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First published online May 11, 2006
doi: 10.1242/10.1242/dev.02379
1 Department of Anatomy and Developmental Biology, University College London,
London WC1E 6BT, UK.
2 MRC Centre for Developmental Neurobiology, King's College London, London SE1
1UL, UK.
3 The University of Maryland School of Medicine, Department of Anatomy and
Neurobiology, and The Program in Neuroscience, Baltimore, MD 21201, USA.
4 Johns Hopkins University School of Medicine, Department of Radiology, Division
of NMR Research and Department of Biomedical Engineering, Baltimore, MD 21205,
USA.
5 F. M. Kirby Research Center for Functional Brain Imaging, Kennedy Krieger
Institute, Baltimore, MD 21205, USA.
6 Osaka University, Osaka, Japan.
7 Department of Cellular Pathology, St Margarets Hospital, The Plain, Epping
CM16 6TN, UK.
8 The University of Queensland, School of Biomedical Sciences and The Queensland
Brain Institute, Brisbane, Queensland, Australia.
Authors for correspondence (e-mail
richards{at}uq.edu.au
and
vasi.sundaresan{at}kcl.ac.uk)
Accepted 23 March 2006
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Thalamocortical axons, Corpus callosum, Hippocampal commissure, Axon guidance, Cell migration, Slit, Mouse
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INTRODUCTION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Marillat et al., 2002) and are
crucially involved in the formation of midline commissures
(Kidd et al., 1998a;
Kidd et al., 1998b;
Brose et al., 1999;
Plump et al., 2002;
Bagri et al., 2002;
Long et al., 2004;
Sabatier et al., 2004).
In the Drosophila nerve cord and in the mammalian spinal cord,
Robo (Roundabout in Drosophila) protein expression is upregulated
after midline crossing, when commissural growth cones become highly responsive
to Slit, preventing them from re-crossing the midline
(Kidd et al., 1998a;
Kidd et al., 1998b;
Zou et al., 2000;
Long et al., 2004;
Sabatier et al., 2004).
However, brain commissural and decussating axons, including the corpus
callosum, optic chiasm and the corticospinal tract express Robo protein
(Sundaresan et al., 2004), and
respond to Slit2 both before and after they cross the midline
(Plump et al., 2002;
Bagri et al., 2002;
Shu et al., 2003a). Thus,
Slit2 may serve a different role than that observed in flies or at the midline
of the spinal cord, probably because brain commissural axons grow away from
the midline after they cross it, rather than remaining in close proximity to
the midline as spinal commissural axons do.
Slit2 and Slit1/2 double knockout animals
display defects in corticothalamic and thalamocortical targeting, callosal and
hippocampal commissure projections (Bagri
et al., 2002), and defects in the formation of the optic chiasm
(Plump et al., 2002). In these
mice, large ectopic commissures are formed at the midline from corticothalamic
axons that would not normally cross the midline. These data suggest that the
Slits normally prevent these axons from crossing the midline and instead guide
them to their respective targets in the thalamus.
In addition to regulating commissural axon guidance and axonal branching
(Wang et al., 1999;
Ozdinler and Erzurumlu, 2002;
Sang et al., 2002), Slit/Robo
signalling also regulates cellular migration
(Hu, 1999;
Wu et al., 1999;
Zhu et al., 1999). Slit
secreted from the ventricular zone of the lateral ganglionic eminence (LGE)
repels cortical interneurons from the subventricular zone of LGE explants and
inhibits tangential migration when added locally at the corticostriatal
boundary of brain slices (Zhu et al.,
1999). However, tangential migration was reported to take place
normally in Slit1/Slit2 double knockout mice, while the
ganglionic eminence (GE) retained its repulsive activity
(Marín et al.,
2003).
In order to examine Slit signalling and its involvement in axonal guidance and neuronal migration, we have generated Robo1 knockout mice by targeted deletion. Here, we analyse the migration of interneurons into the neocortex and the formation of the corpus callosum, hippocampal commissure, corticothalamic and thalamocortical projections. The results reveal striking differences between the phenotypes of Robo1 and Slit knockouts, and suggest that additional mechanisms are involved in Slit/Robo signalling in these systems.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Whole mount in situ hybridization for Robo1 was performed on embryonic day
(E)12.5 intact mouse embryos using a modified protocol
(Wilkinson, 1992;
Henrique et al., 1995) as
described by Camurri et al. (Camurri et
al., 2004). To assess protein expression in the Robo1 knockouts,
gel-electrophoresis and immunoblotting were performed as previously described
(Hivert et al., 2002). Robo1
was detected using a rabbit polyclonal antibody (205) raised against the
C-terminal peptide of DUTT1/ROBO1 (Xian et
al., 2001). To demonstrate equivocal loading, blots were re-probed
using a mouse monoclonal antibody raised against ß-actin (Jackson
Laboratories, MN).
Diffusion tensor magnetic resonance (DTMRI) imaging
Imaging was performed using a General Electric Omega 400 (9.4 Tesla) NMR
spectrometer. A custom-made solenoid volume coil was used as the radio
frequency signal transmitter and receiver. Brains were placed in home-built,
MR-compatible tubes filled with fomblin (Fomblin Profludropolyether, Ausimont,
NJ) to prevent dehydration. Diffusion-weighted images were acquired with a 3D
diffusion weighted multiple echo sequence
(Mori and van Zijl, 1998). The
imaging field of view was 11 x 7.5 x 7.5 mm3. The
imaging matrix had a dimension of 128 x 70 x 72. The spectral data
were apodized by a 10% trapezoidal function and then zero-filled to 256
x 140 x 144. The pixel size after the zero-filling was 43 x
53.5 x 52.1 µm3. Eight diffusion-weighted images with
different diffusion gradient direction and magnitude were acquired for each
sample. A repetition time of 900 ms, an echo time of 37 ms and two signal
averages were used for a total imaging time of 
28-30 hours.
The diffusion tensor was calculated using a multivariate linear fitting
method, and three pairs of eigenvalues and eigenvectors were calculated for
each pixel. The eigenvector associated with the largest eigenvalue was
referred to as the primary eigenvector. For the quantification of anisotropy,
a linear measure (CL) was used (Westin et
al., 2002). Using the primary eigenvector and CL, colour maps were
calculated and the red, green and blue values of each pixel were defined by
the orientation of its primary eigenvector with the intensity proportional to
the CL. Red was assigned to the fibre orientation along the anteroposterior
axis, green to the right-left axis and blue to the dorsoventral axis.
Immunohistochemistry
Brains were collected between E12.5 and E18.5. Embryos were either fixed by
immersion in 4% paraformaldehyde (PFA) or transcardially perfused with saline,
followed by 4% PFA, and then postfixed in the same fixative solution
overnight. Brains were blocked in 3% agar and cut at 40 µm on a Vibratome
(Leica) or 30 µm on a cryostat (Bright). Sections were washed in 1x
phosphate buffered saline (PBS), blocked in a solution of 2% serum (v/v) and
0.2% Triton X-100 (v/v) (Sigma) in PBS for 2 hours. Normal goat serum (S-1000,
Vector Laboratories, Burlingame, CA) or normal donkey serum (017-000-121,
Jackson ImmunoResearch Laboratory, West Grove, PA) was used for primary
antibodies made in rabbit or rat, respectively. Sections were incubated in
either rabbit anti-GFAP (1:30,000; Z0334, Dako, Glostrup, Denmark); rabbit
anti-neurofilament M C-terminal (1:75,000; AB1987, Chemicon, Temecula, CA);
rat anti-L1 (1:5000; MAB5272, Chemicon); rabbit anti-calbindin (1:10000;
D-28K, Swant, Bellinzona, Switzerland); mouse anti-GAD65 (1:200 Affinity
Research Products, Exeter, UK); or rabbit anti-Robo1 or -Robo2 (1:10,000 and
1:5,000, respectively; antibodies prepared by Dr Murakami) overnight. Sections
were washed in PBS and incubated in biotinylated goat anti-rabbit (1:500;
Vector Laboratories) or biotinylated donkey anti-rat (1:500; Jackson
ImmunoResearch Laboratory) for 2 hours, then incubated in avidin-biotin
solution (1:500; Vector Laboratories) and processed as previously described
(Shu et al., 2000).
Quantification of interneuron distribution
Calbindin-positive cells were counted in 200 µm coronal strips of
dorsomedial neocortex at different levels along the rostrocaudal extent of the
cortex at E18.5 (eight sections at each level from each of three animals for
each condition). In all counts, the experimenter did not know the condition of
the animal. Strips were longitudinally divided into six equal bins/sectors,
from bin 1 (ventricular zone) to bin 6 (marginal zone). Interneuron migration
was assessed at E12.5 by counting the total number of calbindin-positive cells
that had crossed the corticostriatal notch and entered the cortex.
Carbocyanine dye tracing
Injections were made using fine-tipped glass pipettes (1-5 µl, Dummond
Scientific Company, Broomall, PA) attached to a pressure injector
(Picospritzer, Parker Instrumentation, NJ). Pipettes were filled with
solutions of either 10% DiI or DiA (D-282 and D3883, Molecular Probes) in
dimethylformamide (data shown in Fig.
5). Other brains (data shown in
Fig. 7) were labelled by
placing a single crystal of either DiI or DiA in the brain as previously
described (Métin and Godement,
1996; Molnár et al.,
1998). DiA crystals were placed in the presumptive somatosensory
cortex to label corticofugal axons, and DiI crystals were placed in the dorsal
thalamus to label thalamocortical axons. To label the corpus callosum and the
hippocampal commissure, injections were first made into the cingulate cortex
(DiI) and then the brains were cut coronally at the level of the hippocampus
to allow the injection of DiA directly into the dentate gyrus. Labelled brains
were stored at 37°C in darkness for 2-6 weeks and then blocked in 4%
agarose and cut at either 40 µm or 100 µm using a Vibratome (40 µm
sections were cut on Leica Vibratome and 100 µm sections were cut on a
Vibroslice, Campden Instruments). Injection sites were verified after
sectioning by the presence of a fluorescent bolus and a pipette track.
Sections were washed and incubated overnight with
4'-6-Diamidino-2-Phenyllindole (DAPI, 1:20,000; D-9542, Sigma) in PBS or
bisbenzimide (10 minutes in 2.5 µg/ml solution in PBS, Sigma). Images were
collected using a confocal microscope (Fluoview FV5000 Olympus, NY or Leica,
Microsystems, UK). Sequential images collected with the Leica microscope were
subsequently reconstructed using Metamorph imaging software (Universal Imaging
Corporation).
Dissociated cell cultures
Dissociated cell cultures were derived from E15 mouse telencephalons
according to the method of Cavanagh et al.
(Cavanagh et al., 1997).
Briefly, GEs were dissected out from embryonic forebrains in Hanks' solution
under a stereo microscope, and isolated tissue was dissociated enzymatically
in Neurobasal media with trypsin (0.1%) and DNase I (0.001%) for 15 minutes at
37°C. Trypsin was inactivated by 10% foetal calf serum (FCS) in Neurobasal
media for 5 minutes and cells dissociated by delicate tritiation with a
sterile pipette tip. The resulting suspension was centrifuged at 1000
g for 3 minutes, the supernatant discarded and cells
resuspended in Neurobasal media containing B27 Supplement, 100 µg/ml
penicillin/streptomycin and 2 mM L-glutamine. They were then plated at a
density of 2x105 cells on poly-L-lysine (10 µg/ml) and
laminin (5 µg/ml) coated 13 mm coverslips in 24-well plates. Cultures
plated were kept in a humidified incubator (95% Air/5%CO2) at
37°C and cells were allowed to attach to the coverslips for 30 minutes.
Fresh medium was then added, and again on the following morning.
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RESULTS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). To achieve this, a targeting vector was
generated using a 9.9 kb genomic fragment identified from a mouse bacterial
artificial chromosome which encompasses exons 4 to 6 of Robo1
(Fig. 1A). Exon 5 was floxed
with a neomycin resistance gene (positive selection) cassette at the 3'
end of exon 5, and a thymidine kinase cassette (for negative selection) was
cloned into the vector arm. 129sv-derived ES were targeted and clones selected
and analysed for homologous recombination by Southern blotting
(Fig. 1B). Three independent
clones demonstrating homologous recombination were propagated and then
injected into C57Bl/6 derived blastocysts and chimeras generated. Two
independently derived chimeras that had transmitted the recombinant allele
were mated with Actin Cre mice (obtained from Dr D. Acampora, Kings College
London) to generate founder mice where the exon5/neo cassette had been
excised. These animals were bred onto a C57Bl/6 background and the resulting
heterozygotes were crossed to generate homozygous deficient mice.
Southern blotting and PCR analysis of E14.5 pup DNA/RNA demonstrated that
exon 5 and the neo cassette had been deleted
(Fig. 1B-D), and sequence
information showed that a frame shift had occurred in mice carrying the
deleted allele (Fig. 1E). We
predicted that the altered RNA species would undergo nonsense message decay
(Li and Wilkinson, 1998), and
homozygous mice would be negative for Robo1 message and, hence, protein. In
situ hybridization studies on wild-type and homozygous mutant E12.5 embryos
revealed that Robo1 mRNA expression was completely absent in the Robo1
knockout, whereas Robo2 and Rig1 expression remained high in the spinal cord
(Fig. 1F). Similarly, western
blot analysis on whole embryo (E14.5) extracts, which were probed with an
antiserum against the C terminus of Robo1 (205)
(Xian et al., 2001), showed
that Robo1 expression was high in the wild-type specimen, reduced in the
heterozygote and completely absent in the knockout
(Fig. 1G). As a control,
western blots were stripped and re-probed with a ß-actin antibody to
demonstrate equal loading. These results indicate that our Robo1
knockout mice produce no Robo1 mRNA or protein and thus should be considered
as complete null mutants.
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Robo1, but not Robo2 is expressed on corpus callosum axons
We have previously shown, using an antibody directed against both Robo1 and
Robo2, that Robo receptors are expressed on callosal axons
(Shu et al., 2003a). However,
the generation of antibodies by F. Murakami that recognize Robo1 and Robo2
independently (Long et al.,
2004; Sabatier et al.,
2004) has demonstrated that Robo1 is expressed at high levels on
callosal axons at E17 (Fig.
2A,C,E), whereas Robo2 is only faintly expressed in this region
(Fig. 2B,D). In the same brain,
Robo2 is expressed at high levels on other axonal tracts within the brain such
as the nigrostriatal pathway, the optic tract and, to a lesser extent, on
axons within the internal capsule (Fig.
2F).
Robo1 knockout mice display unique malformations of the corpus callosum
Robo1 knockout brains from E17 and E18 embryos were first examined
by DTMRI to identify large fibre tracts. This revealed gross abnormalities in
the development of the corpus callosum and hippocampal commissure
(Fig. 3–shown at E17).
DTMRI colour maps demonstrate the diffusion anisotropy of water molecules in
ordered structures (Zhang et al.,
2003) and can be `colour-coded' to demonstrate axonal fibre tracts
running in different orientations within the brain. In the horizontal plane,
the presence of the corpus callosum (Fig.
3A, arrow in A') and hippocampal commissure
(Fig. 3A, arrowhead in
A') are shown in wild-type (n=3) and heterozygote
(n=3) littermates. However, disruption in both of these tracts was
evident in the knockout (n=2; Fig.
3G-I). A large reduction in the size of the corpus callosum and
hippocampal commissure was observed in the mid-sagittal plane (compare green
structures with a large arrow in Fig.
3C' with Fig.
3I'). However, the anterior commissure was present in mice
of each genotype (small arrow in Fig.
3C',F',I'). Coronal sections revealed that
callosal axons in Robo1 knockout mice were blue at the midline
(Fig. 3H, arrow in H')
rather than green (Fig. 3B,
arrow in B'), indicating that axons were coursing in the dorsoventral
plane rather than mediolaterally. This could indicate that when callosal axons
reach the midline, they turn to grow ventrally rather crossing the
midline.
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Development of midline glial structures in Robo1 knockout mice
Previous work has described midline glial structures that guide callosal
axons at the midline (Silver,
1993; Shu and Richards,
2001). In order to examine these structures, we labelled brain
sections with glial fibrillary acidic protein. All three midline glial
populations were present–the glial wedge, indusium griseum glia and the
midline zipper glia (Shu et al.,
2003b)–in both wild-type (n=5;
Fig. 6A,B) and Robo1
knockout brains (n=2; Fig.
6C,D). Although some disruption of the midline zipper glia was
evident (compare Fig. 6B,D), it
appeared that this was secondary to the formation of the large axonal
fascicles described above.
Advanced thalamocortical and corticothalamic projections in Robo1 knockout mice
In Slit2 knockout mice, corticothalamic and thalamocortical projections
deviate within the internal capsule resulting in an ectopic commissure
(Bagri et al., 2002). To
investigate these projections in Robo1 knockout mice, DiI and DiA crystals
were placed in the dorsal thalamus and cortex, respectively, of wild-type and
knockout littermate brains (E12.5-18.5;
Fig. 7). At E12.5, similar to
what has been described in the rat
(Molnár and Cordery,
1999), we observed thalamocortical and corticothalamic fibres,
bearing growth cones at their tips, growing out of their sites of origin and
directed towards the region of the internal capsule. There was no apparent
difference in the pattern or extent of labelling between mutant and Robo1
wild-type mice (n=3 for each condition, data not shown). However, at
E14.5, although both thalamocortical and corticothalamic projections in
knockout brains (n=4) followed paths comparable with those in wild
type (n=4), they were further advanced. Thus, although axons of both
systems in wild-type brains had not advanced past the lateral cortex
(Fig. 7D,E), thalamocortical
projections (DiI, red) in the knockouts were observed well into the cortex
(compare Fig. 7A with 7D) where
a few cortical plate cells had been back-labelled
(Fig. 7B, arrows). Similarly,
the corticothalamic projections in these animals (DiA, green) were already
present in the thalamus (Fig.
7A). Furthermore, DiI-labelled axons crossing transversely over
the thalamocortical fibre bundle were observed at the level of the internal
capsule. This `knot' structure (Fig.
7C, arrow), observed in all knockout brains examined at this age,
could be the result of a misplaced subgroup of thalamic axons or other
misrouted axons such as retrogradely labelled optic tract axons. These data
indicate that early in development, Robo1 plays a role in the timely
projection of thalamocortical and corticofugal axons. Examination of E18.5
brains (n=4 for each condition) showed the advance of thalamocortical
axons persisted in the Robo1 knockouts where the axons projected
further medially into the cortex (Fig.
7G, arrow) compared with controls
(Fig. 7K, arrow). In addition,
back-labelled cells appeared in greater numbers in the thalamus of mutants
following placement of dye in the cortex (compare
Fig. 7I with 7M, arrows),
further indicating that thalamocortical axons had arrived earlier in these
brains. Furthermore, similar to DTMRI analysis, we observed corpus callosum
axons deviating ventrally at the midline
(Fig. 7F, arrow). In addition,
similar to E14.5, DiI-labelled axons coursing transversely at the level of the
internal capsule formed a `knot' structure in all but one of the Robo1
knockout brains examined (Fig.
7H, arrow), while they were absent in the controls
(Fig. 7L, arrow).
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), suggesting that this receptor may regulate the movement of
cortical interneurons from the ventral telencephalon. We examined Robo1
expression in coronal sections of E13.5 and E15.5 mouse brains, ages when
interneuron migration is at its peak. Staining was identified in the mantle
zone of the GE and along the marginal zone (MZ) and lower intermediate zone
(IZ) of the cortex (Fig. 8A),
well-defined routes for tangentially migrating interneurons
(Fig. 8B)
(Anderson et al., 1997;
Parnavelas, 2000). The
staining in these zones was rather dense and it was difficult to discern
individual cell bodies from processes oriented parallel to the ventricular
surface. At high magnification, the Robo1 receptor was clearly visualized in
some individual cells, especially in the GE near the start of the migratory
route (Fig. 8A'). These
cells often displayed elongated somata and leading processes, features typical
of migrating interneurons. Further analysis of dissociated GE cell cultures
prepared from E15 animals showed Robo1 expression in GABAergic cells as
identified by co-labelling with GAD65 (Fig.
8C-E).
A number of studies have reported that interneurons use corticofugal
projections to migrate from the ventral telencephalon to the cortex
(Parnavelas, 2000;
Denaxa et al., 2001;
Morante-Oria et al., 2003;
McManus et al., 2004),
although such an association has not been supported by the work of others
(Tanaka et al., 2003). Thus,
given the defects observed in the corticofugal projections, we wanted to
elucidate the possible role of Robo1 in interneuron migration and distribution
in the cortex. We examined brain sections (E12.5-E18.5) stained for calbindin,
a marker of cortical interneurons. Although not all interneurons express
calbindin (López-Bendito et al.,
2004), this marker has been used routinely to label a large
subpopulation of GABAergic interneurons migrating tangentially to the cortex
(Anderson et al., 1997;
Sussel et al., 1999). Our
results clearly showed that the pattern of calbindin staining closely
resembled that of Robo1 immunoreactivity
(Fig. 8A,B). Analysis of
wild-type E12.5 mice showed relatively few calbindin-positive cells
predominantly in the lateral cortex (Fig.
9D) in agreement with earlier observations
(Anderson et al., 1999;
Anderson et al., 2001).
However, in Robo1 knockout littermates, significantly more calbindin-positive
cells were observed in the cortex (Fig.
9E). Cell counts at E12.5 (n=4 for each condition)
revealed that almost twice as many calbindin-positive neurons had migrated
into the cortices of Robo1 knockouts (220±12,
P<0.001) compared with controls (98±10)
(Fig. 9A,D,E). A comparable
increase in the number of calbindin-positive cells was noted in the cortex of
Robo1 knockouts at E15.5 (n=4 for each condition).
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35% increase in the total number of calbindin-positive cells counted in a
200 µm wide strip of knockout dorsomedial cortex (DMC) compared with counts
in a similar area in wild-type brains (Fig.
9B). Counts at different levels along the rostrocaudal axis showed
that the increased number of calbindin-positive cells was evident in rostral
(Fig. 9C,D,E) and middle
(parietal) cortical areas (Fig.
9C,F,G), but not in the caudal (occipital) cortex
(Fig. 9C). Furthermore, there
was an abundance of calbindin-positive cells in the striatal region
(Fig. 9G,G', arrows), an
area that is normally repulsive to migrating interneurons
(Marín et al., 2001).
The presence of calbindin labelled cells in the striatum was also evident in
E15.5 knockout brains (data not shown). These observations indicate that Robo1
influences both the number of interneurons entering the cortex early and their
migratory route through the ventral telencephalon. |
DISCUSSION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
Thalamocortical axons in these mice display ectopic projections ventrally and
very few enter the cortical plate. However, in Robo1 knockouts,
thalamocortical and corticothalamic axons reach their targets at least one day
earlier than in controls and no ectopic commissures are present in the
diencephalon. Furthermore, previous data show no defect in interneuron
migration into the cortex of Slit or Slit/Netrin (Ntn) mutants
(Marín et al., 2003),
whereas we see an increase, compared with controls, in the number of
interneurons entering the cortex and reaching their targets early. These
differences are significant and imply one of three possibilities for Slit
signalling in this system: (1) both Robo1 and Robo2 are required for Slit
signalling; (2) Robo1, together with an unknown receptor is required for Slit
signalling, or (3) a completely novel Slit receptor is involved in these
processes. An additional possibility is that Robo1 acts alone (possibly as a
homodimer) in one of more of these systems.
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;
Shu et al., 2003a). However,
the Robo receptor involved has been unclear. Our data show that Robo1 is
required for callosal formation, but some subtle differences also exist
between the Robo1 and Slit2 callosal phenotypes. For example, when Slit2 is
removed, axons tend to defasciculate (Bagri
et al., 2002; Plump et al.,
2002; Shu et al.,
2003a), whereas when Robo1 is absent, the axons form tight axon
clusters or bundles that project ectopically en masse (data presented here).
Our data also suggest that Robo1 might be involved in maintaining a crucial
distance between callosal and hippocampal commissure axons. Our finding that
axon bundles from each commissure were mixed together at the midline, rather
than remaining segregated, suggests the involvement of Robo1 in normally
maintaining the separation of these commissures into two distinct fibre
tracts, perhaps either through Robo/Slit signalling or through Robo1
homophilic interactions.
Robo1 regulates the timing of corticothalamic and thalamocortical targeting
Our results demonstrate that corticothalamic and thalamocortical
projections reach their targets prematurely when compared with their wild-type
littermates early in development. This may be interpreted in one of two ways:
either Robo1 normally acts as a growth retardant for these axons in a
Slit-independent manner; or the interactions between Robo1 and either Slit1
expressed in the cortical plate (Whitford
et al., 2002) or Slit1 and Slit2 expressed in the thalamus
(Marillat et al., 2002;
Bagri et al., 2002), prevent
these axons from entering their respective targets until later. Slit2
and Slit1/2 double mutants display major pathfinding defects
in the formation of these projections
(Bagri et al., 2002). However,
the pathfinding defects were not observed in the Robo1 knockouts (other than
overshooting their targets at each age as described). Hence, either these
effects are mediated by a different receptor or by a different combination of
receptors. The finding that Robo2 is not highly expressed on callosal axons
makes it unlikely that elimination of both Robo1 and Robo2 simultaneously
might be required to observe similar defects as those described in Slit
mutants. Regardless, we and others (Long
et al., 2004) have been unable to generate Robo1 and
Robo2 double knockout mice by breeding owing to the proximity of
these two genes on the same chromosome. We found that Robo1 is expressed in
axons within the IZ at a time when both the corticofugal and thalamocortical
axonal systems develop. By E18.5, when these axons have reached their final
targets, Robo1 labelling is downregulated in these axons. Thus, the phenotype
observed in the mutants might be directly mediated by Robo1 on the developing
axons in the cortex. In addition, Robo1 is expressed within the GE and could,
therefore, mediate guidance events on both thalamic and cortical projections
within this region.
Robo1 affects the migration of cortical interneurons from the ventral telencephalon
Our results show a significant increase in the number of interneurons that
enter the cerebral cortex from the ventral forebrain in Robo1-null
mice throughout the period of corticogenesis. Furthermore, we found that these
neurons migrated through the striatum and were not repelled by it. Previous
studies have shown that the striatum expresses Sema3a and Sema3f
(Marín et al., 2001),
and GABAergic interneurons expressing neruopilin 1 (Npn1) are repelled away
from the striatum into the cortex
(Marín et al., 2001;
Morante-Oria et al., 2003).
Slits have also been shown to repel GABAergic interneurons in vitro
(Zhu et al., 1999). However,
in vivo, Slit1/2 double knockout mice and
Slit1/2 and Ntn1 triple knockout mice do not
display defects in cortical interneuron migration
(Marín et al., 2003).
It is difficult to reconcile these data with our findings and explain why in
the Robo1 knockout animals, interneurons migrate through the repulsive
striatal region and enter the cortex earlier and in larger numbers than
normal.
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),
indicating that this might be a Slit-independent event. However, presumably
these neurons still express Npn1 and the striatal region would still express
Sema3a and Sema3f. Thus, perhaps, both Npn1/Sema and Robo1 signalling are
required to steer the cells around the striatum. The second abnormal
developmental event we observed is that more interneurons migrate into the
cortex. The most plausible explanation for this is that these cells follow the
cortical and thalamic axons within the internal capsule that enter their
respective targets earlier than normal in Robo1 knockout mice. A
close association with the TAG-1 expressing axonal bundles of the developing
corticofugal fibre system has been observed, prompting speculation that
interneurons use this system as a scaffold for their migration into the
neocortex (Denaxa et al., 2001;
Morante-Oria et al., 2003;
McManus et al., 2004).
Considering that Robo1 is expressed in the GE, where interneurons originate
from, and in the IZ, a zone that contains the majority of migrating
interneurons to the cortex, it is possible that their trajectory is both
directly delineated by Robo1 and indirectly regulated through fasciculation
and migration along cortical and thalamic axons.
Why was the increase in interneuron number restricted to the rostral and
middle cortical areas of the Robo1 knockout mice? Recent studies by
Yozu et al. (Yozu et al.,
2005) have clearly shown that the sources and mechanisms of
migration of interneurons that populate these areas are different from those
destined for caudal cortical regions. Specifically, these investigators have
demonstrated that interneurons destined for the caudal cortex and hippocampus
arise in the caudal GE and use a novel migratory path, the so-called caudal
migratory stream, whereas those that populate rostral and middle areas arise
predominantly from the MGE/LGE. Finally, we cannot exclude the possibility
that the increase in interneuron numbers in the rostral and middle cortical
areas is due to a differential increase in proliferation in different parts of
the GE.
In summary, we found that Robo1 is required for the correct formation of the corpus callosum and the hippocampal commissure, as well as the timely projection of thalamocortical and corticofugal axons. In addition, the absence of Robo1 led to premature migration of interneurons to the cortex. The differences between the Robo1 knockout phenotype and the phenotypes of Slit-deficient mice suggest that additional components contribute to Robo/Slit signal transduction mechanism.
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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