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First published online 3 May 2006
doi: 10.1242/dev.02382

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Multidirectional and multizonal tangential migration of GABAergic interneurons in the developing cerebral cortex

¹ Graduate School of Frontier Biosciences, Osaka University, and SORST, Japan Science and Technology Corporation (JST), Yamadaoka 1-3, Suita, Osaka 560-8531, Japan.
² Department of Genetic and Behavioral Neuroscience, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maebashi 371-8511, Japan.
³ Neural Circuit Mechanism Group, BSI, RIKEN, Hirosawa 2-1, Wako, 351-0198, Japan.

View larger version (95K): [in a new window]   Fig. 1. Cortical interneurons orient in many directions in multiple zones. (A) Semi-schematic representation of a transverse section of an E15.5 GAD67-GFP mouse cortex indicating the depth of tangential sections. Numbered blue lines represent the approximate level of the cortex from which the corresponding tangential sections in B were obtained. Dotted line indicates the central region of the sections indicated by boxes in B. (B) GFP neurons in E15.5 serial tangential sections, lined from pial to ventricular side. (C) Higher magnification confocal images of the boxed areas in B. Two images were taken from each section by changing the depth of focus. C1, C3, C5, C8 and C10 were taken from the dorsal part of tangential slices numbered 1, 2, 3, 5 and 6 in B, respectively. C2, C4, C6, C9 and C11 were from the ventral part of slices numbered 1, 2, 3, 5 and 6 in B, respectively. GFP neurons orient in many directions in the MZ, CP, SP and VZ. M, medial; V, ventral; R, rostral. Scale bars: A, 200 µm; B, 400 µm; C, 40 µm.

View larger version (144K): [in a new window]   Fig. 2. Time-lapse analysis of MDT migration of interneurons in the VZ. (A) Schematic of time-lapse imaging in a flat-mount preparation. Dorsal cortex was excised and flat-mounted with the ventricular side up. (B) Confocal images of interneurons in the VZ of an E13.5 mouse viewed from the ventricular side. The number in the top right corner is the time in minutes. Neurons indicated by arrowheads of the same colour represent the same neurons. (B') Tracks of the four neurons shown in B. Each dot corresponds to the neurons shown by arrowheads in B and illustrates their positions plotted at 5 minutes intervals. Arrows indicate the direction of migration. R, rostral; L, lateral. Scale bar: 20 µm.

View larger version (40K): [in a new window]   Fig. 3. Quantitative analysis of MDT migration of interneurons in the MZ and VZ. (A,B) Quantification of the migration direction of GFP neurons in the MZ. Most GFP neurons migrated (80±1%; n=1132/1412 neurons, five hemispheres; see Materials and methods for the definition of migrating neurons). 0° represents medial and 90° rostral migration. The horizontal plane was subdivided into 24 (A), four (B, left panel) or two sectors (B, right panel), and the proportion of the neurons migrating in each sector was analysed (n=1132 neurons, five hemispheres). The proportion of neurons in each sector is shown by the histogram (average±s.e.m.). Note that rostrally migrating neurons outnumber those migrating medially (B, left panel). (C,D) As for A and B, but for VZ GFP neurons. Almost all GFP neurons migrated (96±2%; n=474/496 neurons, four hemispheres). In the upper part of A and C, overall distribution is also shown by polar coordinates. All data were from E13.5 mice. *P<0.03, **P<0.02, ***P<0.01 (Mann–Whitney U-test). M, medial; R, rostral; L, lateral; C, caudal.

View larger version (136K): [in a new window]   Fig. 4. DiD-labelled GFP neurons with migrating cell morphology rostral, caudal, medial and lateral to injection site. (A) Schematic of an embryonic mouse brain illustrating DiD injection into the parietal cortex. Injection was performed at E15.5. (B) Transmitted-light image of a sagittal slice from a DiD-injected GAD67-GFP brain one and a half days after injection. (B9) Fluorescent micrograph showing the distribution of DiD-labelled GFP neurons in the boxed area in B. DiD labelling (magenta) was confined to the cerebral cortical wall. Merged view of GFP and DiD fluorescence. Insets show higher magnification views of the DiD-labelled GFP neurons indicated by straight lines. (C,D) Fluorescent micrographs showing DiD-labelled neurons in coronal sections one and a half days after injection. Insets show higher magnification views of the DiD-labelled GFP neurons indicated by straight lines. Both show the morphology of migrating neurons extending leading processes. Ctx, cortex; GE, ganglionic eminence; Hip, hippocampus; R, rostral; M, medial; V, ventral. Dotted lines indicate the slice outline. Scale bars: B, 500 µm; B', 300 µm; C, 100 µm; D, 200 µm; B9,C,D, insets, 10 µm.

View larger version (25K): [in a new window]   Fig. 5. Distribution of DiD-labelled GFP neurons in sagittal and coronal slices, one and a half days after injection. The analysis was carried out for all DiD-labelled GFP neurons located >400 µm from the injection site in sagittal (n=166 neurons, four brains) or coronal sections (n=74 neurons, four brains). (A) Percentage of DiD-labelled GFP neurons distributed in each cortical zone. The neurons were subdivided into four classes based on their position relative to the injection site: medial (purple), rostral (light blue), lateral (orange) or caudal (yellow). MZ, marginal zone; u-CP, upper cortical plate; l-CP, lower cortical plate; SP, subplate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone. (B) DiD-labelled GFP neurons observed rostral and caudal to the injection site in sagittal sections (left), or medial and lateral to the injection site in coronal sections (right). M, medial; R, rostral; L, lateral; C, caudal.

View larger version (67K): [in a new window]   Fig. 6. Dispersion of DiD-labelled GFP neurons in the MZ after DiD injection into the parietal cortex. (A) Schematic of the experimental paradigm used. DiD-labelled GFP neurons in the MZ were analysed after an appropriate survival period by preparing flat-mount cortical preparations. About 70% of DiD-labelled neurons expressed GFP (n=352/485 DiD-labelled cells, 12 brains). The remainder may include Cajal-Retzius cells (Bielle et al., 2005; Takiguchi-Hayashi et al., 2004). (B,C) Distribution of DiD-labelled neurons in flat-mount preparations one and a half days (E17.0; B) or three days (E18.5; C) after injection. (B1) Dorsal view of the area around the injection site. (B2,B3) Higher magnification of the DiD-labelled GFP neurons indicated by straight lines in B1. These neurons extended an unbranched (B2) or branched (B3) leading process and a short trailing process. Comparison of fluorescence in a single optical section demonstrates that this DiD-labelled cell (inset, magenta) expresses GFP (green). (C1) Dorsal view of the area around the injection site. (C2-C4) Higher magnification of the DiD-labelled GFP neurons indicated by straight lines in C1. These neurons showed typical morphology of migrating neurons in the MZ extending a leading process (C2), occasionally extending a thick but short process (C3) that appears to elongate toward the CP (data not shown). Neurons having several long processes, and granular structure in the trailing process were also observed (C4). (D) Distribution of DiD-labelled GFP neurons in flat-mount preparations one and a half (E17.0; black circles) and three days (E18.5; white circles) after injection. The neurons situated >600 µm or >800 µm from the injection site were plotted for E17.0 or E18.5 brains, respectively. Pooled data from two (E17.0) or six (E18.5) independent experiments. The lack of data medial to the injection site is due to folding of the cortex. OB, olfactory bulb; R, rostral; C, caudal; L, lateral. Dotted lines, flat-mount outline. Scale bars: in B1,C1, 200 µm; in B2, B3, 10 µm; in C2-C4, 10 µm.

View larger version (55K): [in a new window]   Fig. 7. Dispersion of DiD-labelled GFP neurons in the MZ after the DiD injection into the frontal or occipital cortex, three days after injection (E18.5). (A,B) Dorsal view of the area around the injection site. Injection was made into the frontal (A) or occipital (B) cortex. Inset diagram illustrates the position of the injection site. (C,D) Distribution of DiD-labelled GFP neurons after injection into the frontal (C) or occipital (D) cortex. Circles show the positions of DiD-labelled GFP neurons 1 mm from injection site. Each graph represents pooled data from two independent experiments. The lack of data in regions rostromedial (A,C) or caudomedial (B,D) to the injection site is due to folding of the cortex. R, rostral; C, caudal; L, lateral. Dotted lines show the flat-mount outline. Scale bars: 500 µm.

View larger version (62K): [in a new window]   Fig. 8. Distribution and characterization of DsRed-labelled cells 3.5 days after electroporation (E15.5). (A) Distribution of DsRed-labelled cells (arrows) in the dorsal cortex in a coronal section. Schematic illustrates GE-directed electroporation at E12.0 (inset). (B,C) Immunohistochemical characterization of DsRed cells (magenta) in the neocortex. Most DsRed cells in the dorsal cortex were positive for both GAD67-GFP (green in B; 98.1±1%; n=207/210 DsRed cells, three brains) and LHX6 (green in C; 91.4±7%; n=154/163 DsRed cells, five brains). Dotted lines indicate the section outline. D, dorsal; L, lateral. Scale bars: A, 200 µm; B,C, 10 µm.

View larger version (154K): [in a new window]   Fig. 9. Time-lapse analysis of MDT migration of DsRed cells in the MZ. (A) Schematic of time-lapse imaging in a flat-mount preparation from DsRed-labelled brain. (B) Confocal images of DsRed cells in the MZ. The number in the top right corner is the time in minutes. Neurons indicated by arrowheads of the same colour represent the same neurons. (B') Tracks of the four neurons shown in B. Each dot corresponds to the neurons shown by arrowheads in B and illustrates their positions plotted at 20 minutes intervals. Arrows indicate the direction of migration. (B'') Migration direction of all of the DsRed neurons shown in B. R, rostral; M, medial. Scale bar: 20 µm.