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Neuronal Migration in the Developing
Cerebral Cortex: Observations Based on
Real-time Imaging
We have used time-lapse imaging of acute cortical slices to study
the migration of neurons from their sites of origin to their positions in
the developing neocortex. We found that two distinct modes of cell
movement, somal translocation and glia-guided locomotion, are
responsible for the radial migration of neurons generated in the
cortical ventricular zone. The former is the prevalent form of radial
movement of the early-born cortical neurons, while the latter is
adopted by those generated later in corticogenesis. Interneurons,
found to originate in the ganglionic eminence, follow tangential
migratory paths to reach the developing cortex. Upon reaching the
cortex, these cells seek the ventricular zone using a mode of
movement that we have termed ‘ventricle-directed migration’, before
they migrate to their positions in the cortical plate. In addition to
these forms of movement, we report here a unique morphological
and migratory behavior for a population of cortical neurons. These
cells are multipolar in form, and are highly motile in the formation
and retraction of their processes. Based on these morphological
features, we refer to this type of cells as ‘branching cells’ and
attribute the phenotype to a subset of cortical interneurons.
B. Nadarajah1, P. Alifragis, R.O.L. Wong2 and J.G. Parnavelas
Department of Anatomy & Developmental Biology, University
College London, London WC1E 6BT, UK and 2Department of
Anatomy & Neurobiology, Washington University School of
Medicine, St Louis, MO 63110, USA
1
Present address: School of Biological Sciences, University of
Manchester, Manchester M13 9PT, UK
other forebrain structures such as the cerebral cortex, olfactory
bulb and hippocampus? How do neurons that are generated in
the subpallial region ‘know’ where to go and ‘what’ guides them
towards the appropriate layers of the developing cortex? Is the
information about their final destination part of their intrinsic
genetic makeup or do these cells ‘acquire’ such information
from guidance cues they encounter en route? While future
investigations may address some of these questions, the demonstration of neurons being generated in pallial and subpallial
regions has led to the study of their various modes of migration
in the developing cortex. Here we review existing evidence for
radial and tangential modes of migration and provide new data
on a form of movement adopted by some young neurons in the
developing cerebral cortex. The significance of the different
modes of migration in building this complex structure is
discussed.
Radial Migration: Somal Translocation and Glial-guided Locomotion
According to the widely accepted model of neocortical development, first documented by the Boulder Committee (Boulder
Committee, 1970), neurons of the cerebral cortex arise in the
germinal ventricular zone (VZ) at the surface of the lateral
ventricles. Newborn neurons migrate towards the margin of the
cerebral wall to form the primordial plexiform layer or preplate
(PP). This zone is then split into the superficial marginal zone
(MZ) and the deeper subplate (SP) by the arrival of the cortical
plate (CP) cells. These neurons accumulate in an ‘inside-out’
sequence, with newly arriving cells migrating radially past the
existing neurons before stopping at the top of the CP (Berry and
Rogers, 1965; Rakic, 1974). The migration of young neurons
from the VZ to the CP is largely dependent on radial glia (Rakic,
1990; Misson et al., 1991). Recent analyses have shown that this
scheme of cortical neuron generation and migration applies only
to pyramidal neurons, the excitator y projection cells of the
neocortex (Mione et al., 1997; Tan et al., 1998; Parnavelas, 2000;
Chan et al., 2001; Anderson et al., 2002).
A number of experimental approaches have clearly demonstrated in recent years that the vast majority of the GABA-containing cortical interneurons arise in the ganglionic eminence
(GE), the primordium of the basal ganglia in the ventral
telencephalon or subpallium (Anderson et al., 1997, 2002;
Lavdas et al., 1999; Wichterle et al., 2001). These neurons
round the corticostriatal notch and follow distinct tangentially
oriented paths to enter the cortex. The demonstration of
neurons generated in two distinct regions of the developing
telencephalon has added a new dimension to the complexity of
the process of cortical formation and, furthermore, has raised
many fundamental questions. What is the evolutionary advantage of having the GE generate interneurons that are parceled to
The early electron microscopical studies in fetal monkey neocortex by Rakic (Rakic, 1972) have shown that migrating
neurons are closely apposed to radial glial fibers, suggesting that
the glial system acts as a scaffold in their movement. Consistent
with these obser vations, more recent immunohistochemical
studies have further revealed that radial glia are present during
corticogenesis and their processes span the full thickness of the
cortical wall (Misson et al., 1991). However, electron microscopical examination of early mouse cortex by Shoukimas and
Hinds (Shoukimas and Hinds, 1978) did not reveal a consistent
association between neurons and glia, even though radial glial
processes were abundantly present. This prompted the
speculation that at the early stages of corticogenesis, when the
cerebral vesicle is relatively thin, radial glia are not required for
guiding neurons. Interestingly, an earlier study using Golgi
staining also suggested a mechanism for radially migrating
neurons in the developing opossum cortex that is independent
of glial guidance (Morest, 1970). In this mode, termed
‘perikaryal translocation’, migrating neuroblasts that initially
maintain processes extending to both ventricular and pial
surfaces loose their ventricular attachments after terminal
division and translocate their somata through pial-directed
processes. However, as hypothesized by Morest (Morest, 1970),
perikaryal translocation does not provide a plausible mechanism
for the migration of later born cortical neurons, particularly at
stages when the cortical anlage is several hundred micrometers
thick. In support of Morest’s earlier work, a more recent
immunohistochemical study (Brittis et al., 1995) has identified
early neuronal populations in the developing rodent cortex with
morphological features characteristic of cells undergoing
perikaryal or somal translocation. In the light of these morphological findings, it seems possible that there may be two distinct
modes of radial migration: an early, glia-independent mode,
© Oxford University Press 2003. All rights reserved.
Cerebral Cortex Jun 2003;13:607–611; 1047–3211/03/$4.00
Introduction
activated during the formation of the PP, and a later one that
utilizes radial glial fibers during CP formation.
In a recent study (Nadarajah et al., 2001), we have used
time-lapse imaging to demonstrate somal translocation as a
distinct mode of radial movement in mouse cortical slices, and
distinguished translocation from glial-guided locomotion by
morphology and migrator y behavior. In describing the two
forms of radial movement we have shown that, during early
corticogenesis, populations of cells undergo long-range somal
translocation from the VZ to their positions beneath the pial
surface. These cells typically showed distinct morphological
features with long radially oriented leading process terminating
at the pial surface and a transient short trailing process. The
migratory behavior of translocating cells is evidently distinct:
firstly, as the soma advances towards the pial surface, the leading
process becomes thicker and progressively shorter, while its
terminal remains attached to the outer surface. Secondly, the
soma of translocating cells displays continuous advancement at
average speeds of 60 µm/h. Based on real-time images, it appears
that somal translocation is a process of nucleokinesis in which
the basal process first extends radially from the VZ to the pial
surface, followed by nucleokinesis together with rapid reorganization of microtubules, resulting in shortening of the basal
process. However, electron microscopical evidence would be
required to demonstrate that the basal process is indeed attached
to the basal lamina at the pial surface prior to translocation.
Locomoting cells, by contrast, have a free motile leading
process that maintains a relatively constant length as the cell
migrates forward. These cells show a characteristic saltator y
pattern of migration — short bursts of forward movements
interspersed with stationary phases, resulting in slower average
speeds of 35 µm/h. In addition, locomoting cells appear to
undergo a short-range translocation in their terminal phase of
movement once their leading process reaches the MZ.
Another line of evidence that lends support for two distinct
modes of radial migration comes from the analysis of mutant
mice that show aberrant cortical layer formation. An anomaly
consistently noted in reeler, mDab1, α3β1 integrin and VLDL
mutant mice is the failure of the PP to be split by migrating CP
neurons, resulting in the accumulation of CP cells beneath the
PP with ill-defined layers [reviewed by Nadarajah and Parnavelas
(Nadarajah and Parnavelas, 2002)]. In mice lacking Cdk5 or its
activator, p35, the PP and the early CP form normally, but
later-generated CP neurons collect below the SP in abnormal
positions (Chae et al., 1997; Gilmore et al., 1998). The normal
formation of PP in all these mutants further indicates that
neurons constituting the PP may use somal translocation, a mode
of migration that is independent of glial guidance and the reelin
signaling pathway. Moreover, PP and early CP neurons that are
generated at the onset of corticogenesis are phylogenetically
older, whereas later generated cells are a more recent evolutionary addition (Marin-Padilla, 1978; Goffinet, 1983). Thus, it is
conceivable that translocation is an older mode of movement in
the evolution of the cerebral cortex for the transfer of PP and
early CP neurons. On the other hand, glial-guided migration that
is dependent on the reelin signaling pathway may have evolved
to guide cells across more complex paths during late stages of
cortical development, thus preser ving the ‘inside-out’ pattern of
corticogenesis.
Tangential and Ventricle-directed Migration in the Developing
Cortex
Contrary to earlier studies that pointed only to radial migration
as the mode of movement adopted by young cortical neurons,
608 Neuronal Migration in the Developing Cortex • Nadarajah et al.
subsequent in vitro experiments, lineage analyses, and mouse
chimeras have provided evidence for distinctly non-radial routes
taken by cortical interneurons from their sites of origin in the GE
(Walsh and Cepko, 1993; Anderson et al., 1997; Mione et al.,
1997; Tan et al., 1998; Lavdas et al., 1999; Sussel et al., 1999).
Tracer-labeling studies have illustrated that the migration of
these neurons occurs during the entire period of corticogenesis
and along multiple tangential routes to their destinations in the
developing cortex (Lavdas et al., 1999; Nadarajah et al., 2002).
Further, while a large contingent of interneurons traverse
through the intermediate zone (IZ), subsets of neurons migrate
through the MZ and at the interface of subventricular zone (SVZ)
and IZ. The need for subpallial neurons to adopt multiple routes
to reach the cortex or the underlying molecular mechanisms that
necessitates such distinct trajectories remains unclear. Interestingly, sections from fixed embryonic brains stained for GABA
or calbindin, markers of interneurons, have shown that while a
large contingent of interneurons are horizontally oriented in
the IZ, the number of labeled cells in the developing CP is
considerably smaller (B. Nadarajah and P. Alifragis, unpublished
observations). Thus, it is plausible that interneurons may need
specifications that would enable them to integrate into the CP. In
this regard, we have recently demonstrated that subsets of
interneurons actively migrate in the direction of the cortical VZ
upon reaching the dorsal telencephalon, a mode of movement
that has been termed ‘ventricle-directed migration’ (Nadarajah et
al., 2002). Interestingly, real-time imaging has demonstrated that
neurons that migrate into the cortical VZ, after pausing for an
extended period of time, resume their migration radially in the
direction of the pial surface to take up their positions in the CP.
The prevalence of interneurons with ventricle-directed morphological features at all stages of corticogenesis has led to the
notion that these cells actively seek the cortical VZ to receive
information, possibly pertaining to their layer position.
Branching Cells in the Developing Cerebral Cortex
A lthough our time-lapse studies have shown that radial and
tangential pathways are the two predominant migratory routes
adopted by cortical neurons (reviewed above), we have also
observed a subset of cells that show distinctive morphological
features and migratory behavior and have referred to them as
‘branching cells’. Using Oregon Green BAPTA-1 to label acute
mouse brain slices as previously described (Nadarajah et al.,
2001, 2002), we have shown that a subset of cells that appear
bipolar at the onset of migration from the VZ, soon show
multipolar form upon reaching the IZ (Figs 1 and 2). Examination of slices taken from mice at E13–E16 (n = 65 slices,
40 embr yos, 320 moving cells) showed that, although the
earliest age at which the branching cells were obser ved was
E14, their characteristic movement was more prevalent in the
later stages of corticogenesis (20% of all moving cells at E15).
Branching cells are morphologically distinct from other types of
migrating neurons, as they are highly motile in the formation
and retraction of their processes. Figure 1 (see supplementary
movie) illustrates the typical migratory behavior of one such cell
that had an unbranched leading process at the onset of
migration, but gave rise to branches with time as it reached the
IZ. In such cases, the soma moved rapidly (1–3 µm/min) up to
the branch point, paused for an extended period to retract one
of the processes before resuming movement in the direction of
the remaining branch which, in turn, continued to branch. The
highly dynamic process of formation and retraction of branches
suggests that these cells are actively exploring their environment
for directional cues. Further, it appears that upon reaching the
Figure 1. Time-lapse images of a branching cell labeled with Oregon Green BAPTA-1. Images were acquired every minute and each frame shows a single optical section. As the tip
of the leading process reaches the IZ, it splits into two distinct branches (arrow). The soma then moves rapidly towards the branching point and pauses for an extended period of time.
In the meantime, one of the two branches retracts, whereas the other splits further (arrowhead). As soon as the retraction is completed, the cell moves again rapidly towards the
second branch point (arrowhead) where it pauses further. Scale bar: 50 µm.
Figure 2. Schematic illustration of a branching cell displaying the typical migratory
behavior.
IZ, these cells either migrate tangentially or continue their radial
ascent with branched leading processes (Fig. 2).
The transient appearance of a thin trailing process (Fig. 1,
t = 20 min) during the movement of branching cells suggests a
process of nucleokinesis that is reminiscent to somal translocation. However, the main feature of neurons that adopt somal
translocation is that the leading process is anchored onto the pial
surface suggesting that their direction of movement is predetermined (Fig. 3a,a′; arrows), in contrast to the exploratory
behavior of branching cells. The appearance of free motile
processes during migration is a feature of branching cells that is
comparable to neurons undergoing glial-guided locomotion.
However, a characteristic feature that distinguishes branching
cells from the locomoting population, is that the latter have
radially oriented leading process that remain unbranched during
the course of migration while maintaining a relatively a constant
length (Fig. 3c,c′; arrows). To investigate whether branching
cells are present in the developing cortex, sections of fixed
embryonic mouse brains (E13–E16) were stained with a panel of
antibodies that label early neuronal populations. These experiments showed that although the IZ contained a subset of neurons
Figure 3. Time-lapse images of cells in the developing cortex illustrating the three distinct modes of migration: somal translocation (a, a′), branching cell movement (b, b′),
and glia-guided locomotion (c, c′). Scale bar: 25 µm.
with many branches that were positive for calbindin, the VZ
was seemingly devoid of such cells (Fig. 4). It is possible that
these cells, whilst being postmitotic, begin to express their
Cerebral Cortex Jun 2003, V 13 N 6 609
Figure 5. Schematic illustration of the various modes of neuronal migration in the
developing cerebral cortex. During early development the prevalent mode of radial
migration is somal translocation (a). As development proceeds and the cortex thickens,
the predominant mode of migration is glia-guided locomotion (b). Cortical interneurons
that arise in the ganglionic eminence follow tangential migratory paths to reach the
cortex (c) and seek the ventricular zone (ventricle-directed migration) (d). A subset of
neurons that switch from radial to tangential modes of movement show active
branching (branching cells) (e).
Figure 4. Neurons with multiple branches (arrows) in fixed brain sections taken
from an E15 mouse are immunopositive for calbindin, a marker of interneurons, in
the early stages of development. VZ, ventricular zone; IZ, intermediate zone. Scale
bar: 50 µm.
phenotype-specific markers only upon exiting the VZ. In
accordance with our observations, previous time-lapse studies
have illustrated that labeled cells that ascend radially from the
cortical VZ display a multipolar phenotype and adopt tangential
migration upon entering the IZ (O’Rourke et al., 1992).
Despite the demonstration that a subset of migrating cortical
neurons are branching cells, their identity or their site of origin
remains unclear. In this regard, earlier electron microscopical
and real-time imaging studies have clearly demonstrated that
pyramidal neurons have unbranched leading processes and
migrate radially from the cortical VZ to the CP (O’Rourke et al.,
1992). Based on these obser vations, it is unlikely that the
branching cells are pyramidal neurons, although one cannot
exclude the possibility of a subset of pyramidal cells that are yet
to be characterized. Alternatively, it is possible that these cells
are a subset of interneurons that are generated in the cortical VZ.
In support of this notion, earlier lineage studies and mouse
chimeras with X-inactivated mosaics (J.G. Parnavelas, unpublished observations) (Tan et al., 1995, 1998) have demonstrated
that neurons that constitute the radial clones in the developing
cortex are glutamatergic, while their related siblings that show
an orthogonal disposition are GABAergic interneurons. Further,
the finding that in mutant mice that lack Dlx1/2 a quarter of the
GABAergic population in the cortex still persists (Anderson et
al., 1997) suggests that a subset of interneurons are likely to be
generated in the cortical VZ. Collectively, these obser vations
together with recent experiments in ferrets (Anderson et al.,
2002) indicate that while the majority of cortical interneurons
are generated in the subpallium, a subset arises in the pallial
proliferative zone. In this context, Rakic and colleagues (Letinic
et al., 2002) have recently demonstrated that 65% of cortical
GABAergic neurons in humans are generated in the proliferative
zones of dorsal forebrain.
In summary, our recent studies have highlighted the various
610 Neuronal Migration in the Developing Cortex • Nadarajah et al.
migrator y modes adopted by developing cortical neurons
(Fig. 5). Although our observations suggest that different modes
of migration are likely to be adopted by distinct populations of
cortical neurons, due to the expansion of the cerebral volume
later born neurons would need to utilize more than one mode of
migration to reach their destinations. Thus, it is likely that later
generated cortical neurons contain the necessary molecular and
cellular machinery to enable them to switch from one mode of
migration to another in a temporal fashion.
Notes
We acknowledge the contributions of Drs Jan Brunstrom, Jamie
Grutzendler and Alan Pearlman, and the support of the Wellcome trust.
Address correspondence to Bagirathy Nadarajah, School of Biological
Sciences, University of Manchester, Manchester M13 9PT, UK. Email:
[email protected].
Supplementary Material
Supplementary material can be found at: http://w w w.cercor.oupjournals.
org
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