RESEARCH ARTICLE 3281
Development 135, 3281-3290 (2008) doi:10.1242/dev.024778
Temporal regulation of ephrin/Eph signalling is required for
the spatial patterning of the mammalian striatum
Lara Passante1, Nicolas Gaspard1, Mélanie Degraeve1, Jonas Frisén2, Klas Kullander3, Viviane De Maertelaer1
and Pierre Vanderhaeghen1,*
Brain structures, whether mature or developing, display a wide diversity of pattern and shape, such as layers, nuclei or segments.
The striatum in the mammalian forebrain displays a unique mosaic organization (subdivided into two morphologically and
functionally defined neuronal compartments: the matrix and the striosomes) that underlies important functional features of the
basal ganglia. Matrix and striosome neurons are generated sequentially during embryonic development, and segregate from each
other to form a mosaic of distinct compartments. However, the molecular mechanisms that underlie this time-dependent process of
neuronal segregation remain largely unknown. Using a novel organotypic assay, we identified ephrin/Eph family members as
guidance cues that regulate matrix/striosome compartmentalization. We found that EphA4 and its ephrin ligands displayed specific
temporal patterns of expression and function that play a significant role in the spatial segregation of matrix and striosome neurons.
Analysis of the striatal patterning in ephrin A5/EphA4 mutant mice further revealed the requirement of EphA4 signalling for the
proper sorting of matrix and striosome neuronal populations in vivo. These data constitute the first identification of genes involved
in striatal compartmentalization, and reveal a novel mechanism by which the temporal control of guidance cues enables neuronal
segregation, and thereby the generation of complex cellular patterns in the brain.
INTRODUCTION
The striatum is a major structure in the mammalian forebrain,
playing a prominent role in the control of movement and emotions.
It displays a unique mosaic organization, made of two major
compartments that can be identifed neuroanatomically and
neurochemically: the matrix and the striosomes (Gerfen, 1992;
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.,
1
Université Libre de Bruxelles (U.L.B.), IRIBHM (Institute for Interdisciplinary
Research), 808 Route de Lennik, B-1070 Brussels, Belgium. 2Karolinska 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: [email protected])
Accepted 12 August 2008
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 birthdatebased 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.
DEVELOPMENT
KEY WORDS: Forebrain, Neuronal migration, Ephrin, Striatum, Guidance
3282 RESEARCH ARTICLE
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.
MATERIALS AND METHODS
Transgenic mice, breeding and genotyping
Timed-pregnant mice were obtained from Harlan and from local colonies of
mutant and wild-type mice. The plug date was defined as embryonic day
E0.5, and the day of birth as P0. Ephrin A5, EphA4 and ephrin A5/EphA4
double knockout mutants have been described previously (Frisen et al.,
1998; Kullander et al., 2001; Dufour et al., 2003).
Development 135 (19)
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 ttest.
In situ hybridization probes have been described previously (Dufour et al.,
2003; Vanderhaeghen et al., 2000). In situ hybridization using digoxigeninlabeled 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 500⫻103 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 (EphA3Fc, 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
RESULTS
A novel organotypic assay to study striatal
compartmentalization
To address the issue of the cellular and molecular mechanisms of
striatal compartmentalization, we set up a novel organotypic assay
to study in vitro the sorting of striatal neurons. In particular we
sought to recapitulate the temporal pattern observed in vivo, where
neurons born at a different time end up in distinct compartments.
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 GFPexpressing 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
DEVELOPMENT
In situ RNA hybridization
Ephrins pattern the striatum
RESEARCH ARTICLE 3283
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).
When plating E15-LGE derived cells, we found that the GFP+
cells were not distributed uniformly over the striatal surface (Fig.
1C), but rather showed a preference for the DARPP32-negative
matrix compartment (Fig. 1C-H) with a mean M/S value greater
than 1 (1.65±0.106, P<0.001, Student’s t-test). By contrast, when the
same experiment was performed with E12-LGE derived cells, they
were found to settle preferentially in the DARPP32+ striosomal
compartment (Fig. 1I-N), with a mean M/S value less than 1
(0.58±0.061, P<0.001, Student’s t-test), which is very different from
the M/S value observed for the E15-derived cells (P<0.001,
Student’s t-test, Fig. 1O). As a control cell population for
compartment sorting, we used physiologically irrelevant E14 dorsal
thalamic cells in the same striatal overlay assays. Quantification
showed that they were randomly distributed with a mean M/S value
not significantly different from 1 (1.05±0,067, P=403, Student’s ttest).
These results indicate that presumptive matrix- and striosomedestined 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
DEVELOPMENT
Fig. 1. In vitro recapitulation of the striatal
sorting. (A) Schematic model of development of
matrix and striosome compartments in the mouse
striatum. Striosomal neurons (in red) are generated
first and migrate from the lateral ganglionic
eminence (LGE) to the striatal mantle, followed by
matrix neurons (in blue). The two populations
transiently mix within the striatal mantle, then
segregate from each other to form a mosaic of
matrix/striosome compartments. (B) A novel
organotypic assay to recapitulate the sorting of
matrix versus striosome neurons. GFP+ cells are
dissociated from the LGE at distinct embryonic ages
(E12/E15), and plated onto postnatal striatal slices.
The relative distribution of the GFP+ cells is assessed
by an M/S value that represents the ratio of the
densities of cells that settled in the DARPP32negative matrix (M) and DARPP32-positive striosome
(S) compartments. (C-H) Overlay with E15-LGE
derived cells. GFP+ cells preferentially settled in the
DARP32-negative matrix compartment (arrows in FH), while avoiding the DARPP32-positive striosomes
(arrowheads). (I-N) Overlay with E12-LGE derived
cells. GFP+ cells preferentially settled in the DARP32positive striosome compartments (arrowheads).
(O,P) Quantification of the relative distribution of the
GFP+ cells between the striatal compartments.
(O) Schematics of the quantification of
matrix/striosome cell sorting. The number of GFP+
pixels counted in each striosome (S) and within
corresponding areas in adjacent matrix compartment
(M) enable to determine a M/S ratio, reflecting the
relative distribution of the GFP+ cells in each
compartment. (P) E15-LGE and E12-LGE derived cells
display very different M/S values (*P<0.0001),
respectively greater than and less than 1, reflecting
their preference for complementary compartments,
contrary to a control thalamic cell population. Scale
bar: 200 μm.
3284 RESEARCH ARTICLE
Development 135 (19)
temporal pattern of differential expression in early- versus lategenerated striatal neurons at embryonic stages; and/or (2) a spatial
pattern of differential expression within striosome/matrix
compartments at early postnatal stages.
Fig. 2. Temporal and spatial pattern of expression of ephrin/Eph
genes. (A-I) In situ hybridization for EphA4 (A,D,G), ephrin A5 (B,E,H)
and ephrin B2 (C,F,I) in mouse embryos. (A-C) At E12.5, ephrin A5 and
ephrin B2 are expressed in the ventricular zone (VZ), as well as in the
presumptive striatum (Str, arrows in B,C), whereas EphA4 is expressed
only weakly in the VZ (arrows in A). (D-F) At E14.5, ephrin A5 and
ephrin B2 are expressed throughout the striatal mantle. EphA4
expression also starts to be detected, although mostly remains in the
VZ. (G-I) At E16.5, the striatum is diffusely stained for both ephrin
ligands and for EphA4. (J-O) In situ hybridization for EphA4 (J,M) and
ephrin A5 (K,N) and ephrin B2 (L,O), at peri-natal stages. EphA4
receptor displays a matrix-like pattern of expression surrounding
patches of negative expression (arrows in M), ephrin A5 is expressed in
a progressively more restricted pattern, resulting in a distribution in
patches (arrows in N), whereas ephrin B2 is expressed in a diffuse and
progressively weaker pattern.
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
DEVELOPMENT
Differential temporal and spatial patterns of
expression of ephrin/Eph genes in the developing
striatum
Given their prominent role in compartmentalization in the hindbrain,
ephrin/Eph genes constituted logical candidates in our search for
matrix/striosome-sorting factors. The EphA4 receptor has
previously been shown to be expressed in the matrix compartment
at postnatal stages [P6 (Janis et al., 1999)], 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 striosomedestined 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 earlygenerated 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
Ephrins pattern the striatum
RESEARCH ARTICLE 3285
Fig. 3. Matrix/striosome pattern of expression of ephrin A5 and
EphA4. (A-C,G-I) Comparison between EphA4 and DARPP32
distribution on alternate sections of P2 (A-C) and P4 (G-I) mouse
striatum. EphA4 expression (determined by in situ hybridization) was
converted into false green (A,G) and DARPP32 immunostaining into
false red (B,H). Overlays (C,I) reveal that the majority of the EphA4negative patches appear positive for DARPP32 (arrowheads in C,I),
indicating that the EphA4 receptor strictly belongs to the matrix
compartment. (D-F,J-L) Comparison between EphA4 and ephrin A5
staining on alternate sections. EphA4 expression was converted into
false green (D and J) and ephrin A5 expression into false red (E,K).
Overlays (F,L) reveal a partially complementary pattern, with ephrin A5
being preferentially expressed in a subset of EphA4-negative patches,
particularly in the centre of the striatum (arrowheads in F and L), but
also in the EphA4+ compartment, and progressively downregulated
between P2 and P4.
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.
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), preincubation 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 ttest), 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.
DEVELOPMENT
Altogether, these results suggest a complex and highly dynamic
spatiotemporal pattern, where ephrin A5/B2 levels of expression are
highest in the early striatal population and EphA4 levels are highest
in later-generated population at embryonic ages. At early postnatal
stages, EphA4 is expressed at high levels and is mostly restricted to
the matrix compartment, whereas ephrin A5 retains a partially
complementary pattern and ephrin B2 becomes gradually
downregulated. This distinctive temporal and spatial pattern of
distribution in the developing striatum is compatible with an
involvement of EphA4-ephrin signalling in the control of
matrix/striosome compartmentalization. Specifically, it leads to the
hypothesis that the differential, partially complementary, expression
of EphA4 and ephrin ligands by the two striatal populations could
trigger a bi-directional repulsive guidance response that would then
cause the sorting of the matrix and striosome neurons and, as a
consequence, the segregation of the two striatal populations into
distinct compartments.
Fig. 4. Ephrin/Eph inhibitors disrupt the sorting of the matrix and
striosomal neurons in vitro. (A-F) When mouse E16 LGE-derived cells,
enriched for the matrix neurons, are overlaid on postnatal striatal slices
preincubated with control Fc reagents (A-C) they display a preference for
the DARPP32-negative matrix compartment (arrows). This preference is
partially lost when slices are preincubated with soluble ephrin inhibitors
EphA3-Fc/EphB2-Fc (D-F), revealing more GFP+ cells in DARPP32-positive
compartments (arrowheads). (G-L) When E12-LGE derived cells, enriched
for the striosome neurons are overlaid on postnatal striatal slice
preincubated with control Fc reagents (G-I), they display a preference for
the DARPP32-positive striosome compartments (arrowheads). This
preference is partially lost when slices are preincubated with soluble
ephrin inhibitors (J-L), revealing more GFP+ cells in DARPP32-negative
compartments (arrows). (M) Quantification of the relative densities of
GFP+ cells in matrix (M) and striosome (S) compartments. Treatment
with ephrin soluble inhibitors results in a decrease of matrix preference
for E16 cells, and conversely a decreased striosome preference for E12
cells (*P=0.001, **P=0.0047). Scale bar: 100 μm.
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
Development 135 (19)
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).
To determine whether the compartment formation was affected in
the mutant mice, we again took advantage of the temporal pattern of
matrix/striosome formation. We treated embryos with BrdU pulses
at embryonic days E16 and E17 to label a majority of endogenous
matrix neurons, then sacrificed the newborn pups at P2 and
performed a double immunofluorescence against BrdU and
DARPP32, in order to visualize the matrix neurons and the
striosomal compartments, respectively.
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
DEVELOPMENT
3286 RESEARCH ARTICLE
Ephrins pattern the striatum
RESEARCH ARTICLE 3287
Fig. 5. Altered vivo striatal patterning in ephrin
A5/EphA4 mutants. (A-C) When injected with BrdU at
E16/E17 and sacrificed at P2, control animals (double
heterozygotes for ephrin A5 and EphA4, n=9) display a matrixlike distribution of BrdU. (D-F) Ephrin A5 KO animals (n=5)
display a similar distribution of BrdU+ neurons. (G-I) EphA4 KO
animals (n=4) display a much more uniform distribution of the
BrdU+ matrix neurons across the striatum and more diffuse
DARPP32 staining. In addition, many more BrdU+ neurons are
found at ectopic locations within DARPP32-positive striosomes.
(J-L) Ephrin-A5/EphA4 DKO animals (n=6) show an almost
completely uniform distribution of the BrdU+ matrix neurons
across the striatal mantle (J), a more diffuse DARPP32 staining
(K) and numerous ectopic BrdU+ matrix neurons within the
striosomes (inset in L). Arrowheads show DARPP32+ positive
striosomes. Scale bar: 100 μm.
These data thus indicate that EphA4 signalling is a major player
in vivo for the normal segregation between the two striatal
populations and, as a consequence, for the correct formation of the
striatal compartments during development.
DISCUSSION
The functions of the mammalian striatum rely on its mosaic
compartmentalized structure, including the matrix and striosome
neurons (Gerfen, 1992; 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).
DEVELOPMENT
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).
3288 RESEARCH ARTICLE
Development 135 (19)
Fig. 6. EphA4 signalling is required for striatal neuron sorting in
vivo. The mean M/S values determined for control (n=9), ephrin A5 KO
(n=5), EphA4 KO (n=4) and ephrin-A5/EphA4 DKO (n=6) littermates
reveal an abnormal, more even distribution of presumptive matrix
neurons in EphA4 KO and DKO genotypes, whereas their distribution in
ephrin A5 KO remains similar to controls.
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
Fig. 7. Schematic comparison of cell sorting processes occuring
during hindbrain segmentation and striatal
compartmentalization. (A) In the developing hindbrain, neural cells
generated from adjacent rhombomeric segments express repulsive
ephrin ligands (in red) or Eph receptors (in green) in a mutually exclusive
pattern. This allows a strict restriction of cell intermingling that results
in the characteristic segmented pattern of the rhombomeres. (B) In the
developing striatum, striosome neurons (S) are generated first (t1) and
express high levels of ephrin ligands (in red) and low levels of Eph
receptors (in green). Matrix neurons are generated later (t2) and express
high levels of receptors and low levels of ligands. Matrix and striosome
cells first intermix and then partially segregate, through the effects of
bidirectional interactions, to form the mature mosaic pattern of the
striatum (t3). See text for further discussion.
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
DEVELOPMENT
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.
This model thus strongly implies the involvement of bidirectional signalling, mediated not only by Eph receptors (forward
signalling) but also by ephrin ligands (reverse signalling), in
accordance with our in vitro and in vivo data, where disruption of
ephrin/Eph signalling also results in the mislocalization of matrix
and striosome neurons.
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 homoadhesion 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).
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; SnyderKeller, 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 contactdependent 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.
RESEARCH ARTICLE 3289
Temporal patterning of guidance cues and spatial
patterning
The matrix/striosome compartmentalization relies highly on
temporal patterning, whereby neurons generated at different timepoints 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.
We thank Gilbert Vassart for continuous support and interest, members of the
laboratory and IRIBHM for helpful discussions and advice. We are grateful to
Jean-Marie Vanderwinden for precious help with confocal microscopy, and to
Franck Polleux for expert advice on overlay assays. This work was funded by
grants from the Belgian FNRS and FRSM, by the Belgian Queen Elizabeth
Medical Foundation, by a UCB Neuroscience Award, by the Action de
Recherches Concertées (ARC) Programs, by the Interuniversity Attraction Poles
Program (IUAP), by the Belgian State Federal Office for Scientific Technical and
Cultural Affairs, and by the Programme d’Excellence CIBLES of the Walloon
Region. P.V. is a Senior Research Associate of the FNRS, and N.G. and L.P. were
funded as Research Fellows of the FNRS. K.K. is a Royal Swedish Academy of
Sciences Research Fellow supported by a grant from the Knut and Alice
Wallenberg Foundation.
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