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Absence of Cajal-Retzius cells and subplate neurons associated with defects of tangential cell migration from ganglionic eminence in Emx1/2 double mutant cerebral cortex

Koji Shinozaki^1,*, Toshihiko Miyagi^1,*, Michio Yoshida¹, Takaki Miyata³, Masaharu Ogawa³, Shinichi Aizawa^1,2, and Yoko Suda^1,2

¹ Department of Morphogenesis, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811, Japan
² Vertebrate Body Plan Group, RIKEN Center for Developmental Biology, 2-2-3 Minatojima Minami, Chuou-ku, Kobe 650-0047, Japan
³ Laboratory for Cell Culture Development, RIKEN Brain Science Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
^* The first two authors contributed equally to this work

View larger version (108K): [in a new window]   Fig. 1. Morphological features of Emx1/2 double mutant telencephalon. Coronal views at (A-C) E18.5, (D-F) E15.5, (G-I) E13.5 and (J-L) E11.5. (A,D,G,J) wild-type, (B,E,H,K) Emx2–/– and (C,F,I,L) Emx1–/– Emx2–/– telencephalon. LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence; MP, medial pallium; NC, neocortex; PA, pallidium; ST, striatum. Scale bars: (A-F) 450 µm; (G-L) 300 µm.

View larger version (53K): [in a new window]   Fig. 2. BrdU incorporation. (A) Boxed areas indicate 100 µm wide sampling areas. (B,C) Numbers of total cells (B) and BrdU-positive cells (C) were counted in nine sections obtained from three embryos of each genotype. (D) The labeling index, or BrdU-positive cells per total cells. Scale bars, 100 µm.

View larger version (22K): [in a new window]   Fig. 3. Frequency of tetraploid cells in the neocortex. The frequency was determined for BrdU-incorporating (R1) and non-incorporating (R2) populations by 7-AAD staining. The averages of R1 and R2 in three independent analyses were 7.3 and 6.6% in wild type (A), 8.2 and 7.3% in Emx2 single mutants (B) and 7.9 and 5.8% in Emx1/2 double mutants (C), respectively.

View larger version (83K): [in a new window]   Fig. 4. Cell influx from the ganglionic eminence. DiI was placed in the E13.5 medial ganglionic eminence (A-C). Subsequently, slices were cultured for 1 (D-F) and 2 days (G-I). (J-L) Enlarged views of the boxed areas in G-I, respectively. (A,D,G,J) wild type, (B,E,H,K) Emx2 single and (C,F,I,L) Emx1/2 double mutants. Yellow dotted lines show the cortical/subcortical borders. Scale bars: (A-I) 100 µm; (J-L) 400 µm.

View larger version (40K): [in a new window]   Fig. 5. Transplantation analysis of migration defects. To determine that migration defects in Emx1/2 double mutants do not reside in ganglionic eminence cells, three assays were conducted (A). In the first assay (1), E13.5 medial ganglionic eminences were dissected and dispersed with trypsin. Then, the dispersed cells were labeled with DiI and injected into E13.5 medial ganglionic eminence of the host telencephalon slices. The donor cells were also BrdU-labeled, by injecting the drug into the pregnant mother intraperitoneally at E11.5, to rule out the possible secondary DiI-staining of host cells. BrdU-positive donor cells in the cortex were detected by immunohistochemical staining with the antibody against BrdU after vibratome sectioning. In the second assay (2), explants of about 150 µm diameter were prepared from E13.5 medial ganglionic eminences and labeled with DiI. The donor explant was placed into a hole cut through the medial ganglionic eminence of the E13.5 host telencephalic slice. In the third assay (3), cortices and medial ganglionic eminences were surgically separated obliquely (green line) at the lateral ganglionic eminence level. A wild-type cortex piece was then recombined with a mutant medial ganglionic eminence piece and vice versa. Subsequently, ganglionic eminence cells were labeled with either DiI or Venus. Venus is a EYFP variant (Nagai et al., 2002). The plasmid DNA solution was injected in the medial ganglionic eminences and introduced into the cells by electroporation (Marin et al., 2001). Typical examples of each assay are shown in B. Red arrows indicate the labeled cells in the front of migration; yellow dotted lines show the cortical/subcortical borders. Scale bars: 100 µm. (C) A summary odf the assay results. indicates the donor to host relationship. Each column of the table indicates the number of slices that showed the migration of donor ganglionic eminence cells into host cortex per total slices examined.

View larger version (116K): [in a new window]   Fig. 6. Marker analyses of neuronal differentiation. (A-C) NEX expression at E15.5; (D-F) Lhx2 expression at E15.5; (G-I) RC2 staining at E16.5; (J-L) GFAP staining at E18.5. (A,D,G,J) Wild-type, (B,E,H,K) Emx2–/– and (C,F,I,L) Emx1–/– Emx2–/– cortices. Scale bars, 100 µm.

View larger version (108K): [in a new window]   Fig. 7. Cortical lamination. (A-H) Nissl’s staining; (E-H) enlarged views of marginal zone. (I-L) MAP2 staining. (M-O) GABA staining. (P-R) Glutamate staining. (S-U) SCIP expression. (A,E,I,M,P,S) Wild-type, (B,F,J) Emx1–/–, (C,G,K,N,Q,T) Emx2–/– and (D,H,L,O,R,U) Emx1–/– Emx2–/– cortices at E16.5 (M-R) and E18.5 (A-L,S-U), respectively. CP, cortical plate; IZ, intermediate zone; SP, subplate; SV, subventricular zone; MZ, marginal zone; VZ, ventricular zone. Scale bars, (E-L) 50 µm; others 100 µm.

View larger version (96K): [in a new window]   Fig. 8. Birthdate analysis. BrdU was injected at E13.5 (A-D) or E15.5 (E-H), followed by examination of the distribution of BrdU-positive cells at E19.5. (A,E) Wild-type, (B,F) Emx1–/–, (C,G) Emx2–/– and (D,H) Emx1–/– Emx2–/– cortices. Brackets indicate the areas where BrdU-positive cells are distributed. Abbreviations as in Fig. 7. Scale bars, 100 µm.

View larger version (84K): [in a new window]   Fig. 9. Reelin expression. Reelin expression at (A-C) E13.5 and (D-I) E15.5. (G-I) Enlarged views of the marginal zone of D-F, respectively. (A,D,G) Wild-type, (B,E,H) Emx2–/– and (C,F,I) Emx1–/– Emx2–/– cortices. Scale bars, 100 µm.

View larger version (116K): [in a new window]   Fig. 10. Marker analyses for CR cells and subplate neurons. (A-C) GAP43 staining, (D-F) CSPGs staining, (G-I) calretinin staining, (J-L) L1 staining and (M-O) TAG1 staining at E16.5 (A-I) and E17.5 (J-O). Inserts in G-I provide enlarged views of the staining. (A,D,G,J,M) Wild-type, (B,E,H,K,N) Emx2–/– and (C,F,I,L,O) Emx1–/– Emx2–/– cortices. Scale bars, (A-I) and (M-O) 100 µm; (J-L) 400 µm.

View larger version (96K): [in a new window]   Fig. 11. Onset of Emx1/2 double mutant neocortical defects. (A-C) Emx1 and (D-F) Emx2 expression in wild-type cortex at E11.5 (A,D) and at E15.5 (B,C,E,F). (C,F) Enlarged views of B,E at the marginal zone. (G-I) MAP2, (J-L) reelin and (M-O) calretinin expression in E12.5 (G-I) and E11.5 preplate (J-O). (P-R) Birthdate analysis of E11.5 pioneer neurons. BrdU was injected at E11.5, subsequently, the distribution of BrdU-positive cells was examined at E18.5. (G,J,M,P) Wild-type, (H,K,N,Q) Emx2–/– and (I,L,O,R) Emx1–/– Emx2–/– cortices. Abbreviations as in Fig. 7. Scale bars, C and F 25 µm; others 100 µm.

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