First published online 8 October 2003
doi: 10.1242/dev.00825
Multimodal tangential migration of neocortical GABAergic neurons independent of GPI-anchored proteins
Daisuke Tanaka1,*,
Yohei Nakaya1,*,
,
Yuchio Yanagawa2,3,4,
Kunihiko Obata2,3,4, and
Fujio Murakami1,4,5,
1 Graduate School of Frontier Biosciences, Osaka University, Machikaneyama 1-3,
Toyonaka, Osaka 560-8531, Japan
2 Laboratory of Neurochemistry, National Institute for Physiological Sciences,
Myodaiji, Okazaki, 444-8585, Japan
3 The Graduate University for Advanced Studies, Hayama, Kanagawa 240-0193,
Japan
4 SORST, Japan Science and Technology Corporation, Kawaguchi, 332-0012,
Japan
5 Division of Behavior and Neurobiology, Department of Regulation Biology,
National Institute for Basic Biology, Myodaiji-cho, Okazaki 444-8585,
Japan
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Fig. 1. GFP+ neurons in the dorsal cortex of heterozygous
(Gad67gfp/+) mice. Insets are high-magnification views of migrating
cells identified by numbered arrows. Corresponding insets are arranged from
left to right. Note that there are many cells that appear to be migrating
tangentially (B-1, C-1) or obliquely to the tangential axis (B-2). Some cells
are oriented radially (C-2,3). GFP+ neurons can also be found in
the VZ. (A) E12; (B) E13.5; (C) E15.5. GFP+ neurons penetrated into
the lateral cortex at E12 and are distributed in more medial parts at later
stages. VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone;
SP, subplate; CP, cortical plate; MZ, marginal zone. Scale bar: 100 µm.
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Fig. 2. Migration of GFP+ neurons in cortical slices. (A) The time
sequence of migrating neurons in a coronal slice taken over a time interval of
35 minutes in an E13.5 mouse embryo. (B,C) Similar to A but from an E15.5
mouse and taken at time intervals of 40 and 25 minutes, respectively. (D) A
merged view of a time sequence taken at a 30 minutes interval from an E15.5
mouse. The image taken at an earlier time of observation was converted to
green and later captured to red. Thus, cells colored yellow mostly represent
cells that stayed in the same position during the intervals, although part of
them could represent two different cells, one of which migrated to original
position of the other. Those in red or green represent cells that had migrated
during the interval. Note that most MZ neurons are yellow, suggesting no
migration. (E,F) Enlarged view of an image generated by merging the two panels
of A after color conversion described above. (E) Two tangentially migrating
cells indicated by arrowheads in A are shown by a pair of arrows. The neuron
labeled as `1' is deflected towards the MZ, whereas neuron `2' migrated
tangentially. (F) Enlarged view of the area shown by a rectangle in A. Note
that there are many yellow dots near the surface, suggesting that many cells
in this region may be stationary. (G) A tangentially migrating neuron in the
IZ. (H) A radially migrating neuron in the cortical plate towards the MZ.
(G,H) Enlarged views of a merged image generated from B and C, respectively
and the positions of these cells are indicated by arrowheads. (I) Radially
migrating neuron away from the MZ. (J) A radially migrating neuron, with some
angle, in the IZ over a 25 minutes interval. (K) A tangentially migrating
neuron in the SVZ but in a reversed direction over a 30 minutes interval.
(I-K) Merged view from an E15.5 mouse embryo. Medial is towards the left and
dorsal is upwards. Scale bars: 100 µm in A–C; 50 µm in D; 25 µm
in E-J. For animated versions of the complete time-lapse sequence, see Movies
1 and 2 at
http://dev.biologists.org/supplemental/
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Fig. 3. Migration of GFP+ neurons observed in flat-mount preparation of
the neocortex. (A,B) A is an image from E13 mouse dorsal cortex and B is the
same cortex taken 50 minutes later. (C) Merged view of images in A,B. The
images were taken from the leading edge of migratory streams. Most cells stay
almost in the same position (yellow). Although they show some motility,
migration toward a specific direction was not observed. Occasionally, some
cells near the leading edge showed medially directed migration (arrows). *A
neuron moving laterally. The white rectangle in the inset illustrates
approximate position of the image. (D) The same as A but from an E13.5 mouse
dorsal cortex. (E) An image captured 50 minutes later (red) was merged with D
(green) as in C. The white rectangle in the inset illustrates approximate
position of the image. (F) The movement of 85 arbitrarily selected cells in D,
taken during a 20 minutes interval, was plotted. Yellow dots indicate
stationary cells and green dots with arrows indicate migrating cells and their
direction of migration. X symbols and double circles indicate where cells
disappeared or appeared, respectively. Medial is upwards, lateral is downwards
and anterior is towards the left. For an animated version of the complete
time-lapse sequence, see Movies 3 and 4 at
http://dev.biologists.org/supplemental/.
Scale bar: 50 µm.
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Fig. 4. The relationship between the position of GFP+ neurons in the
cerebral cortex and their rate or orientation. (A,B) The average speed of
migration for individual cells plotted against the distance from the
ventricular surface. Each point represents a migrating neuron at (A) E13.5 and
(B) E15.5 mouse. (C,D) The orientation of migration as measured from
deflection from a line drawn vertical to the ventricular surface was plotted
against the distance from the ventricular surface at (C) E13.5 and (D) E15.5.
Inset illustrates the method of angle measurement. The green line represents
90°. Note that a substantial proportion of cells in the IZ, SP and CP
migrate with angles of more than 90°, indicating that they deflect towards
the pial surface. Negative angles indicate the occurrence of medial-to-lateral
migration. All cells that could be traced were plotted. The high density of
GFP+ neurons, particularly in the SVZ and the MZ, allowed
measurement of only a fraction of the cells. The speed of migration is
underestimated, because rapidly migrating cells in cell-dense regions could
not be followed and curved trajectories of migrating cells were approximated
by straight lines. Although most MZ neurons were stationary, some slight
dislocations can be observed. This is largely due to a drift of the slice
during observation. Thus, in C and D, cells that moved less than 10 µm per
hour were excluded from the analysis.
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Fig. 5. Distribution and morphology of GFP+ neurons in a transgenic
mouse homozygous for the Gad67-GFP allele (Gad67gfp/gfp). (A,B)
GFP+ neurons in fixed sections of the dorsal cortex from an E13.5
(A) or an E15.5 (B) homozygous (Gad67gfp/gfp) mouse embryo. There
is no notable difference in distribution and morphology of GFP+
neurons between heterozygous (Gad67gfp/+) and homozygous
(Gad67gfp/gfp) mice (compare with
Fig. 1B,C). As in heterozygous
mice, many neurons with a leading process are found in the lower IZ. (C,D)
Quantitative representation of GFP+ migrating cells from an E13.5
homozygous mouse. There is no noticeable difference in direction or average
speed between heterozygous and homozygous mice (compare with
Fig. 4A,C). Scale bar: 50
µm.
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Fig. 6. Expression of Tag1 in the cortex and the distribution of GFP+
neurons. Coronal sections of an E13.5 (A) or E15.5 (B) mouse cortex were
immunostained for Tag1 (red). Only a small fraction of GFP+ cells
migrate in the region where Tag1 immunoreactivity can be observed. (C)
High-magnification view of the area shown by a rectangle in B. Arrows indicate
radially oriented Tag1+ fibers. Note while the fibers are strictly
oriented radially (see Fig. 6A also) in the cortical plate, only a fraction of
GFP+ neurons are. Scale bar: 50 µm.
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Fig. 7. The effect of PI-PLC treatment on migration of GFP+ neurons in
cortical slices. (A) Tag1 immunostaining of a cortical slice incubated in a
control solution for 5 hours. (B) Tag1 immunostaining of a cortical slice
after 2 hours incubation in a PI-PLC-supplemented solution. No immunostaining
can be observed. (C) The time sequence of migrating neurons in an E15.5
coronal slice taken over a time interval of 60 minutes. (D,E) The relationship
between the position of GFP+ neurons in the cerebral cortex and
their direction of migration or the rate of migration. (D) The average speed
of migration for individual cells plotted against the distance from the
ventricular surface. (E) The direction of migration as measured from
deflection from a line drawn vertical to the ventricular surface was plotted
against the distance from the ventricular surface. See legend to
Fig. 4 for further explanation.
Scale bars: 125 µm in A,B; 50 µm in C.
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Fig. 8. Summary diagram of the present study and a model for intracortical
migration of GABAergic neurons. (A) The migratory behavior of GABAergic
neurons in E13.5 neocortex. Dorsomedially directed tangential migration was
most prominent in the lower intermediate zone (IZ), but some of the neurons
deflected towards the marginal zone (MZ). Neurons in the MZ tangentially
migrated in many directions (4), although many of them are stationary (blue
spheres). (B) The modes of migration of GABAergic neurons observed in the
coronal plane of E15.5 neocortex. Tangentially migrating IZ/SVZ neurons
continue to migrate towards the hippocampus (1), whereas those deflected
towards the pial surface migrate radially (2) or with some angle to the radial
axis (3), towards the MZ. In the MZ, a part of these neurons may migrate on
the tangential plane in many directions and spread into the whole cortical
areas (see neurons labeled by 4). A part of these neurons may descend from the
MZ to be integrated into CP (5). Intracortical migration occurs independent of
Tag1+ axons (yellow). Red spheres represent motile cells.
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© The Company of Biologists Ltd 2003
