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First published online 28 August 2008
doi: 10.1242/dev.024778
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1 Université Libre de Bruxelles (U.L.B.), IRIBHM (Institute for
Interdisciplinary Research), 808 Route de Lennik, B-1070 Brussels,
Belgium.
2 Karolinska Institute, Department of Cell and Molecular Biology, SE-171 77
Stockholm, Sweden.
3 Uppsala University, Department of Neuroscience, 75123 Uppsala, Sweden.
* Author for correspondence (e-mail: pierre.vanderhaeghen{at}ulb.ac.be)
Accepted 12 August 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Forebrain, Neuronal migration, Ephrin, Striatum, Guidance
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Graybiel and Ragsdale, 1978;
Johnston et al., 1990). The
matrix compartment contains the majority (80-85%) of the striatal medium spiny
(MS) projection neurons and is organized around the striosomes, which contain
the rest of the MS neurons and appear as patches of neurons scattered in the
striatum.
The matrix and the striosomes can be distinguished on the basis of various
neurochemical markers selectively enriched in one of the compartments, such as
calbindin in the matrix and µ-opioid receptor in the striosomes
(Gerfen et al., 1987;
Herkenham and Pert, 1981;
Liu and Graybiel, 1992;
Nastuk and Graybiel, 1985).
Importantly, the striatal compartmentalization is also related to the
organization of cortical inputs to the striatum, with matrix and striosome
neurons receiving preferential inputs from distinct cortical layers and areas
(Gerfen, 1989;
Gerfen, 1992;
Kincaid and Wilson, 1996).
These anatomical differences are tightly linked to functional differences that
have started to be unravelled recently. Neurons from striosome and matrix
compartments display differential activity during natural behaviour or
following psychomotor stimulant treatments
(Brown et al., 2002;
Canales and Graybiel, 2000).
Similarly, the specific loss of neurons from either compartment has been
correlated with distinct clinical features in Huntington's disease
(Tippett et al., 2007), while
selective impairment of the function of either neuronal compartment can have
different effects on motor function and behaviour
(Tappe and Kuner, 2006).
The development of striatal compartments is a highly orchestrated process.
Striatal projection neurons are generated in the lateral ganglionic eminence
(LGE) in the ventral forebrain (Marin and
Rubenstein, 2003; Wilson and
Houart, 2004; Wilson and
Rubenstein, 2000), but interestingly the commitment to one
specific striatal compartment is linked to the developmental stage at which
embryonic neurons are generated (Song and
Harlan, 1994; van der Kooy and
Fishell, 1987). Early-generated neurons are destined to the
striosomal compartment, whereas neurons exiting the cell cycle at mid and late
embryogenesis are committed to the matrix compartment
(Mason et al., 2005;
van der Kooy and Fishell,
1987; Yun et al.,
2002). This birthdate-based spatial confinement is reminiscent of
the layered organisation found in the cerebral cortex, where neurons populate
non-overlapping layers depending on their birthdate
(Bayer and Altman, 1991).
However, in the case of the striatum, neurons that are generated sequentially
first migrate to the same domains of the striatal mantle (the striatal
primordium), where they intermix at embryonic stages, before segregating
during early postnatal periods, forming a mosaic pattern rather than distinct
layers (Krushel et al., 1995;
Lanca et al., 1986)
(Fig. 1A). In vitro assays
showed that the striosome neurons display homophilic adhesive properties,
providing a first hint about potential underlying cellular mechanisms
(Krushel et al., 1995;
Krushel and van der Kooy,
1993). Although earlier in vivo studies tended to minimize the
role played by extrinsic factors such as the projections from the cortex and
substantia nigra (Snyder-Keller,
1991; van der Kooy and
Fishell, 1992), recent in vitro studies have suggested that the
early-generated striosomal neurons may cluster around corticostriatal fibres
(Snyder-Keller et al., 2001;
Snyder-Keller, 2004) and that
striatal compartmentalization may thus rely on such extrinsic cues.
Striatal development thus constitutes an original model of pattern
generation through cell sorting, the mechanisms of which may be quite
different from those operating during hindbrain segmentation, for example
(Poliakov et al., 2004).
Although some of the genes implicated in the specification of striatal MS
neurons have been identified (Arlotta et
al., 2008; Mason et al.,
2005; Yun et al.,
2002), the molecular cues involved in striatal neuron sorting
remain completely unknown. Cadherins have been found previously to be
expressed differentially between the murine striatal compartments
(Redies et al., 2002), as are
some Eph receptors, which are selectively enriched in the matrix compartment
(Janis et al., 1999); however,
the functional involvement of these molecules in striatal patterning has not
been explored. Ephrins and Eph receptors have been involved in the generation
of distinct developmental compartments, such as the segmentation of the
hindbrain into distinct rhombomeres, making them attractive candidates for the
segregation of striatal compartments
(Barrios et al., 2003;
Klein, 2004;
Pasquale, 2008;
Poliakov et al., 2004;
Swartz et al., 2001).
Using in vivo analyses of mutant mice and a novel organotypic assay that recapitulates striatal development in vitro, we have identified ephrin/Eph family members as guidance cues that control matrix/striosome compartmentalization. These data constitute the first identification of genes involved in the formation of the mosaic pattern of the striatum, supporting a model whereby the temporal control of membrane-bound cues is tightly linked to the spatial organization of this structure.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Kullander et al., 2001;
Dufour et al., 2003).
In situ RNA hybridization
In situ hybridization probes have been described previously
(Dufour et al., 2003;
Vanderhaeghen et al., 2000).
In situ hybridization using digoxigenin-labeled RNA probes was performed as
described (Vanderhaeghen et al.,
2000). All hybridization results obtained with antisense probes
were compared with control sense probes. Pictures of the in situ RNA
hybridization were acquired with Axioplan2 Zeiss microscope and a Spot RT
camera, and converted in false colours and overlayed using Adobe Photoshop
software.
Organotypic overlay assay
Vibratome coronal slices (250 µm) were isolated from transgenic embryos
ubiquitously expressing GFP at E12 or E15
(Okabe et al., 1997). The
lateral ganglionic eminence (LGE) was dissected out in ice-cold L15 and
mechanically dissociated. Up to 500x103 cells were laid down
on top of postnatal striatal vibratome slices (P0-P2) and cultured within cell
culture inserts (1 µm pore size PET membranes; Becton Dickinson), as
previously described (Polleux and Ghosh,
2002). Organotypic co-cultures were performed using an
air-interface protocol and were maintained in a 5% CO2 humidified
incubator for 20 hours in vitro. Ephrin/Eph inhibitors (EphA3-Fc, EphB2-Fc)
and control reagent (Fc) were purchased from R&D Systems. GFP and DARPP32
were detected by immunofluorescence as previously described
(Dufour et al., 2003), and
imaged using a Bio-Rad MRC1024 or Zeiss LSM510 confocal microscope.
BrdU incoroporation and immunofluorescence
For BrdU labelling, timed-pregnant female mice were injected
intraperitoneally, with four pulses (50 mg kg-1 body weight) every
2 hours, of 5-bromo-2'-deoxyuridine (Sigma) dissolved in physiological
sterile solution, at E16 and E17. Newborns were sacrificed 2 days after birth,
fixed by perfusion with PFA 4%, followed by overnight immersion in the same
fixative. The forebrain was vibratome sectioned at 50 µm. Sections were
then processed for double-immunofluorescence against BrdU (mouse antibody,
1/1000, Becton Dickinson) and DARPP32 (rabbit antibody, 1/500, Chemicon).
Quantification methods of matrix/striosome distribution in vitro and in vivo
To determine the matrix/striosome (M/S) values in organotypic assays, the
area of each single DARPP32-positive striosome was determined in Photoshop
using the Lasso tool, and GFP-positive pixels (representing the GFP+ cells)
were quantified by selection with the Magic Wand tool in each DARPP32-positive
striosome, giving a first value of GFP+ cell density in the striosome (S).
Next, the same selected area corresponding to the striosome was moved into
three different adjacent matrix regions outside the striosome, where
GFP-positive pixels were quantified similarly. A mean value for the pixels
counted in the three matrix regions outside the striosome was then calculated
(as M, the mean density of GFP+ cells in the adjacent matrix) and divided by
the number of pixels counted in the adjacent striosome (S) to obtain a
matrix/striosome value that reflects the ratio of GFP+ cell densities between
each striosome and adjacent matrix area. Thus, the M/S value reflects a cell
density and is independent of the size of the area where cells are counted,
and an M/S value of 1 is obtained if cells are distributed in a uniform
fashion across striosome and matrix compartments. This procedure was applied
to all the striosomes and to the corresponding adjacent matrix areas of all
the striatal sections to obtain a mean M/S value for each condition.
The distribution of BrdU cells in matrix versus striosome compartments in wild-type and ephrin A5/EphA4 mutants were quantified on all visible striosomes of brain sections (n=5 sections for each animal) using similarly determined M/S values, by quantifying the distribution of BrdU-positive cells within DARPP32-positive striosomes (S) and neighbouring DARPP32-negative matrix domains (M) (owing to the single cell resolution of the BrdU staining, BrdU-positive cells were counted manually).
M/S means were compared using classical Student's t-test with Welch's correction to account for unequal variances. The hypothesis that the mean value of M/S could be equal to 1 was tested with compatibility Student's t-test.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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This assay consisted of a heterochronic co-culture where striatal neurons
generated at different embryonic ages were confronted with and allowed to sort
out between nascent matrix and striosome compartments. First, cells were
dissociated from the LGE (where all striatal projection neurons are generated)
of ubiquitously GFP-expressing embryos
(Okabe et al., 1997) at
embryonic stages E12 or E15-16, to obtain a population enriched for either the
striosome or the matrix neurons, respectively
(Fig. 1B). The dissociated cell
suspensions were then plated onto organotypic slices of the striatum at early
postnatal stages (P0-P2), the stage at which the two compartments just start
to emerge clearly in vivo (Fig.
1A). The culture was stopped after 20 hours in vitro, and the
slices were processed to allow examination of the distribution of the GFP+
neurons throughout the striatum, in comparison with the pattern of DARPP32,
which marks selectively the striosomes at early stages of striatal development
(Anderson et al., 1997)
(Fig. 1B).
The distribution of the GFP+ cells in each compartment was quantified by comparing the density of GFP+ cells that settled in each DARPP32-positive striosome compartment (S) with the density that settled in the adjacent DARPP32-negative matrix compartments (M). The relative distribution of the GFP+ cells in each compartment was then expressed as the ratio between M and S cellular densities as an M/S value (Fig. 1B; see also Materials and methods). Thus, M/S values of 1 indicate that the cells are distributed in a random fashion across striosome and matrix compartments, whereas M/S values less than 1 indicate a preferential distribution in the striosome compartment and, conversely, M/S values greater than 1 indicate a preferential distribution in the matrix compartment (Fig. 1O).
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These results indicate that presumptive matrix- and striosome-destined neurons (isolated at E15 and E12, respectively) can respond differentially to local cues present in the early postnatal striatal slice, and thereby can segregate into the appropriate compartment. This quantitative assay thus recapitulates a major feature of striatal patterning: its time dependence. It enables us to reveal the differences in responsiveness of striatal progenitors to local cues (depending on their age), and also that early striatal cells preferentially set on striosome compartments while later striatal cells preferentially set on the matrix compartment.
These results prompted us to use this system to test for the involvement of candidate molecules in matrix/striosome patterning. To this end, we first tried to identify which chemoaffinity molecules might be differentially expressed between striatal compartments and could control the sorting of the two striatal populations. In view of our results, we looked for guidance cues that might show: (1) a temporal pattern of differential expression in early-versus late-generated striatal neurons at embryonic stages; and/or (2) a spatial pattern of differential expression within striosome/matrix compartments at early postnatal stages.
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)],
although its expression at time points relevant to matrix/striosome
establishment was not explored, nor was the expression of candidate ligands
for EphA4. To explore the involvement of EphA4 in matrix/striosome formation, we performed in situ hybridization for this receptor, as well as for putative ligands known to be expressed in the developing forebrain (ephrin A2, ephrin A3, ephrin A5, ephrin B1, ephrin B2 and ephrin B3), focusing on their expression at embryonic and early postnatal stages, covering the whole period of genesis, migration and sorting of the two striatal populations. This analysis revealed striking temporal patterns of expression of EphA4 and its ligands ephrin A5 and ephrin B2 (Fig. 2), and ephrin B3 (similar to ephrin B2, data not shown).
By E12.5, when the genesis and the migration of the striosome-destined neurons have started, EphA4 was detected only in the ventricular zone and not in the striatum per se (Fig. 2A), whereas expression of ephrin A5 and ephrin B2 was already detected both in the ventricular zone (VZ) and the striatal mantle (Fig. 2B,C, arrows). By E14.5, when most of the striosomal neurons have migrated in the striatal mantle, expression of ephrin A5 and ephrin B2 was detected throughout the striatal mantle. At this time, most of EphA4 receptor expression still remained mostly confined to the ventricular zone, with some expression found in the mantle (Fig. 2D-F, arrows). By E16.5, when many matrix neurons have migrated in the striatal mantle, expression of EphA4 could be detected throughout the striatal mantle, similar to ephrin A5 and ephrin B2 (Fig. 2G-K). These data indicate that EphA4 and its ligands ephrin B2 and ephrinA5 display a differential temporal pattern of expression, suggesting that ligands are preferentially expressed in early-generated striatal neurons, and that EphA4 is preferentially expressed in later-generated neurons.
At early postnatal stages (P0-P2), when matrix/striosome patterns first appear visible, the EphA4 receptor showed a matrix-like distribution all over the striatum (Fig. 2J,M), with small and evenly distributed patches devoid of staining that become more and more distinct from P0 to P2. Ephrin A5 exhibited a differential expression profile, with a progressively lower expression level and a more restricted patchy distribution, reminiscent of the striosomal compartment (Fig. 2K,N), whereas ephrin B2 was expressed at progressively weaker levels (Fig. 2L,O).
To compare more precisely the distinct patterns of expression of ephrin A5
and EphA4 with matrix and striosome compartments at postnatal ages, we
performed, on alternate striatal sections, immunohistochemistry for DARPP32,
an early marker for the striosomal compartment
(Anderson et al., 1997), and
compared it with the patterns of the ligand-receptor pair.
EphA4 receptor distribution across the striatum was found to complement the DARPP32-positive striosomes, from postnatal stage P2 (Fig. 3A-C, arrowheads) to P4 (Fig. 3G-I, arrowheads), implying that the receptor belongs mainly to the matrix compartment, although also to some striosomal cells at a lower level. At P0, EphA4 expression already exhibited a matrix-like distribution, but failed to strictly complement the DARPP32 staining, consistent with the notion that the process of compartmentalization between the two striatal population is not completed at that stage (data not shown).
Next, we compared the EphA4 staining with the ephrin A5 distribution on alternate sections, and found a partial complementarity between the two that was dependent on the striatal subdomain considered. Within the peripheral regions of the striatum, ephrin A5 and EphA4 were partially co-expressed (Fig. 3D-F,J-L), whereas within the central-most region of the striatum, a complementary expression was observed, with patches of ephrin A5 devoid of EphA4 expression (Fig. 3F,L, arrowheads). Similarly, a partial complementarity was observed when comparing ephrin A5 with DARPP32 (data not shown), suggesting that ephrin A5 expression is enriched in a subpopulation of striosomal neurons, but also present at lower levels in some parts of the matrix compartment.
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Disruption of ephrin-Eph signalling partially alters the matrix/striosome neuronal sorting in vitro
To test directly our hypothesis on the involvement of ephrin/Eph signaling
in striatal compartmentalization, we first used the organotypic overlay assays
described above, in combination with specific soluble inhibitors of ephrin/Eph
signalling added to the culture media
(Dufour et al., 2003). Given
the unique functional promiscuity of EphA4 [it can bind and activate most
ligands of ephrin A and ephrin B subfamilies
(Flanagan and Vanderhaeghen,
1998)], we used EphA3-Fc and EphB2-Fc inhibitors in combination,
to provide efficient inhibition of both ephrin A and ephrin B signalling.
Control conditions consisted of addition of Fc proteins only.
When performing these experiments with cells derived from E16 LGE cells (presumptive matrix neurons), pre-incubation of the receiving striatal slices with EphA3/B2-Fc resulted in a significant decrease of the preference of these cells for any specific striatal compartment (Fig. 4A-F,M), as assessed by a decreased M/S value (1.15±0,105), which was not different from 1 (P=0.151, Student's t-test), and different from the mean M/S value obtained for the control condition (incubation with control Fc reagents; P=0.001, unpaired t-test with Welch's correction). Similarly, when performing these experiments with cells derived from E12 LGE cells (presumptive striosome neurons), pre-incubation of the receiving striatal slices with EphA3/B2-Fc resulted in a significant decrease of the preference of these cells for any striatal compartment (Fig. 4G-L,M), with a mean M/S value (0.99±0.117) not different from 1 (P=0.945, Student's t-test), and different from the mean M/S value obtained under the control conditions (incubation with control Fc reagents, P<0.0047, unpaired t-test with Welch's correction).
Interestingly, when EphA3-Fc or EphB2-Fc were used alone, they did not affect the matrix/striosome preference of LGE embryonic cells (data not shown), thus providing evidence that a combination of ephrin A and ephrin B signalling is required for proper striatal neuron sorting in vitro.
These data indicate that acute inhibition of ephrin/Eph interactions normally present in the striatum results in a partial loss of the appropriate sorting of matrix and striosome neuronal populations, consistent with the hypothesis that ephrin/Eph signalling is indeed involved in this process, at least in the context of the organotypic assay.
EphA4 signalling is required for the normal patterning of striatal compartments in vivo
According to the expression patterns and in vitro analysis, the loss of
either ephrin ligands or EphA4 receptor, or both, would impair the neuronal
segregation during striatal development and, as a consequence, the correct
formation of striatal compartments. To further test this hypothesis, we
analyzed ephrin A5 knockout mice, EphA4 receptor knockout mice
(Frisen et al., 1998;
Kullander et al., 2001) and
ephrin A5/Eph4 double knockout (DKO) mice, given their known interactions in
other systems in vivo (Dufour et al.,
2003; Eberhart et al.,
2004; Marquardt et al.,
2005; Swartz et al.,
2001).
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The relative distribution of the presumptive matrix neurons between the two striatal compartments was assessed by comparing the density of BrdU+ cells in matrix and striosomal compartments. The data were quantified as in the in vitro assay, by determining the cell density within each compartment, and expressed as an M/S value (as for in vitro assays), which would be equal to 1 if the matrix neurons were homogeneously distributed, and greater than 1 if BrdU+ neurons were preferentially located in the matrix compartment. This value, thus, reflects the degree of segregation between the two striatal populations. In addition, the DARPP32 staining enabled the qualitative determination of the spatial distribution of striosomal neurons across the striatum.
Control animals showed a distribution of BrdU+ neurons that was typical of the matrix neurons across the striatum: a dense matrix of BrdU+ cells (Fig. 5A) with interspersed zones almost devoid of BrdU+ cells (Fig. 5A, arrowheads and insert), corresponding to the DARPP32+ striosomes (Fig. 5B, arrowheads and insert), from which the BrdU+ cells were largely excluded (Fig. 5C, arrowheads and insert). The mean M/S value was significantly greater than 1 (1.3±0.069, P=0.001, Student's t-test, Fig. 6), indicating that, as expected in control animals, the BrdU+ neurons were significantly enriched in the matrix compartment. Thus, this labelling method appeared to be a sensitive and specific method to determine the level of matrix/striosome segregation in vivo, which we next applied to the analysis of ephrin A5, EphA4 and compound ephrin A5/EphA4 mutants.
When analysed and compared with control animals, the single ephrin A5 knockout mice (n=5) displayed a similar distribution of BrdU+ matrix neurons across the striatum, with a preferential distribution of BrdU neurons in the matrix compartment (Fig. 5D-F, Fig. 6). In addition, examination of the DARPP32 pattern did not reveal any obvious difference compared with control animals, suggesting that all compartments are overall properly formed in this mutant. Consistent with this qualitative analysis, the M/S value was significantly greater than 1 (1.37±0.083, P<0.001; Student's t-test, Fig. 6)
By contrast, a similar analysis performed on EphA4 receptor knockout mice
revealed several striking defects in matrix/striosome organization. First, the
BrdU+ cells appeared to be distributed more uniformly across the striatum
(Fig. 5G), with much less
distinct zones devoid of BrdU+ cells (Fig.
5G, inset). In addition, the DARP32 staining appeared much more
diffuse (Fig. 5H, arrowheads
and insert) than in the control animals, suggesting an alteration in the
distribution of presumptive striosomal neurons. Finally, BrdU+ cells were
found to be distributed at ectopic locations within the remaining striosomes
(Fig. 5I, arrowheads and
insert). Consistent with these observations, the M/S value was not different
from 1 in these mutants (0.99±0.055, P=0.798 Student's
t-test, Fig. 6),
indicating that the matrix neurons were distributed much more uniformly
between the two compartments. Given the known interactions between ephrin A5
and EphA4, and the likelihood that other redundant ephrin/Eph genes are
involved in the system, we next turned our analysis to ephrin A5/EphA4 DKOS,
reasoning that, as in other systems (Dufour
et al., 2003), this may reveal further the involvement of these
genes in striatal development. The analysis of DKO mice (n=6)
revealed several qualitative and quantitative changes in the striatal
patterning. First, the BrdU+ cells were more uniformly distributed over the
striatal mantle (Fig. 5J);
second, the DARPP32+ cells were less densely packed into well-defined
striosomes (Fig. 5K); and
finally, ectopic BrdU+ matrix cells were found abundantly in the striosomal
compartment (Fig. 5L, arrowhead
and inset). These qualitative defects were further quantified by determining
the M/S values in DKOs, which revealed an M/S value that was not different
from 1 (0.96±0.045, P=0.421, Student's t-test,
Fig. 6). Although the analysis
of the DKOs thus clearly confirmed the defects observed in the single EphA4
KOs, it failed to reveal a quantitative interaction between ephrin A5 and
EphA4 genes, suggesting compensatory mechanisms by the other ligands
(including ephrin Bs) acting in combination with ephrin A5 in this context.
Importantly, the absolute density of BrdU neurons throughout both striatal
compartments was similar in all mutants analysed (data not shown), indicating
that the changes observed in EphA4 and ephrin A5/EphA4 mutants was not due to
a change in the absolute number of striatal neurons, related, for example, to
changes in proliferative or apoptotic patterns
(Depaepe et al., 2005;
Holmberg et al., 2005).
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Johnston et al., 1990). Here,
we report that ephrin/Eph family members act as guidance factors that regulate
the compartmentalization of the striatum, both in vitro and in vivo. Our data
shed new light on the molecular and cellular mechanisms that control the
cytoarchitecture of this important forebrain structure. In addition they
reveal a novel developmental mechanism by which the temporal control of
guidance cues can control the spatial patterning of brain nuclei.
Ephrin/Eph and other guidance cues controlling matrix/striosome sorting: a combination of repulsion and adhesion
Although the development of the unique cytoarchitecture of the striatum
must rely on differential cell guidance and adhesion, the underlying
mechanisms have remained essentially unknown. Our data identify the first
molecular cues involved in striatal compartmentalization, and point to a
cellular mechanism involving bidirectional repulsion, according to the
following scenario (Fig.
7B).
At early stages of striatal neurogenesis, ephrin A5/B2+ neurons start to be generated and progressively migrate to populate uniformly the striatum. Later on, the expression profile of the newly generated striatal neurons progressively shifts to the EphA4 receptor, and these late-generated EphA4+ neurons mix with the resident ephrin A5/B2+ neurons. Following cell-cell interactions, ephrin/Eph signalling will result in mutual segregation of the two cell populations, which will cause the EphA4+ neurons to adopt a matrix distribution around the clustered ephrin A5/B2+ neurons.
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Although, in principle, such a model could fully account for the
segregation of matrix/striosome neurons, it is very likely that other cues act
in concert, in particular to enable the preferential homo-adhesion between
striosome neurons, as suggested by previous in vitro studies
(Krushel and van der Kooy,
1993; Krushel,
1989). In this context, an attractive set of cues would be
cadherins, which have previously been shown to be expressed in the developing
striatum (Redies et al.,
2002), and control the compartmentalization of neuronal
populations within the spinal cord (Price
et al., 2002). In addition, other Eph receptors, in concert with
EphA4, could also be involved in homophilic cellular adhesion processes
(Holmberg et al., 2000). Such
a scenario would be reminiscent of other complex systems involving ephrins,
such as the retinotectal system, hindbrain patterning or motoneuron guidance,
where ligands and receptors control differentially the guidance of distinct
neuronal populations through bi-directional signalling
(Eberhart et al., 2004;
Flanagan, 2006;
McLaughlin and O'Leary, 2005;
Pasquale, 2005;
Pasquale, 2008;
Poliakov et al., 2004).
Generating mosaic patterns of cytoarchitecture: the importance of partially overlapping expression of repulsive cues
The patterns of expression of EphA4 and its ephrin ligands in the striatum
may appear to be only partially consistent with a simple model of cell
repulsion. Indeed, their temporal and spatial patterns are not strictly
complementary, contrary to other systems of ephrin/Eph-dependent cell
segregation [such as hindbrain rhombomere formation
(Fig. 7A)
(Wilkinson, 2001)].
Interestingly, the striatal architecture constitutes a unique model of
organization, very distinct from the strictly segmented pattern observed for
hindbrain rhombomeres. Instead, they form compartments that are intermingled
with each other to form a mosaic pattern, and the formation of such patterns
may require the use of partially complementary labels, as recently suggested
by theoretical modelling of ephrin/Eph patterning mechanisms
(Honda and Mochizuki, 2002).
According to these models, if two cellular populations express reciprocally a
ligand and its receptor, following a strict mutually exclusive expression
pattern, they will segregate into two adjacent distinct compartments, similar
to hindbrain rhombomeres, for example (Fig.
7A). Most interestingly, however, if the two populations
co-express the ligand and the receptor but differ in the relative levels of
ligand/receptor in a complementary fashion, then a very different pattern can
be achieved, resembling a mosaic pattern strikingly similar to a
matrix/striosome organization (Fig.
7B).
Such a mixed distribution of guidance cues is highly reminiscent of the expression pattern observed for striatal ephrin/Eph genes, where the two striatal populations express, in a partially complementary pattern, ephrin ligands and the EphA4 receptor. Thus, the fact that each striatal population expresses ligands and receptors, but at different relative levels, might contribute to the formation of a mosaic matrix-striosome organization, as opposed to a strictly segmented structure such as the hindbrain, where cell populations express repulsive cues in a mutually exclusive pattern (Fig. 7A,B).
Intrinsic and extrinsic mechanisms of striatal compartmentalization
The cellular mechanisms orchestrating the compartmentalization of the
striatum remain almost completely unknown. In particular, it has remained
unclear whether the two neuronal populations segregate from each other through
direct interactions, or through the influence of extrinsic factors, such as
the nigrostriatal and corticostriatal projections. Earlier in vivo experiments
tended to minimize the role of the nigrostriatal afferences in the segregation
of the striatal populations (van der Kooy
and Fishell, 1992;
Snyder-Keller, 1991), whereas,
more recently, organotypic assays involving co-cultures of the striatum with
cortex or substantia nigra suggested that the afferents from this structure
had a prominent influence on the emergence of striatal patterning
(Snyder-Keller, 2004;
Snyder-Keller, 2001).
Here, we provide direct in vitro and in vivo evidence that intrinsic cues expressed within the striatum contribute importantly to compartment formation, by a mechanism relying on contact-dependent cell sorting. Indeed, the organotypic assays that we used included striatal tissue only, in absence of nigral or cortical afferents, and yet they allow a faithful recapitulation of the cell sorting that occurs normally in vivo, achieving an M/S density score strikingly similar to the one measured in vivo.
In addition, the pattern of ephrin/Eph expression also suggests that EphA4 and ephrin ligands act, at least in part, locally in the striatum to contribute to the sorting of the two main striatal neuron populations. However, although the phenotype observed in ephrin A5/EphA4 DKOs and EphA4 KOs provides clear indication for a key role of this receptor in striatal patterning, the contribution of ephrin A5 appears to be more limited. Indeed, the absence of any striatal phenotype in the ephrin A5 KO mice strongly suggests that other ligands are involved in the EphA4-mediated neuronal sorting, including ephrin B2/B3. This is also consistent with the expression data, where striatal ephrin A5 expression is only partially complementary to EphA4 expression, with preferential expression in a subset of the striosomes. The requirement for combined signalling involving ephrin A5 and ephrin B proteins is also consistent with the in vitro data, where only the combination of EphA3/B2-Fc inhibitors was able to inhibit efficiently the matrix/striosome sorting.
Finally, other ligands or receptors enriched in the striosomes may be
brought by the afferent innervations, such as the nigrostriatal projections,
as such extrinsic cues would not be detected at the transcript level in the
striatum. Consistent with this hypothesis, earlier studies revealed that
dopaminergic neuron innervation coming from the substantia nigra leads to
increased striatal expression of the EphB1 receptor
(Halladay et al., 2000).
Similarly, it is clear that, as in any other system, there must be genes and
mechanisms other than ephrin/Eph genes involved in this process, which would
include other types of cues, acting locally or from the afferents from the
cortex and substantia nigra. In any case, the identification of the role of
ephrin/Eph signalling in matrix/striosome formation provides an important
framework for identifying these other cues and the pathways involved.
Temporal patterning of guidance cues and spatial patterning
The matrix/striosome compartmentalization relies highly on temporal
patterning, whereby neurons generated at different time-points end up in
different spatial compartments. This is highly reminiscent of the formation of
distinct layers within the cerebral cortex, each of which consists of distinct
populations generated at different time points
(Bayer and Altman, 1991). The
process by which neurons born at a different time end up in different cortical
layers remains unclear, although the reelin signalling pathway has emerged as
an important player in this process
(Tissir and Goffinet, 2003).
Our data provide evidence for a model of spatial segregation that relies on
the temporal control of neuronal guidance cues: it will be interesting to test
whether a similar mechanism may underlie the establishment of other patterned
brain structures.
Implication of matrix/striosome disruption for striatal connectivity and function
Several mouse mutants have been reported to display alterations in the
specification of matrix or striosome neurons, including Mash1
(Ascl1), Notch1 and CTIP2 (Bcl11b) mutant mice,
where early striatal populations are reduced in size or lost
(Arlotta et al., 2008;
Casarosa et al., 1999;
Mason et al., 2005), and
Dlx1/2 and Ebf1 mutants, where the matrix
population is mostly affected (Anderson et
al., 1997; Garel et al.,
1999). However, these mutants display no defect in striatal
compartmentalization per se, so that EphA4 mutants constitute the first model
of selective disruption of the cytoarchitecture of the striatum.
Matrix/striosome organization has been shown to be important for several
aspects of striatal function, but the exact relationships between striatal
function and cytoarchitecture remain poorly known. Ephrin A5/EphA4 mutants
constitute the first example of genetic disruption of the cytoarchitecture of
the striatum, and thereby constitute a unique model with which to test how the
disruption of matrix/striosome compartments might be associated with
abnormalities in connectivity and function of the basal ganglia, and how these
may be related to abnormal behavioural traits.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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