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First published online 21 January 2004
doi: 10.1242/dev.00984

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Misrouting of mitral cell progenitors in the Pax6/small eyerat telencephalon

Division of Developmental Neuroscience, Tohoku University Graduate School of Medicine, 2-1, Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan

View larger version (71K): [in a new window]   Fig. 1. The OBLS forms ectopically at the lateral part of the telencephalon of Pax6 mutants. (A-D) Lateral views and (E-L) coronal sections of wild-type (WT, A,C,E,F,I,J) and Pax6-mutant telencephalons (-/-, B,D,G,H,K,L), stained with anti-neruopilin 1 (Nrp, A,B,I,K), anti-Calretinin (CR, I,K), anti-GAD67 antibodies (J,L) or hybridized with specific probes for neuropilin 1 (Nrp, E,G), netrin G1 (C,D,F,H). A-H and I-L show E16.5 and E18.5 embryos, respectively. Nrp and netrin G1 are expressed in the protruding OB in the wild type (arrowheads in A,C), whereas these markers are observed at the lateral part of the mutant telencephalon (arrowheads in B,D). Note that Nrp protein is localized mostly at the neuropiles and axons of mitral cells (arrowheads and arrows in A,B). The distribution of Nrp and netrin G1 overlap in the OB and the OBLS (E-H). Nrp, CR and GAD67 are expressed in the different types of neurons of the OB and the OBLS (I-L). egl, external granule cell layer; epl, external plexiform layer; grl, glomerular layer; mcl, mitral cell layer. Scale bars: A-D, 1 mm; E-L, 50 µm.

View larger version (77K): [in a new window]   Fig. 2. The pattern of cell migration is abnormal in the telencephalon of Pax6 mutants. (A) Experimental procedures for cell tracing in the telencephalon. DiI solution was injected into the rostral-most telencephalic wall of E12.5 embryos and whole embryos cultured for 48 hours. (B) Migration of the DiI-labeled cells in the telencephalon of wild type (WT, n=4) and Pax6 mutants (-/-, n=4). Compared to wild type, the mutant cells migrate caudally from the injection point. * P<0.05, t-test. (C,D) Wild-type (C) and mutant (D) embryos just after injection of DiI into the rostral-most telencephalon. Arrowhead indicates the injection points. (E,F) Migration patterns of labeled cells in the wild-type (E) and mutant (F) telencephalon. In the wild type, the majority of labeled cells are distributed at the rostral part of the telencephalon (C). In the Pax6 mutant, many labeled cells migrate caudally into the lateral part of the telencephalon (arrowheads in F). Scale bars: C,D, 1 mm; E,F, 200 µm.

View larger version (71K): [in a new window]   Fig. 3. Mitral cells are derived from the rostral part of the wild-type telencephalon. (A) Experimental procedures for cell tracing of the mitral cell progenitors by electroporation of GFP-expression vector. (B-D) A rostral-lateral view (B) and parasagittal sections (C,D) of the head of electroporated, wild-type embryos. At 24 hours after electroporation, strong GFP-fluorescence is seen at the rostral area of the telencephalon that expresses Pax6 (C,D). (E-E'',F-F'') Distribution of GFP-positive cells in the telencephalon after 2 days of TOC. The OB evaginates at the rostral-most area of the telencephalic explants. F-F'' are transverse sections of a TOC sample. GFP-positive cells contribute to the OB marked by CR and Nrp staining (E',F'). (G-G'') Confocal images of GFP-positive cells in the OB. Nrp is expressed at the neuropiles of the GFP-labeled cells (arrowheads). (H-H'') BrdU incorporation in the GFP-positive cells in the OB. tel, telencephalon; LV, lateral ventricle. Scale bars: B-D, 500 µm; E-F'', 100 µm; G-G'', 20 µm; H-H'', 10 µm.

View larger version (120K): [in a new window]   Fig. 4. Misrouting of mitral cell-precursors in the telencephalon of Pax6 mutants. (A-C) Lateral view (A) and parasagittal sections (B,C) of the head of electroporated, mutant embryos. At 24 hours after electroporation, there is GFP fluorescence in the rostral-most telencephalon. (D-D'',E-E'') Distribution of GFP-positive cells following TOC for 3 days. E-E'' are transverse sections of a TOC sample. GFP-positive cells migrate caudally (arrowheads in D-D''), and constitute the OBLS, which is marked by CR and Nrp immunostaining (arrowheads in D',E'). (F-F'') Confocal images of GFP-positive cells in the OBLS. Nrp protein is localized at the neuropiles of GFP-positive cells (arrowheads). (G-G'') GFP-positive cells are also labeled with BrdU (arrowheads). tel, telencephalon; LV, lateral ventricle. Scale bars: A-C, 500 µm; D-E'', 100 µm; F-G'', 10 µm.

View larger version (61K): [in a new window]   Fig. 5. Misrouting is caused by non-cell autonomous defects for the mitral cell precursors. (A) Experimental procedures for cell transplantation. The rostral part of the telencephalon of donor embryos was isolated, and neuroepithelial cells dissociated with enzymes and transplanted into the rostral part of the host telencephalon. Donor cells were isolated from GFP-transgenic rat embryos. (B,C) Distribution pattern of cells derived from homozygous mutants (-/-) transplanted into heterozygous (+/) embryos after 48 hours of WEC followed by TOC. Donor cells contribute to the host OB and express Nrp (C,C'). (D,E) Distribution pattern of +/- derived cells in -/- embryos after 48 hours in WEC (D) and 2 days in TOC (E). Donor cells migrate caudally toward the lateral telencephalon and invade the OBLS, which is marked by Nrp immunostaining (E). A donor-derived cell in the OBLS expresses Nrp (E'). (F) The distribution of donor cells after transplantation. The white bar indicates that donor cells are located only at the rostral part of the telencephalon. Black bars indicate that donor cells are located in both rostral and lateral parts of the host telencephalon. Scale bars: B,D, 500 µm; C,E, 100 µm; C',E', 10 µm.

View larger version (50K): [in a new window]   Fig. 6. Misrouting of the mitral cell precursors in Pax6 mutants is not caused by lack of olfactory nerve innervation. (A) Experimental procedures for brain culture. After removal of the olfactory epithelium and head mesenchyme, brain tubes were cultured in rotating bottles (whole brain culture, WBC) and in TOC for 2 days. (B,C) Distribution of netrin G1-positive cells in the wild-type (WT; B) and the mutant (-/-; C) telencephalon after WBC. In the wild-type telencephalon, netrin G1-positive cells are located in the rostral-most telencephalon (arrowhead in B), whereas in the mutant they are in the lateral part of the telencephalon (arrowhead in C). Scale bar: 500 µm.

View larger version (41K): [in a new window]   Fig. 7. Misrouting of mitral cells is restored by transfection of Pax6 into the telencephalon of Pax6 mutants. (A) Expression vectors were electroporated into the rostral half of the mutant telencephalon. (B-F) Pax6-mutant embryos/brains transfected with either GFP alone (B) or GFP plus Pax6-expression vector (C-F). (B,C) lateral views of cultured brains. After 48 hours of WEC followed by 2 days of TOC, netrin G1-positive cells are distributed at the lateral surface of the telencephalon in the mutant introduced GFP-gene alone (arrowhead in B). By contrast, netrin G1-positive cells (arrowhead in C) are localized in the rostral-most telencephalon in the embryo electroporated with Pax6. (D-F) horizontal sections of the rostral part of the brains transfected with Pax6-expression vector. Nrp-positive cells are localized at the rostral-most telencephalon, which corresponds to the netrin G1-positive area (E). Pax6-immunopositive cells are distributed at the rostral part of the telencephalon after 2 days of TOC (F). (G) The location of the accumulation of netrin G1-positive cells in the mutant telencephalon after electroporation. tel, telencephalon; r, rostral; c, caudal. Scale bars: A, 500 µm; B,C, 1 mm; D-F, 50 µm.