental procedure. The red spots represent DiI crystals placed at E13.5 in an organotypic slice culture assay. (Bottom) Bright-field images of wild-type (C) and mutant (F) slices corresponding to those shown in the scheme. Sections are taken at comparable rostrocaudal levels. (D,E,G,H) Fluorescent images of the same slices from wild-type (D,E) and Vax1-/- (G,H) embryos; (E,H) high-magnification photographs of the region boxed in D and G, respectively. Cells that migrate along a lateral-tangential pathway are present in the wild-type slice (E, white arrows) but there is complete loss of this stream in the mutant brain (H). lv, lateral ventricle; lge, lateral ganglionic eminence; sa, septal area; sa*, presumptive septal area.