1 Department of Psychiatry, Nina Ireland Laboratory of Developmental
Neurobiology, Langley Porter Psychiatric Institute, 401 Parnassus Avenue,
University of California San Francisco, CA 94143, USA
2 Department of Biological Sciences, Howard Hughes Medical Institute, 371 Serra
Mall, Stanford University, Stanford, CA 94305, USA
* Present address: Instituto de Neurociencias, CSIC-Universidad Miguel
Hernández, 03550 San Juan, Alicante, Spain
Present address: Merck & Co., 126 East Lincoln Avenue, Rahway, NJ 07065,
USA
Present address: Instituto de Neurobiología Ramón y Cajal, Avda.
Doctor Arce, 37, 28002 Madrid, Spain
Author for correspondence (e-mail:
jlrr{at}cgl.ucsf.edu)
Accepted 30 January 2003
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SUMMARY |
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Key words: Cell migration, Interneuron, Cholinergic neurons, Basal magnocellular complex, Telencephalon, Cortex, Basal telencephalon, medial ganglionic eminence, Mouse, Slit1, Slit2, netrin 1
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INTRODUCTION |
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Neurons with disparate migratory behaviors may arise from common progenitor
zones within the neural tube, suggesting that distinct cell populations have
intrinsic mechanisms to distinguish the molecular cues that are relevant to
their directional guidance. An extreme example of this circumstance is found
in the subpallial telencephalon, where the lateral and medial ganglionic
eminences (LGE and MGE, respectively) give rise to multiple neuronal
populations with disparate migratory patterns. During mid-embryonic stages,
for example, the LGE gives rise primarily to cells that migrate radially to
differentiate as -aminobutyric-containing (GABAergic) projection
neurons in the striatum, whereas the basal telencephalon (MGE and adjacent
regions of the telencephalon stalk) are the source of cells that migrate
tangentially towards the striatum, neocortex and hippocampus, where they
differentiate as GABAergic interneurons
(Anderson et al., 1997
;
Anderson et al., 2001
;
Lavdas et al., 1999
;
Pleasure et al., 2000
;
Sussel et al., 1999
;
van der Kooy and Fishell,
1987
; Wichterle et al.,
1999
; Wichterle et al.,
2001
). In addition, the LGE appears to simultaneously contribute
to cells that migrate rostrally into the developing olfactory bulb, whereas
the MGE also gives rise to neurons that remain within the basal telencephalon
(Marín et al., 2000
;
Wichterle et al., 2001
). It is
clear, therefore, that a precise control of the migratory behavior of each one
of these neuronal populations must exist to guarantee the correct wiring of
the telencephalon.
The mechanisms that control the migration of the different cell populations
derived from the subpallium are poorly understood. Repulsion from the
ventricular zone of the subpallium has been suggested to play a role both in
the tangential migration of interneurons to the cortex and olfactory bulb
(Hu, 1999;
Wu et al., 1999
;
Zhu et al., 1999
), as well as
in the radial migration of projection neurons into the developing striatum
(Hamasaki et al., 2001
). It
seems unlikely, however, that the same mechanism of repulsion from the
ventricular zone can account alone for such extremely divergent migratory
patterns in vivo, and it is therefore expected that additional mechanisms
exist to delineate each of the different routes of migration. In line with
this expectation, a repulsive activity for migrating cortical interneurons in
the developing striatum has been shown to contribute to the channeling of
migrating cells into specific paths on their way towards the cortex
(Marín et al.,
2001
).
As for the mechanisms involved, the molecular nature of the cues that
direct neuronal migrations in the subpallium is largely unknown. Slit1, a
diffusible guidance protein, has been shown to repel GABAergic cells derived
from the LGE in vitro, and it has been suggested that this repulsion provides
the directional guidance for neurons migrating from the LGE to the cortex
(Zhu et al., 1999). Another
diffusible guidance protein, netrin 1, has similarly been implicated in the
repulsion of cells from the ventricular zone of the LGE towards the developing
striatum (Hamasaki et al.,
2001
). Hepatocyte growth factor (HGF) has been shown to act as a
motogen (i.e. a factor that stimulates migration) for cells tangentially
migrating to the cortex (Powell et al.,
2001
), although it is not known whether the same factor provides
any directional guidance to this migration. Antibody-blocking experiments
suggest that interaction between migrating interneurons and cell adhesion
molecules (including TAG1) may contribute to regulating the migrations
(Denaxa et al., 2001
).
Finally, repulsion from the developing striatum by class 3 (secreted)
semaphorins helps sort subsets of tangentially migrating interneurons towards
the cortex (Marín et al.,
2001
). However, these insights fail to provide a cohesive
understanding about the mechanisms that set the direction of the migration
from the subpallium towards the cortex.
These studies have provided several important candidates for molecules directing subpallial migrations and, in the case of class 3 semaphorin and cell adhesion molecule involvement, direct tests of the functions of these molecules have been obtained. Nonetheless, the full complement of cues directing these migrations is still undefined, and the specific involvement of Slit and netrin proteins, which has been suggested to be key to directing these cells, has not been tested directly. The goals of the present study were therefore to define the developmental mechanisms that direct the migration of interneurons from the basal telencephalon to the cortex, and to test the roles of the Slit and Netrin proteins in this process. Through the development of new slice culture assays that test the behavior of tangentially migrating cells, we show that the basal telencephalon contains a repulsive activity for these cells whereas the cerebral cortex contains an activity that attracts them. Analysis of mice carrying loss-of-function alleles for Slit1, Slit2 and netrin 1 (Ntn1) demonstrate that, contrary to expectation, these proteins are not necessary parts of the repulsive activity found in the basal telencephalon and, in addition, to not appear to play a significant role in controlling tangential migration of interneurons to the cerebral cortex. However, Slit proteins are important regulators of neuronal positioning within the basal telencephalon, controlling cell migration across the midline and establishing the bilateral location of specific cell groups, such as the cholinergic basal magnocellular complex.
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MATERIALS AND METHODS |
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Slice culture experiments
Organotypic slice cultures of embryonic mouse telencephalon were prepared
as previously described (Anderson et al.,
1997). Briefly, embryos (E12.5-16.5) were removed by Caesarean
section and decapitated. Brains were removed, embedded in 4% low-melt point
agarose, and 250 µm thick coronal sections were cut on a vibratome. The
sections were then transferred to polycarbonate culture membranes (13 mm
diameter, 8 µm pore size; Corning Costar, Cambridge, MA) in organ tissue
dishes containing 1 ml of medium with serum (Gibco MEM with glutamine, 10%
fetal calf serum, penicillin, and streptomycin). They were subsequently
incubated for 1 hour in a sterile incubator (37°C, 5% CO2),
after which the medium was changed to Neurobasal/B-27 (Gibco BRL, Life
Technologies Inc, Gaithersburg, MD). Transplantation was performed immediately
after this step, as described before
(Marín et al., 2001
).
In other cases, DiI placements were used to study tangential migration, as
described elsewhere (Anderson et al.,
1997
). DiI crystals (C-16 DiI; Molecular Probes) were placed into
the tissue with an insect pin and slices were returned to the incubator for
the appropriate time, then fixed with 4% PFA and mounted on slides.
Matrigel explants
For co-culture experiments, E13.5 brains were embedded in 4% low melting
point agarose in PBS and vibratome sections were obtained as described above.
Small pieces of the cortex and MGE were dissected from approximately the same
rostrocaudal level of the telencephalon and incubated for 1 hour in 1 ml of
medium with serum. To set up the co-cultures, 25 µl of Matrigel®
solution (BD Biosciences, NJ) was pipetted onto the bottom of four-well dishes
(Nunc, Roskilde, Denmark) and allowed to gel for about 45 minute. Explants
were then placed onto this base and 25 µl of collagen were added on top.
Collagen co-cultures consisted of a piece of neocortex and a piece of MGE
separated by approximately 400 µm. After a period of 45 minutes, to allow
the matrigel to gel, Neurobasal/B-27 medium was added. Explants were cultured
for 36 hours in a sterile incubator (37°C, 5% CO2). Cell
migration from MGE explants was semiquantified as described before
(Zhu et al., 1999).
In situ hybridization
35S-riboprobes were used for in situ hybridization as described
previously (Marín et al.,
2000). Probes used for GAD67, Lhx6, Slit1, Slit2, Ntn1
and Isl1 have been previously described
(Brose et al., 1999
;
Grigoriou et al., 1998
;
Pfaff et al., 1996
;
Serafini et al., 1994
).
Immunohistochemistry
Embryos were obtained by Caesarean section, anesthetized by cooling,
perfused with 4% PFA in PBS and postfixed in PFA for 2-8 hours. Following
postfixation, brains were cryoprotected in 30% sucrose and cut in a freezing
sliding microtome at 40 µm. Free-floating sections were preincubated in 1%
bovine serum albumin (BSA) and 0.3% Triton X-100 in phosphate-buffered saline
(PBS) for 1 hour at room temperature, and subsequently incubated with the
primary antisera for 24-36 hours at 4°C in 0.5% BSA and 0.3% Triton X-100
in PBS. The following antibodies were used: rabbit anti-calbindin (Swant,
Bellinzona, Switzerland; diluted 1:5000), rabbit anti-calretinin (Chemicon;
diluted 1:5000), rabbit anti-NPY (Incstar; diluted 1:3000), and rabbit
anti-GABA (Sigma; diluted 1:2000). Sections were then incubated in
biotinylated secondary antibodies (Vector; diluted 1:200) and processed by the
ABC histochemical method (Vector). The sections were then mounted onto
Superfrost Plus slides (Fisher), dried, dehydrated, and coverslipped with
Permount (Fisher).
Cell counting
Cells were counted on images obtained from 40-60 µm sections acquired in
a Spot2 cooling CCD camera attached to a conventional microscope. For cell
counting in the cerebral cortex, a standardized box (40,000 µm2)
was used to delineate the appropriate areas. Three sections at different
rostrocaudal levels in the telencephalon were used in each case, whereas two
different levels were employed for cell counting in the hippocampus, striatum
and basal magnocellular complex. For slice migration experiments,
quantification of the different cell populations was expressed as a percentage
of the total number of cells per slide. For the inverted cortex experiments
illustrated in Fig. 3, cells
were counted in two standardized areas (22,500 µm2) of the
medial and lateral regions of the cortex at the same distance (600-850 µm,
depending on the case) from the center of the MGE graft. One-way ANOVA was
used to estimate significant differences among cell populations in all
experiments.
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RESULTS |
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The basal telencephalon contains a repulsive activity for cells
tangentially migrating to the cortex
In a first series of experiments we sought to determine whether the basal
telencephalon contains a repulsive activity for tangentially migrating cells.
To study the tangential migration of cells, we prepared slice cultures from
E13.5 wild-type mouse embryos, and transplanted into these hosts portions of
the MGE from green fluorescent protein (GFP)-expressing transgenic mice; to
ensure synchrony, GFP and non-GFP embryos were always littermates
(Marín et al., 2001).
We first tested the behavior of tangentially migrating cells in the absence of
the cortex. The entire cortex was removed unilaterally and a piece of the MGE
obtained from GFP-expressing slices (MGEGFP) was transplanted
homotypically and ipsilaterally into the host slice
(Fig. 1A). After 48 hours in
culture, GFP cells had migrated dorsally, accumulating close to the edge of
slice (n=19). As in control experiments, GFP cells never migrated
ventrally (Fig. 1B,C and data
not shown). Thus, information present in the subpallial telencephalon is
sufficient to direct tangential migration of MGE cells towards the cortex.
|
To test whether the repulsive activity found in the most ventral aspect of the subpallium was present in a gradient, we designed an experiment in which MGE cells were forced to migrate towards the ventral midline. The cortex was removed ipsilateral to the side where a piece of MGEGFP was transplanted into the pallial-subpallial boundary (Fig. 1G). After 48 hours in culture, GFP cells had invaded the subpallium, although they did not migrate uniformly within this tissue (Fig. 1H). Thus, most GFP cells remained within the mantle of the LGE, with very few cells migrating into the MGE mantle and virtually none close to the ventral midline of the basal telencephalon (Fig. 1H,I; n=25). The distribution of GFP cells in the slice cultures did not vary significantly when the slices were maintained for up to four days in vitro (n=8; data not shown), suggesting that tangentially migrating cells derived from the MGE respond to a graded repulsive activity with its peak concentration in the most ventral region of the basal telencephalon.
The cerebral cortex contains an attractive activity for cells
tangentially migrating to the cortex
Tangential migration from the basal telencephalon to the cortex could also
require attraction from the cerebral cortex. To test this hypothesis, we
analyzed the effect of an ectopic cortex on the tangential migration of cells
derived from the MGE. In these experiments, we removed the entire
contralateral subpallium and placed the contralateral cortex close to the
basal telencephalon. To study the behavior of MGE cells, a piece of
MGEGFP was homotypically transplanted into the intact side of the
slice (Fig. 2A; n=22).
After 48 hours in culture, GFP cells migrated normally on the intact side of
the slice, with many cells reaching the ipsilateral cortex
(Fig. 2B). As in previous
experiments (Fig. 1), however,
GFP cells derived from the MGE did not migrate ventrally into the POa. Thus,
this initial experiment failed to reveal an attractive activity in cortex, but
it was not conclusive since attraction could have been obscured by the
repulsive activity in the POa. In a second series of experiments, we therefore
tested the behavior of the tangentially migrating cells derived from the MGE
in a similar paradigm but in the absence of the POa. We removed the entire
contralateral subpallium as well as the ipsilateral POa and placed the
contralateral cortex close to the MGE, which contained a piece of
MGEGFP (Fig. 2C;
n=14). After 48 hours in culture, GFP cells migrated normally on the
intact side of the slice, with many cells reaching the ipsilateral cortex
(Fig. 2D). In addition,
however, many GFP cells also migrated into the ectopic cortex
(Fig. 2D). These experiments
reinforced the notion that the POa contains a repulsive activity for
tangentially migrating cells.
|
We performed additional experiments to further characterize the apparent cortical attractive activity. First, we transplanted MGEGFP into the neocortex of wild-type slices (Fig. 3D; n=16), and studied the behavior of the tangentially migrating cells after 48 hours in culture. MGE cells disperse in both lateral and medial directions, but virtually none of them migrated into the subpallium (Fig. 3E,F). Interestingly, GFP cells tended to migrate preferentially towards the medial cortex rather than to the lateral cortex (13 out of 16 cases). This experiment suggest that the attractive activity present in the cortex may be distributed in a gradient increasing from lateral to medial regions of the cortex. Alternatively, the medial cortex is more permissive than the lateral cortex for the tangentially migrating cells.
If the cortex contains an attractive activity that is expressed in a medial to lateral gradient, then MGE cells would preferentially migrate towards the medial cortex if both regions of the cortex were found at the same distance from the source of migrating cells. To explore this possibility, we inverted the orientation of the neocortex (i.e., excluding the piriform cortex and the hippocampus) in wild-type slices and transplanted MGEGFP homotypically to study the behavior of MGE cells (Fig. 3G; n=12). Quantification of the number of migrating cells found in medial and lateral regions of the inverted cortex located at the same distance from the center of the source of migrating cells (m and 1 boxes in Fig. 3H) revealed that significantly more MGE cells migrated to medial than lateral regions of the cortex [Fig. 3H,I; n=12, cells per 22,500 µm2; medial (m) 237.25 (±67.35 s.d.), lateral (1) 149.92 (±69.46), P=0.0049]. The fact that more cells preferentially migrate towards the medial cortex in this experiment suggests that the cortex not only constitutes a highly permissive substratum for migration but also that it contains a chemoattractive activity that influences the behavior of tangentially migrating cells.
To provide direct evidence for the existence of a diffusible cortical chemoattractant for cells migrating from the MGE, we co-cultured small explants of MGE and neocortex on a permissive substratum and analyzed the distribution of cells that migrated out of the MGE explants after 36 hours in culture (Fig. 4A). In matrigel matrix, cells migrating out the MGE are preferentially oriented toward the cortical explant in co-culture experiments (n=38 explants; Fig. 4B,C). In contrast, migration of cells was similar from all sides of the explant when isolated pieces of the MGE were cultured (data not shown). Thus, the developing cortex releases a diffusible attractive activity that influences the migration of MGE cells.
|
To directly address the role of Slit proteins in the guidance of cells
tangentially migrating from the subpallium to the cortex, we studied mice
carrying loss-of-function alleles for both Slit1 and Slit2
(Plump et al., 2002). We first
examined the distribution of tangentially migrating interneurons in the
embryonic cortex, as revealed by the expression of GAD67, Lhx6 and
Dlx2, three genes that identify embryonic GABA interneurons
(Anderson et al., 1997
;
Lavdas et al., 1999
).
Comparison of the expression of these markers at different rostrocaudal levels
within the cortex of wild-type and Slit1;Slit2 double mutants, and at
different embryonic stages (E12.5 and E14.5) showed no obvious differences in
the number or laminar distribution of GAD67, Lhx6 and Dlx2
expressing cells within the cortex (n=3;
Fig. 5A-D and data not shown),
suggesting that Slit1 and Slit2 are not necessary for the
migration of interneurons to the embryonic cortex. In agreement with these
experiments, analysis of tangential migration in slice cultures using DiI
crystals to label cells derived from the basal telencephalon revealed no
significant differences between slices obtained from wild-type or
Slit1;Slit2 double mutants (n=17 for each case; data not
shown).
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Simultaneous loss of Slit1, Slit2 and Ntn1 does not prevent
interneuron migration to the cortex
The previous experiments suggest that molecules other than Slit1 and Slit2
account for the repulsive activity present in the subpallium. Recently, it has
been shown that, in vitro, Ntn1 can repel GABAergic cells derived from the
ganglionic eminences (Hamasaki et al.,
2001). Ntn1 is abundantly expressed in all regions of the
subpallium, including the POa (Tuttle et
al., 1999
), suggesting that it may have a synergistic effect with
Slit proteins in controlling cell migration in the basal telencephalon. To
test this hypothesis, we first generated Slit1;Ntn1 double mutants.
In contrast to Slit1 mutants, which survive into adulthood and have
roughly normal telencephalic development
(Bagri et al., 2002
;
Plump et al., 2002
),
Slit1;Ntn1 double mutants died at birth. Analysis of cortical
interneuron markers at E18.5, however, failed to reveal a difference in the
number of cortical interneurons between wild type, Slit1 and
Slit1;Ntn1 mutants. For example, despite the smaller size of the
cortex in Slit1;Ntn1 double mutants, the density of calbindin
immunoreactive cells in the cortex was similar in the two genotypes
[Fig. 8A-F; n=3. Cells
per 40,000 µm2; control 91.45 (±2.27s.d.),
Slit1/2-/- 97.78 (±7.08), P=0.214].
Similar results were obtained when the distribution of GABA-, GAD67-,
Lhx6- or Dlx2-expressing cells was analyzed (n=3; data
not shown). In addition, calbindin immunohistochemistry also showed that there
was not a severe defect in the generation of striatal neurons in
Slit1;Ntn1 mutants.
|
Slit proteins control neuronal migration close to the midline in the
basal telencephalon
Our previous analysis on the role of Slit1 and Slit2 in
the guidance of forebrain axons in vivo suggested that, despite their broad
expression within the telencephalon, Slit proteins appear to exert their
primary function close to the midline
(Bagri et al., 2002). To
determine whether Slit proteins play a role in the guidance of any neuronal
populations within the basal telencephalon, we analyzed the distribution of
specific neuronal populations that are normally located close to the ventral
midline of the telencephalon in mice deficient in Slit2 or in both
Slit1 and Slit2. One of these neuronal populations is the
basal magnocellular complex, which contains large cholinergic neurons that
distribute in bilateral groups in the preoptic area
(Fig. 9A,G,H). In
Slit2 and Slit1;Slit2 mutants there is a periventricular
ectopic collection of cholinergic neurons, with some of their processes
crossing the midline (Fig. 9B,E
and data not shown). Normally, cholinergic neurons and their processes are not
present in these locations (Fig.
9A,C). These defects are more prominent in Slit1;Slit2
double mutants than in Slit2 mutant mice, suggesting that
Slit1 and Slit2 have partially redundant contributions in
controlling cell positioning close to the ventral midline. The collection of
ectopic cholinergic neurons appear to be misplaced from the basal
magnocellular complex, since the number of neurons remaining within this
nucleus was reduced, in particular at caudal levels
[Fig. 9G,J; n=3. Cells
per 40,000 µm2; control 170.17 (±17.46),
Slit1/2-/- 69.5 (±5.63), P<0.001]. In
contrast, the number of cholinergic neurons within the striatum was similar in
control and Slit1;Slit2 mutant mice
[Fig. 9D,F; n=3. Cells
per 40,000 µm2; control 103 (±6.56),
Slit1/2-/- 96 (±16.52), P=0.532].
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DISCUSSION |
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Little is know about the nature of the cues that provide directionality to
the migration of interneurons from the basal telencephalon to the cortex. In
vitro experiments have demonstrated that Slit proteins repel GABA neurons
derived from the subpallium, leading to the suggestion that these molecules
guide interneurons from the subpallium into the cortex
(Zhu et al., 1995). In an
attempt to clarify the source and nature of the cues that control this
process, we have used slice culture experiments as well as analysis of mice
carrying loss-of-function alleles for Slit1, Slit2 and Ntn1
to study interneuron migration to the cortex. Three main conclusions can be
drawn from our experiments: (1) both attractive and repulsive activities
direct interneuron migration to the cortex; (2) Slit1, Slit2 and Ntn1 are not
required in vivo for interneuron migration; and (3) Slit proteins control
neuronal positioning near the midline in the basal telencephalon.
Coordinate attractive and repulsive activities control interneuron
migration to the cerebral cortex
Our experiments indicate that both attractive and repulsive cues exert
considerable influence on the guidance of tangentially migrating cells from
the subpallial telencephalon to the cortex. In slice culture experiments, we
have shown that the most ventral region of the telencephalon is repulsive for
tangentially migrating cells (Fig.
1), whereas the developing cortex is attractive for cells directed
towards this region (Figs 2,
3). Early migration in the
subpallium may be more dependent upon repulsion from the basal telencephalon,
whereas extension through the pallium may rely more directly on cues present
in the cerebral cortex. In line with this hypothesis, initial migration
towards the cortex is largely independent of the presence of the cortex
itself, since cells migrate dorsally in the absence of its target and they
reach the pallial/subpallial boundary (the dorsal border between the striatum
and the cortex) roughly at the same time in the presence or absence of the
cortex (Fig. 1).
It has been previously suggested that migration of interneurons from the
LGE to the cortex is mediated by a repulsive activity present in the
ventricular zone of the subpallium (Zhu et
al., 1999). Zhu et al. hypothesized that a gradient of repulsive
activity, with the strongest repulsion at the medial side of the striatal
primordium, drives GABAergic interneurons to migrate laterally into the
neocortex. However, since most cortical interneurons appear to derive from
subpallial regions ventral to the LGE, such as the MGE
(Anderson et al., 2001
;
Lavdas et al., 1999
;
Sussel et al., 1999
;
Wichterle et al., 1999
;
Wichterle et al., 2001
), with
the LGE giving rise primarily to neurons that remain in the striatum
(Hamasaki et al., 2001
;
Wichterle et al., 2001
), it
seems more reasonable that the repulsive activity for cells tangentially
migrating to the cortex should be located ventral to the MGE. In agreement
with this notion, our experiments suggest that tangentially migrating cells
derived from the MGE are repelled by an activity present in the mantle of the
most ventral region of the basal telencephalon (Figs
1,
2,
6). This activity appears to
inhibit the motility of tangentially migrating cells, directing their
migration towards the cortex. However, repulsion from the ventricular zone
might be necessary to facilitate radial migration of cells away from the
progenitor zones of the subpallium towards the developing basal ganglia, as
suggested for the striatum (Hamasaki et
al., 2001
).
Our experiments also suggest that the cortex influences tangential
migration from the basal telencephalon (Figs
2,3,4).
Since migrating cells are able to reach the pallial/subpallial boundary in the
absence of the cortex (Fig.
7B), the activity present in the cortex may primarily function to
facilitate the lateral to medial dispersion of tangentially migrating cells. A
good candidate molecule for this attractive activity is HGF, which acts as a
motogen (i.e. a factor that stimulates migration) for tangentially migrating
cells (Powell et al., 2001).
It is still unknown whether HGF can also function as a chemoattractive
molecule for cortical interneurons, but the fact that it can function as a
chemoattractant for developing motor axons
(Ebens et al., 1996
) is at
least consistent with this possibility. Ntn1, a guidance molecule that has
been shown to be attractive for migrating cells in other systems
(Alcántara et al., 2000
;
Bloch-Gallego et al., 1999
;
Yee et al., 1999
), is
expressed at low levels in the developing hippocampus, but is completely
absent from the rest of the cortex
(Livesey and Hunt, 1997
;
Serafini et al., 1994
).
However, both Ntn1 and DCC mutants seem to have normal
numbers of cortical interneurons (Anderson
et al., 1999
). Other molecules that are known to be expressed in
the developing cortex have been reported to be attractive for migrating cells
in a variety of different systems. These factors include chemokines, EGF, FGF
and TGFß-related molecules (Branda and
Stern, 2000
; Caric et al.,
2001
; Lehmann,
2001
; Zou et al.,
1998
), but their potential role in controlling the tangential
migration of interneurons remains to be investigated.
Our slice experiments suggest that the cortical attractive activity is
present in a gradient with the strongest attraction in the medial cortex
(Fig. 3). A gradient of
increased attraction from lateral to medial regions of the cortex would direct
migration towards medial regions of the cortex, suggesting that the cortex
fills up with incoming interneurons from medial to lateral regions. This
hypothesis is consistent with the observation that interneurons in the
hippocampus are generally born earlier than interneurons in the neocortex
(Soriano et al., 1989a;
Soriano et al., 1989b
),
although additional experiments would be required to confirm this observation.
Despite the presence of an attractive activity controlling the direction of
migration within the cortex, an additional mechanism seems necessary to ensure
balanced distribution of interneurons throughout the cortex, e.g. to prevent
all interneurons from accumulating in the hippocampus. The nature of such a
mechanism remains to be elucidated.
Roles of Slit1, Slit2 and Ntn1 in cell migration in the
telencephalon
In this study, we have focused on the initial characterization of cues
responsible for the repulsive activity present in the basal telencephalon.
Candidate molecules for this activity included Slit1 and Slit2, which are
expressed in the subpallium during the time of interneuron migration and in
vitro can repel GABAergic cells derived from the ganglionic eminences
(Zhu et al., 1999). Our
analysis of Slit1;Slit2 double mutants suggests, however, that these
proteins are not necessary for tangential migration from the basal
telencephalon to the cortex (Figs
5,
6). Moreover, despite the fact
that Ntn1 can also repel subpallial GABAergic cells
(Hamasaki et al., 2001
),
analysis of Slit1;Slit2;Ntn1 triple mutants demonstrates that a
cooperative action of these three proteins is not required for the
subpallial-to-pallial interneuron migrations
(Fig. 8). Thus, it is likely
that other factor(s) provide the repulsive information.
How can one reconcile the apparently contradictory results obtained in
vitro [Slit repulsion of GABAergic cells derived from the subpallium
(Zhu et al., 1999)] and in
vivo (lack of migration defects in the absence of Slit1 and Slit2; present
study)? One possibility would be that other molecules cooperate with Slits in
repelling interneurons towards the cortex. Our slice experiments, however,
suggest that the repulsive activity found in the basal telencephalon is not
altered in Slit1;Slit2 mutants, suggesting that the contribution of
Slits to this activity is not significant
(Fig. 7). A second possibility
is that the results obtained in vitro do not reflect the effect of Slit
proteins on cells tangentially migrating to the cortex, but rather on neurons
that normally remain within the basal ganglia. Indeed, our experiments show
that Slit proteins are required for the migration of subsets of subcortical
GABAergic (NPY) and cholinergic neurons. This interpretation would suggest
that the heterogeneity of cell populations present in migration assays should
be taken into account in future in vitro experiments. Finally, it is also
possible that tangentially migrating cells indeed respond to Slits, but they
only normally do so once the cells arrive in the cortex. For example,
Slit1, Slit2 and Slit3 are expressed in very restricted
laminar patterns in the early postnatal cortex
(Marillat et al., 2001
),
suggesting that Slits may play a role in controlling the layer destination of
GABAergic interneurons, a possibility that we have not yet explored.
Previous experiments have suggested a role for Slits and Ntn1 in neuronal
migration in the striatum. Projection neurons in the striatum are largely
derived from the LGE (Wichterle et al.,
2001), with early-born cells destined for the patch compartment
and later-generated cells directed towards the matrix
(van der Kooy and Fishell,
1987
). Interestingly, Slit1 and Ntn1 are
co-expressed in the ventricular zone of the LGE, and in vitro experiments have
shown that both molecules influence the migration of cells derived from the
LGE (Hamasaki et al., 2001
;
Zhu et al., 1999
). In
particular, Slit1 and Ntn1 mimic the repulsive activity present in the
ventricular zone of the LGE, leading to the suggestion that these molecules
may play a crucial role in the outward migration of postmitotic cells away
from the ventricular zone towards the developing striatum
(Hamasaki et al., 2001
).
Nevertheless, our analysis of Slit1;Ntn1 double mutants shows that a
striatum of roughly normal appearance forms in the absence of these cues
(Fig. 8). This does not exclude
the possibility of more subtle defects in the development of the striatum and
other subpallial structures that could be revealed through a more detailed
analysis of Slit1;Ntn1 mutants.
Despite the lack of evidence supporting a role in vivo for Slit1 and Slit2
in the tangential migration of cells from the basal telencephalon to the
cortex, analysis of Slit mutants demonstrates that these proteins
play a significant role in controlling cell positioning in the mammalian
telencephalon. The distribution of specific neuronal populations, such as the
cholinergic basal magnocellular complex, is affected in mice lacking
Slit2, and even more so in mice lacking both Slit1 and
Slit2 (Fig. 9). Thus,
loss of Slit function appears to impair the ability of some neurons to migrate
away from their progenitor zone particularly in the region of the ventral
telencephalic midline. Alternatively, since several axonal pathways are
disrupted in the telencephalon of Slit2 and Slit1/2 mutants
(Bagri et al., 2002), it is
conceivable that the defects in the position of cholinergic and NPY neurons
could be secondary to alterations of the ventral midline caused by the
accumulation of ectopic fibers. This possibility seems less likely, however,
because the cholinergic neurons of the basal forebrain are born at least 2
days before the arrival of cortical and thalamic axons to the basal
telencephalon (Brady et al.,
1989
). Thus, in the telencephalon, Slits appear to be required to
regulate the guidance of neurons at the midline, a function that parallels
previous observations in Drosophila, where Slit was shown to be
required for the migration of muscle precursors away from the midline
(Kidd et al., 1999
); so, the
role of Slit proteins in controlling cell migration appears to be highly
conserved throughout evolution.
A model for the directional guidance of cortical interneurons
As it has been demonstrated for growing axons
(Zou et al., 2000), long-range
neuronal migrations, such as the migration of cells from the subpallium to the
cerebral cortex, appear to be controlled through a number of carefully
choreographed guidance commands
(Marín and Rubenstein,
2003
). In this case, for example, interneurons migrating from the
MGE are first directed dorsally through repulsion by an unknown activity.
Cortical interneurons are then instructed to avoid the developing striatum
through repulsion involving semaphorin/neuropilin interactions
(Marín et al., 2001
).
In addition, interneurons are drawn towards the cortex by a graded attractive
activity that may also facilitate their dispersion through all cortical
territories. Finally, additional cues may be necessary to direct other aspects
of interneuron patterning in the cortex, such as lamina-specific positioning.
Considering the impact that defects in radial migration of cortical projection
neurons have in the etiology of multiple human neurological conditions
(Ross and Walsh, 2001
), it is
reasonable to hypothesize that disruption of the tangential migration of
interneurons may also underlie some human neurological disorders.
Identification of the attractive and repulsive factors that direct this
migration will help test this hypothesis.
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ACKNOWLEDGMENTS |
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REFERENCES |
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---|
Alcántara, S., Ruiz, M., de Castro, F., Soriano, E. and
Sotelo, C. (2000). Netrin 1 acts as an attractive or as a
repulsive cue for distinct migrating neurons during the development of the
cerebellar system. Development
127,1359
-1372.
Anderson, S., Mione, M., Yun, K. and Rubenstein, J. L. R.
(1999). Differential origins of neocortical projection and local
circuit neurons: role of Dlx genes in neocortical interneuronogenesis.
Cereb. Cortex 9,646
-654.
Anderson, S. A., Eisenstat, D. D., Shi, L. and Rubenstein, J. L.
R. (1997). Interneuron migration from basal forebrain to
neocortex: dependence on Dlx genes. Science
278,474
-476.
Anderson, S. A., Marín, O., Horn, C., Jennings, K. and
Rubenstein, J. L. R. (2001). Distinct cortical migrations
from the medial and lateral ganglionic eminences.
Development 128,353
-363.
Bagri, A., Marín, O., Plump, A. S., Mak, Y., Pleasure, S. J., Rubenstein, J. L. R. and Tessier-Lavigne, M. (2002). Slit proteins prevent midline crossing and determine the dorsoventral position of major axonal pathways in the mammalian forebrain. Neuron 33,233 -248.[Medline]
Bloch-Gallego, E., Ezan, F., Tessier-Lavigne, M. and Sotelo,
C. (1999). Floor plate and netrin-1 are involved in the
migration and survival of inferior olivary neurons. J.
Neurosci. 19,4407
-4420.
Brady, D. R., Phelps, P. E. and Vaughn, J. E. (1989). Neurogenesis of basal forebrain cholinergic neurons in rat. Brain Res. Dev. Brain. Res. 47, 81-92.[Medline]
Branda, C. S. and Stern, M. J. (2000). Mechanisms controlling sex myoblast migration in Caenorhabditis elegans hermaphrodites. Dev. Biol. 226,137 -151.[CrossRef][Medline]
Brose, K., Bland, K. S., Wang, K. H., Arnott, D., Henzel, W., Goodman, C. S., Tessier-Lavigne, M. and Kidd, T. (1999). Slit proteins bind Robo receptors and have an evolutionarily conserved role in repulsive axon guidance. Cell 96,795 -806.[Medline]
Caric, D., Raphael, H., Viti, J., Feathers, A., Wancio, D. and
Lillien, L. (2001). EGFRs mediate chemotactic migration in
the developing telencephalon. Development
128,4203
-4216.
Corbin, J. G., Nery, S. and Fishell, G. (2001). Telencephalic cells take a tangent: non-radial migration in the mammalian forebrain. Nat. Neurosci. 4,1177 -1182.[CrossRef][Medline]
Denaxa, M., Chan, C. H., Schachner, M., Parnavelas, J. G. and Karagogeos, D. (2001). The adhesion molecule TAG-1 mediates the migration of cortical interneurons from the ganglionic eminence along the corticofugal fiber system. Development 128,4635 -4644.[Medline]
Ebens, A., Brose, K., Leonardo, E. D., Hanson, M. G., Jr, Bladt, F., Birchmeier, C., Barres, B. A. and Tessier-Lavigne, M. (1996). Hepatocyte growth factor/scatter factor is an axonal chemoattractant and a neurotrophic factor for spinal motor neurons. Neuron 17,1157 -1172.[Medline]
Grigoriou, M., Tucker, A. S., Sharpe, P. T. and Pachnis, V.
(1998). Expression and regulation of Lhx6 and Lhx7, a novel
subfamily of LIM homeodomain encoding genes, suggests a role in mammalian head
development. Development
125,2063
-2074.
Hamasaki, T., Goto, S., Nishikawa, S. and Ushio, Y. (2001). A role of netrin-1 in the formation of the subcortical structure striatum: repulsive action on the migration of late-born striatal neurons. J. Neurosci. 21,4271 -4280.
Hu, H. (1999). Chemorepulsion of neuronal migration by Slit2 in the developing mammalian forebrain. Neuron 23,703 -711.[CrossRef][Medline]
Kidd, T., Bland, K. S. and Goodman, C. S. (1999). Slit is the midline repellent for the robo receptor in Drosophila. Cell 96,785 -794.[Medline]
Lavdas, A. A., Grigoriou, M., Pachnis, V. and Parnavelas, J.
G. (1999). The medial ganglionic eminence gives rise to a
population of early neurons in the developing cerebral cortex. J.
Neurosci. 19,7881
-7888.
Lehmann, R. (2001). Cell migration in invertebrates: clues from border and distal tip cells. Curr. Opin. Genet. Dev. 11,457 -463.[CrossRef][Medline]
Livesey, F. J. and Hunt, S. P. (1997). Netrin and netrin receptor expression in the embryonic mammalian nervous system suggests roles in retinal, striatal, nigral, and cerebellar development. Mol. Cell. Neurosci. 8,417 -429.[CrossRef][Medline]
Marillat, V., Cases, O., Nguyen Ba-Charvet, K. T., Tessier-Lavigne, M., Sotelo, C. and Chédotal, A. (2001). Spatio-temporal expression patterns of slit and robo genes in the rat brain. J. Comp. Neurol. 442,130 -155.[CrossRef]
Marín, O., Anderson, S. A. and Rubenstein, J. L. R.
(2000). Origin and molecular specification of striatal
interneurons. J. Neurosci.
20,6063
-6076.
Marín, O. and Rubenstein, J. L. R. (2001). A long, remarkable journey: tangential migration in the telencephalon. Nature Rev. Neurosci. 2, 780-790.[CrossRef][Medline]
Marín, O. and Rubenstein, J. L. R. (2003). Cell migration in the forebrain. Annu. Rev. Neurosci. (in press).
Marín, O., Yaron, A., Bagri, A., Tessier-Lavigne, M. and
Rubenstein, J. L. (2001). Sorting of striatal and cortical
interneurons regulated by semaphorin/neuropilin interactions.
Science 293,872
-875.
McBain, C. J. and Fisahn, A. (2001). Interneurons unbound. Nat. Rev. Neurosci. 2, 11-23.[CrossRef][Medline]
Pfaff, S. L., Mendelsohn, M., Stewart, C. L., Edlund, T. and Jessell, T. M. (1996). Requirement for LIM homeobox gene Isl1 in motor neuron generation reveals a motor neuron-dependent step in interneuron differentiation. Cell 84,309 -320.[Medline]
Pleasure, S. J., Anderson, S., Hevner, R., Bagri, A., Marín, O., Lowenstein, D. H. and Rubenstein, J. L. (2000). Cell migration from the ganglionic eminences is required for the development of hippocampal GABAergic interneurons. Neuron 28,727 -740.[Medline]
Plump, A. S., Erskine, L., Sabatier, C., Brose, K., Epstein, C. J., Goodman, C. S., Mason, C. A. and Tessier-Lavigne, M. (2002). Slit1 and Slit2 cooperate to prevent premature midline crossing of retinal axons in the mouse visual system. Neuron 33,219 -232.[Medline]
Polleux, F., Whitford, K. L., Dijkhuizen, P. A., Vitalis, T. and
Ghosh, A. (2002). Control of cortical interneuron migration
by neurotrophins and PI3-kinase signaling. Development
129,3147
-3160.
Powell, E. M., Mars, W. M. and Levitt, P. (2001). Hepatocyte growth factor/scatter factor is a motogen for interneurons migrating from the ventral to dorsal telencephalon. Neuron 30,79 -89.[Medline]
Ross, M. E. and Walsh, C. A. (2001). Human brain malformations and their lessons for neuronal migration. Annu. Rev. Neurosci. 24,1041 -1070.[CrossRef][Medline]
Serafini, T., Colamarino, S. A., Leonardo, E. D., Wang, H., Beddington, R., Skarnes, W. C. and Tessier-Lavigne, M. (1996). Netrin-1 is required for commissural axon guidance in the developing vertebrate nervous system. Cell 87,1001 -1014.[Medline]
Serafini, T., Kennedy, T. E., Galko, M. J., Mirzayan, C., Jessell, T. M. and Tessier-Lavigne, M. (1994). The netrins define a family of axon outgrowth-promoting proteins homologous to C. elegans UNC-6. Cell 78,409 -424.[Medline]
Soriano, E., Cobas, A. and Fairen, A. (1989a). Neurogenesis of glutamic acid decarboxylase immunoreactive cells in the hippocampus of the mouse. I: Regio superior and regio inferior. J. Comp. Neurol. 281,586 -602.[Medline]
Soriano, E., Cobas, A. and Fairen, A. (1989b). Neurogenesis of glutamic acid decarboxylase immunoreactive cells in the hippocampus of the mouse. II: Area dentata. J. Comp. Neurol. 281,603 -611.[Medline]
Sussel, L., Marín, O., Kimura, S. and Rubenstein, J.
L. (1999). Loss of Nkx2.1 homeobox gene function results in a
ventral to dorsal molecular respecification within the basal telencephalon:
evidence for a transformation of the pallidum into the striatum.
Development 126,3359
-3370.
Tessier-Lavigne, M. and Goodman, C. S. (1996).
The molecular biology of axon guidance. Science
274,1123
-1133.
Tuttle, R., Nakagawa, Y., Johnson, J. E. and O'Leary, D. D.
(1999). Defects in thalamocortical axon pathfinding correlate
with altered cell domains in Mash-1-deficient mice.
Development 126,1903
-1916.
van der Kooy, D. and Fishell, G. (1987). Neuronal birthdate underlies the development of striatal compartments. Brain Res. 401,155 -161.[CrossRef][Medline]
Wichterle, H., Garcia-Verdugo, J. M., Herrera, D. G. and Alvarez-Buylla, A. (1999). Young neurons from medial ganglionic eminence disperse in adult and embryonic brain. Nat. Neurosci. 2,461 -466.[CrossRef][Medline]
Wichterle, H., Turnbull, D. H., Nery, S., Fishell, G. and
Alvarez-Buylla, A. (2001). In utero fate mapping reveals
distinct migratory pathways and fates of neurons born in the mammalian basal
forebrain. Development
128,3759
-3771.
Wu, W., Wong, K., Chen, J., Jiang, Z., Dupuis, S., Wu, J. Y. and Rao, Y. (1999). Directional guidance of neuronal migration in the olfactory system by the protein Slit. Nature 400,331 -336.[CrossRef][Medline]
Yee, K. T., Simon, H. H., Tessier-Lavigne, M. and O'Leary, D. M. (1999). Extension of long leading processes and neuronal migration in the mammalian brain directed by the chemoattractant netrin-1. Neuron 24,607 -622.[Medline]
Zhu, W., Dahmen, J., Bulfone, A., Rigolet, M., Hernandez, M. C., Kuo, W. L., Puelles, L., Rubenstein, J. L. and Israel, M. A. (1995). Id gene expression during development and molecular cloning of the human Id-1 gene. Mol. Brain Res. 30,312 -326.[CrossRef][Medline]
Zhu, Y., Li, H., Zhou, L., Wu, J. Y. and Rao, Y. (1999). Cellular and molecular guidance of GABAergic neuronal migration from an extracortical origin to the neocortex. Neuron 23,473 -485.[Medline]
Zou, Y., Stoeckli, E., Chen, H. and Tessier-Lavigne, M. (2000). Squeezing axons out of the gray matter: a role for slit and semaphorin proteins from midline and ventral spinal cord. Cell 102,363 -375.[Medline]
Zou, Y. R., Kottmann, A. H., Kuroda, M., Taniuchi, I. and Littman, D. R. (1998). Function of the chemokine receptor CXCR4 in haematopoiesis and in cerebellar development. Nature 393,595 -599.[CrossRef][Medline]