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The Journal of Neuroscience, February 1, 2000, 20(3):1109-1118
The Lack of Emx2 Causes Impairment of
Reelin Signaling and Defects of Neuronal Migration in
the Developing Cerebral Cortex
Antonello
Mallamaci1,
Sara
Mercurio1,
Luca
Muzio1,
Chiara
Cecchi1,
Celia Leonor
Pardini1,
Peter
Gruss2, and
Edoardo
Boncinelli1, 3
1 Department of Biological and Technological Research
(DIBIT), Istituto Scientifico H. San Raffaele, 20132 Milano, Italy,
2 Max-Planck Institute of Biophysical Chemistry,
37018 Goettingen, Germany, and 3 Molecular and Cellular
Pharmacology, Consiglio Nazionale delle Ricerche, 20129 Milano,
Italy
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ABSTRACT |
Neocorticogenesis in mice homozygous for an Emx2
null allele is the topic of this article. The development of both main
components of neocortex, primordial plexiform layer derivatives and
cortical plate, was analyzed, paying special attention to radial
migration of neurons forming the cortical plate. The products of the
Reelin gene, normally playing a key role in
orchestrating radial migration of these neurons, display normal
distribution at the beginning of the cortical neuronogenesis but are
absent in the neocortical marginal zone of the mutant mice at the time
when the cortical plate is laid down. As a consequence, the development
of radial glia is impaired, and neurons making up the cortical plate
display abnormal migration patterns. In addition, restricted defects
along the rostrocaudal and the mediolateral axes are present in the subplate, suggesting an Emx2-specific role in priming
the proper development of this layer.
Key words:
neocortex; Emx2; Cajal-Retzius cells; reeler; radial glia; subplate
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INTRODUCTION |
Emx2 is a vertebrate
homeobox gene related to the Drosophila gap gene
ems (Dalton et al., 1989 ). It was originally found in mouse
and in man (Simeone et al., 1992a,b) and subsequently isolated in
chicken (A. Mallamaci, unpublished results), frog (Pannese et al.,
1998), and fish (Morita et al., 1995; Patarnello et al., 1997). Its
expression in the anterior CNS of the developing mouse embryo has been
extensively described (Simeone et al., 1992a,b; Gulisano et al., 1996;
Mallamaci et al., 1998). Emx2 has been knocked-out in mice
by homologous recombination in embryonic stem (ES) cells.
Homozygous mutant mice die perinatally, probably because of the absence
of kidneys. The archicortex of these animals is heavily affected; the
dentate gyrus is missing, and the hippocampus and the medial limbic
cortex are greatly reduced in size. The olfactory bulb is also
disorganized, and the olfactory nerve fails to project to it.
Lamination defects have been reported to occur in the neocortex
(Pellegrini et al., 1996; Yoshida et al., 1997).
It has been suggested that Emx2 plays an important role in
the formation of the abstract areal code specifying the dorsal telencephalon regional identity (Simeone et al., 1992b; Shimamura et
al., 1995; Yoshida et al., 1997). Ablation of this early basic function
could account for some of the most relevant features of the mutant
mice, such as defects in the limbic area (Yoshida et al., 1997).
Additionally, more specific functions have been proposed to be acted by
Emx2 later in development. Their impairment might result in
more subtle traits of the mutant phenotype, e.g., poorly characterized
neocortical lamination defects (Yoshida et al., 1997). This late
contribution of Emx2 to neocortical development was the main
object of our study.
The formation of the cerebral cortex is a biphasic process. Early
postmitotic neurons accumulate at the marginal edge of the cortical
wall, forming the primordial plexiform layer (PPL). Then, later born
neurons climb along fascicles of radial glia and infiltrate the PPL.
They split it into the more superficial marginal zone (MZ) and the
deeper subplate (SP) and accumulate between them, making up the
cortical plate (CP) (Marin-Padilla, 1978). Careful analysis of the
development of both PPL derivatives and CP in mutant embryos allowed us
to clarify the key role played by Emx2 in orchestrating the
process of radial migration, as well as in priming the development of
the SP.
Hints about a possible association between Emx2 and neuronal
migration recently came from the finding of large amounts of EMX2
protein in Cajal-Retzius (CR) cells in the MZ of late gestation mouse
embryos (Mallamaci et al., 1998). CR cells are among the most relevant
players in orchestrating the radial migration of CP neurons
(Marin-Padilla, 1988, 1998; Del Rio et al., 1995), partly through the
protein product of the Reelin gene (Reln)
(D'Arcangelo et al., 1995, 1997; D'Arcangelo and Curran, 1998; Ogawa
et al., 1995), and it is conceivable that EMX2 protein is necessary to allow CR cells to perform this function. As expected, we found that
late gestation Emx2 / embryos lacked
Reelin mRNA in their MZ and consequently displayed severe
impairment of neuronal radial migration.
The SP is a heterogeneous cortical layer (Antonini and Shatz, 1990),
critical for the proper development of the cerebral wiring. It is
involved in establishing transient, both corticopetal and cortifugal
initial connections, which forerun and allow the proper development of
the permanent ones (Ghosh, 1995 and references therein). Very little is
known about the genetic control of the SP development at the moment.
Interestingly, we found that specific neuronal subpopulations of the
SP, which normally does not express Emx2, are selectively
affected in Emx2/ embryos at axial
locations just corresponding to areas of the ventricular zone (VZ) that
normally express Emx2 at the highest levels. This suggested
that Emx2 could play an essential, specific role in priming
the proper development of these neurons.
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MATERIALS AND METHODS |
Animal husbandry, bromodeoxyuridine labeling, and embryo
harvesting. Emx2/+ female
mice of mixed C129Sv/J-C57Bl6 genetic background (Pellegrini et al.,
1996) were mated overnight with
Emx2/+ males of similar genetic
background and inspected at 9.00 A.M. on the following day for the
presence of vaginal plug; noon of this day was assumed to correspond to
embryonic day 0.5 (E0.5). In a similar way, C57Bl6 mice heterozygous
for the Reeler Edimburgh null allele were intercrossed. When
appropriate, at selected times, pregnant dams were injected
intraperitoneally with 100 µg of bromodeoxyuridine (BrdU) per gram of
body weight. At scheduled times, pregnant females were anesthetized by
CO2 and killed by cervical dislocation.
Embryos were harvested, genotyped according to Pellegrini et al. (1996) and D'Arcangelo et al. (1995), and further processed.
Immunohistochemistry. Embryos were treated in two different
ways depending on the kind of analysis to be performed. In the case of
immunohistochemistry with the monoclonal antibody against the glial RC2
epitope, they were embedded fresh in OCT, frozen on dry ice, and cut by
cryostat at 50 µm. Sections were mounted on Fischer SuperFrost Plus
slides, air dried for 30 min at room temperature (RT), and
stored at  80°C. Subsequently, they were washed in PBS and
post-fixed in absolute MeOH at 20°C for 8 min. Slides were
rehydrated at RT by descending methanolic series and finally processed
for immunohistochemistry. In the case of other immunohistochemistries,
embryos were fixed upon harvesting in paraformaldehyde 4% PBS
overnight at +4°C and then washed, dehydrated, and embedded in wax
according to standard protocols. In these cases, embryos were
subsequently cut at 10 µm by a microtome, and sections were mounted
on Fischer SuperFrost Plus slides. Subsequently, samples were dewaxed
by xylene, rehydrated in descending ethanolic series, and further
processed. Specific treatments preceded the detection of some antigens.
Before  -EMX1 immunohistochemistry, the antigen was unmasked by
boiling samples in 10 mM sodium citrate, pH 6.0, for 5 min and allowing them to cool down slowly. In the case of BrdU
detection to depurinate genomic DNA and make the epitopes accessible,
slides were kept in 2 M HCl for 30 min at 60°C
and then neutralized in 0.1 M borate buffer, pH
8.5, for 15 min at RT. In general, subsequent steps of
immunohistochemistry were performed according to Mallamaci et al.
(1998). The following primary antibodies were used: -BrdU, mouse
monoclonal (Becton Dickinson, Mountain View, CA), 1:100;
-calretinin, rabbit polyclonal (Swant), 1:100; -EMX1, rabbit
polyclonal (Briata et al., 1996), 1:400; -neuron-specific class III
 -tubulin, mouse monoclonal (clone TuJ1; BabCo, Richmond, CA), 1:100;
-microtubule-associated protein 2 (MAP2), mouse monoclonal
(clone AP20; Boehringer Mannheim, Indianapolis, IN) 1:100; and -RC2,
monoclonal, 1:1 (Misson et al., 1988).
In situ hybridization. Radioactive and nonradioactive
in situ hybridizations were performed according to Gulisano
et al. (1996) and Bovolenta et al. (1997), respectively. For the
detection of 0ct6, an Oct6 radiolabeled probe
corresponding to the entire coding region (Meiyer et al., 1990) was
used. For Reln, a Reln antisense digoxygenated
probe corresponding to nucleotides 5818-5973 (D'Arcangelo et al.,
1995) was used.
EMX1 cell counting. Three pairs of
Emx2/ and wild-type E19.0 embryos were
frontally sectioned at 10 µm, and for each embryo, every 13th section
was kept. For each section, the cortical sector was partitioned in 16 equally spaced bins, which were numbered from ventricular to marginal;
each bin was further divided in two hemibins, one medial (M) and one
lateral (L), by the radial line intersecting the corticostriatal notch.
Heavily -EMX1-reactive cells located in hemibins 5, 6, and 7 M and
5, 6, and 7 L were counted by two different operators. Data were
averaged, and the total numbers of cells located in all 5, 6, and 7 M
and all 5, 6, and 7 L hemibins of
Emx2/ and wild-type embryos were
calculated and compared.
Graphics. BrdU-labeled cells were counted as explained in
the legend of Figure 4. Graphics were generated on a MacIntosh G3 computer (Apple Computers, Cupertino, CA) by Microsoft (Seattle, WA)
Excel software and processed by Adobe Photoshop 5.0 software (Adobe
Systems, San Jose, CA).
Photography. Photos were taken by a Nikon (Tokyo, Japan)
Eclipse 600 microscope and an SV MICRO CV3000 digital microscope camera. Electronic files were processed on a MacIntosh G3 computer by
Adobe Photoshop 5.0 software.
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RESULTS |
Development of marginal zone and subplate
The development of the PPL derivatives, MZ and SP, was analyzed by
scoring the expression of several specific markers: among them, CaR,
EMX1, and Reln mRNA.
At E15.5, the Ca-binding protein calretinin (CaR) is normally
restricted to CR cells of layer I and to a cellular subpopulation of
the SP (Fig. 1a-c). No
-CaR immunoreactivity was detected in the MZ of mutant embryos,
except for a very few labeled cells located in the medialmost part of
the mantle, in the presumptive cingulate cortex and hippocampal anlage
(Fig. 1d-f). The intermediate, SP-associated CaR
stripe, normally detectable along the entire rostrocaudal length of the
developing cortex (Fig. 1a-c), was restricted in the mutant
animals to the rostralmost pallium, being absent at the level of the
foramen of Monro and more posteriorly (Fig. 1d-f).
Similar differences were found between wild-type and mutant animals at
approximately the end of the gestational life (data not shown).
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Figure 1.
CaR and EMX1 in the telencephalon of wild-type and
Emx2/ late gestation embryos.
a-f, Here is reported the distribution of CaR protein
on frontal rostral (a, d), intermediate
(b, e), and caudal (c,
f) sections of E15.5 wild-type
(a-c) and Emx2/
(d-f) telencephalons. Medial is to the
right. In wild-type embryos, CaR is detectable in septum
(a), cingulate cortex (a),
and presumptive hippocampus (b, c). CaR
is also present in the neocortex throughout its entire anteroposterior
length. Here, it is distributed in two bands, corresponding to subplate
and marginal zone (a-c).
Emx2/ embryos express CaR in septum,
cingulate cortex (d, arrows), and, to
lesser extent, in the hippocampal anlage (e,
f, arrows). In their neocortex, CaR
expression is confined to the rostralmost subplate (d,
arrowheads); no signal can be detected in the midcaudal
subplate (e, f) or in the entire
marginal zone (d-f). g,
h, Distribution of EMX1 on midfrontal sections of E19
wild-type (g) and
Emx2/ (h)
cerebral cortexes. Medial is to the right. In both
cases, the protein is scarce in the ventricular zone and abundant in
the deep part of the transitional field (g,
h). In the wild-type cortex, a row of heavily labeled
cells is detectable in the subplate, extending from the lateral cortex,
which overlies the striatum up to the medial wall of the telencephalon
(g, arrowheads). In the mutant
animals, similarly labeled cells can be found at the same depth;
however, they are much less numerous and are prevalently restricted to
the lateralmost part of the wall (h,
arrowheads). Plenty of weakly labeled cells,
approximately arranged in two broad stripes, can be found in the
cortical plate of wild-type embryos (g); only a
few lightly stained cells can be detected at corresponding locations in
mutant brains (h). AH, Anterior
hippocampus; CC, cingulate cortex; CP,
cortical plate; DG, dentate gyrus; HI,
hippocampus; MZ, marginal zone; PH, posterior
hippocampus; SE, septum; SP (or
SbP), subplate; ST, striatum;
TF, transitional field; VZ, ventricular
zone. Scale bar, 100 µm.
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In E19.0 wild-type embryos, -EMX1 stains three main cortical
domains. Plenty of lightly labeled cells are in the CP in which they
form two broad and quite distinct bands. Numerous immunoreactive cells
lie in the transitional field (TF), displaying a graded signal, higher
medially than laterally. And finally, a very thin band, formed by
heavily stained cells, can be easily distinguished in the SP (Fig.
1g, arrowheads). Few EMX1-positive cells were present in the Emx2/ CP; conversely,
in the Emx2/ TF, the graded expression
of the protein was retained (Fig. 1h). Remarkably, less
heavily -EMX1-immunoreactive cells were detectable in the
Emx2/ SP and were mainly
restricted to the lateralmost part of the telencephalic wall (Fig.
1h, arrowheads). In knock-out embryos, the total
number of these cells was reduced to 40% compared with wild-type
animals. This reduction was not uniform. Cells located laterally to the corticostriatal notch were reduced to 65%, and those located medially to this notch were reduced to ~25% (data not shown).
In the cerebral cortex of wild-type embryos, since E11.5 and up to
birth, Reln mRNA is specifically detectable in CR cells, located in the MZ just underneath the pia and spread along the entire
cortex (Fig.
2a,c,e-g,k);
starting from E19, additional small Reln-expressing cells
can be found in the deep CP (Fig. 2k). The distribution of
Reln-expressing cells in E11.5
Emx2/ embryos basically paralleled
that of wild-type ones (Fig. 2b). Differences between
knock-out and wild-type animals were detectable starting from E13.5. At
this stage, in mutant animals, both lateral neocortex and presumptive
paleocortex overlying the ganglionic eminence were almost completely
free of any Reln RNA, whereas plenty of tightly clustered
Reln-expressing cells could be detected in the MZ of the
medial cortical primordium (Fig. 2d). At E15.5, differences
were even more pronounced. At this stage, only a few Reln-expressing cells were present in the cortical MZ of
Emx2/ embryos, reproducing the
distribution of -CaR-immunoreactive cells in the same animals (Fig.
2h-j). Reln-expressing cells were restricted to
septum, cingulate cortex, and hippocampal anlage, whereas in neocortex
they were absent. Ectopic, scattered Reln mRNA-positive
cells were found in the TF of mutant embryos, being more numerous
caudally than rostrally (Fig. 2h-j). Similar distribution of Reln-expressing cells was also found in the MZ of E19.0
Emx2/ embryos. In these animals, like
in wild-type ones at the same stage, it was possible to detect plenty
of cortical nonmarginal small Reln-expressing cells;
however, differently from wild-type ones, these cells were not
clustered in a narrow radial band but were dispersed throughout the
entire width of the cerebral wall (Fig. 2l).
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Figure 2.
Reln mRNA in the telencephalon of
wild-type and Emx2/ embryos.
a-d, Reln mRNA on midfrontal sections of
E11.5 (a, b) and E13.5 (c,
d) wild-type (a, c) and
Emx2/ (b,
d) embryos. In wild-type embryos of both stages,
Reln is expressed in a ring of cells located in the
marginalmost part of the telencephalic wall, including the ganglionic
eminence. In Emx2 knock-out mice, the distribution of
Reln-positive cells, basically normal at E11.5
(b, arrowheads), looks substantially
altered at E13.5 (d); at this stage, almost no
Reln-positive cell is detectable in the lateral
presumptive cortex, except a few of them located above the ganglionic
eminence (d, arrowheads), whereas plenty
of them are tightly clustered in the marginal medial cortical wall
(d, arrows). e-j, Here is
reported the distribution of Reln mRNA on frontal
rostral (e, h), intermediate
(f, i), and caudal
(g, j) sections of E15.5 wild-type
(e-g) and Emx2/
(h-j) telencephalons. Medial is to the
right. In wild-type embryos, marginal rings of
Reln-expressing cells can be detected at all
rostrocaudal levels (e-g); specific clustering of these
cells takes place in the hippocampus (g). The
telencephalon of Emx2/ embryos is
severely deprived of Reln mRNA. In these animals,
Reln-positive neurons are still detectable in the
marginal zone of septum (h, small black
arrow), cingulate cortex (h, i,
open arrow), and hippocampus (j,
large black arrow), but almost completely not in the
neocortical marginal zone (h-j). Ectopic, scattered
positive cells can be found in the developing neocortex, more numerous
at the caudalmost levels (h-j,
arrowheads). k, l,
Reln mRNA on midfrontal sections of neocortices of E19.0
wild-type (k) and
Emx2/ (l)
embryos. Large Reln-positive Cajal-Retzius neurons can
be found in the marginal zone of the wild-type cortex
(k, white arrows) but not in the
corresponding layer of the mutant one (l). Small
labeled cells are in the outer half of the wild-type cortical plate
(k, white arrowheads); small
Reln-positive cells are irregularly spread throughout
the entire width of the mutant cortex (l, white
arrowheads). CC, Cingulate cortex;
CP, cortical plate; CX, cortex;
GE, ganglionic eminence; HI, hippocampus;
MZ, marginal zone; NC, neocortex;
SE, septum; SP, subplate;
TF, transitional field; VZ, ventricular
zone. Scale bar, 100 µm.
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In summary, in late Emx2/ embryos, CaR
and Reln mRNA were absent in the entire neocortical MZ, CaR
was undetectable in the caudal SP, and EMX1 was prevalently reduced in
the medial SP. Downregulation or disappearance of these markers could
be consequence of the absence of cells normally expressing them;
alternatively, it could arise from abnormal behavior of these cells.
This argument will be analyzed further in Discussion.
Radial migration of neurons belonging to the cortical plate
The finding that late Reln expression in the
neocortical MZ of Emx2/ mice was
absent strengthened our expectation that radial migration of late born
neurons toward the CP could be deeply perturbed in these mice, possibly
in a reeler-like way. It was for this reason that we decided
to study neuronal radial migration in the neocortex of Emx2
mutant mice.
This problem was first approached by monitoring the distribution of two
molecules: the neuron-specific class III -tubulin and
Oct-6 mRNA.
The monoclonal antibody TuJ1 selectively recognizes the neuron-specific
class III -tubulin, one the early markers expressed by postmitotic
neuronal cells (Lee et al., 1990; Easter et al., 1993). In E15.5
wild-type animals, it stains the layers of the cerebral cortex from the
subventricular zone (SVZ) to the MZ, with different intensities; almost
no reactivity can be found in the VZ (Fig.
3c,e). In the case
of Emx2/ embryos, numerous
TuJ1-positive cells were present in the cortical VZ; the TF displayed a
conspicuous accumulation of intensely immunoreactive cells (Fig.
3d, f) that was absent in wild-type
embryos. Even if we cannot rule out alternative explanations (for
example, accumulations of TuJ1 in the SVZ could correspond to neurons
tangentially migrating from the ganglionic eminence to the cortex; see
Anderson et al., 1997 and references therein), it is conceivable that
at least ectopic TuJ1-positive cells found in the VZ could include
neuronal cells that locally came out of the mitotic cycle and were
migrating, slower than normal, from the VZ toward their final radial
location.
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Figure 3.
Neuron-specific class III -tubulin and
Oct6 mRNA in the telencephalon of wild-type and
Emx2/ late gestation embryos.
a, b, Distribution of Oct6
mRNA in the telencephalon of E16.0 wild-type (a)
and Emx2/ (b)
embryos; frontal sections, slightly caudal to the level of the foramen
of Monro. Medial is to the right. In both cases, the RNA
is in the developing striatum, as well as in the pallium. In the
cerebral cortex, the signal is detectable throughout the transitional
field. It gives rise to an very intense band in the outer part of this
layer, just underneath the subplate (a,
b). In the outer lateral cortical plate of the wild-type
cortex, an additional thin Oct6 band can be found,
possibly corresponding to the first neurons belonging to the
prospective layer V, which overcame the subplate and settled at their
final radial level (a, white arrowheads).
No trace of this additional signal can be detected at corresponding
locations in the Emx2/ brain
(b). c-f, Distribution of the
neuron-specific class III -tubulin on middle frontal sections of
dorsal telencephalon in wild-type (c) and
Emx2/ (d)
embryos, revealed by the monoclonal antibody TuJ1. In e
and f, magnifications of boxed areas of
c and d are represented, respectively. In
normal embryos, immunoreactivity is detectable throughout the entire
width of the cortical wall, except the ventricular and the inner
subventricular zones (c, e). In
Emx2/ animals, several scattered
TuJ1-positive cells can be found in the ventricular zone, and numerous
clustered immunoreactive cells lie around the border between
subventicular and intermediate zones (d,
f). CP, Cortical plate;
HI, hippocampus; IZ, intermediate zone;
MZ, marginal zone; SP, subplate;
ST, striatum; SVZ, subventricular zone;
TF, transitional field; VZ, ventricular
zone. Scale bar, 100 µm.
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Oct-6 has been described to be specifically expressed in the
rat by neurons of layer V, from the middle of their migration up to the
adult stage (Frantz et al., 1994). At E15.5, in both Emx2/ and wild-type mouse embryos,
Oct-6 RNA was detectable throughout the TF, especially in
the outer part of this layer (Fig. 3a,b). The
wild-type cortex displayed an additional Oct6 signal in the outer lateral CP, possibly corresponding to the first neurons belonging
to the perspective layer V, which overcame the SP and settled at their
final position (Fig. 3a). No trace of these positive cells
could be found in Emx2/ brains (Fig.
3b), in agreement with the hypothesis of a possible impairment of neocortical radial migration.
To systematically compare the migratory behavior of cells fated to form
the neocortex in Emx2 null versus Emx2 wild-type
embryos, we pulse-labeled neurons born at E12.0, E13.5, and E15.0 by
BrdU and scored their radial distributions through the neocortical wall
of E19 Emx2/ and wild-type
embryos (Fig. 4a-h). Data
were collected, processed, and graphically synthesized as explained in
the legend of Figure 4. Reln/ embryos
were included in this analysis as well, as useful paradigms.
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Figure 4.
Radial distributions of E12.0, E13.5, and E15.0
BrdU pulse-labeled cells in the neocortex of E19 wild-type,
Emx2/, and
Reln/ mice. a-h,
Photographic examples of radial distribution of BrdU on midfrontal
neocortical sectors of E19 wild-type (a,
c, e) and
Emx2/ (b,
d, f) mouse embryos, pulse-labeled
at E12.0 (a, b), E13.5 (c,
d), and E15.0 (e,
f); in g and h,
there are magnifications of boxed areas in
a and b, respectively.
i-k, Graphic synthesis of radial distributions of
E12.0, E13.5, and E15.0 BrdU pulse-labeled cells in the neocortex of
E19 wild-type (i),
Emx2/ (j),
and Reln/ (k)
mice. Mice embryos were pulse-labeled at E12.0, E13.5, and E15.0 and
harvested at E19.0. For each genotype and each labeling time, four animals were analyzed. Telencephalons were
frontally sectioned, and the distribution of BrdU-labeled cells was
scored in neocortical sectors at five standard rostrocaudal levels:
anterior edge of the corpus callosum, rostral edge of the foramen of
Monro, caudal edge of the foramen of Monro, anterior hippocampus, and
posterior hippocampus. At each level, the neocortical sector was
partitioned in 16 bins of equal radial width, which were numbered from
ventricular to marginal, and the percentage of immunoreactive cells
located in each bin was calculated. For all animals of a given genotype
and BrdU-pulsed at a given time, we integrated data relative to these
five rostrocaudal levels and represented the radial distribution of
labeled cells by plotting the average percentage of labeled cells
located in each bin against the bin number. Three triplets of colored
graphs were obtained: green was used for representing
E12.0 born cells, blue for E13.5 cells, and
red for E15.0 cells. Finally, for each genotype, the
three graphs were superimposed onto the same Cartesian plane, and the
three pictures in the figure were obtained. In E19.0 normal embryos
(i), the graph referring to E12.0 born neurons
gave rise to two peaks, a sharp marginal one corresponding to
Cajal-Retzius cells, and a smooth intermediate one at the level of the
SP. In the same embryos, both E13.5 and E15.0 graphs display one peak,
falling between the two E12.0 peaks; the E15.0 peak is marginal to the
E13.5 peak. In the case of Emx2/
embryos (j), the E12 graph gave rise only to a
smooth peak, lying at the presumptive SP level; no trace of the normal
marginal peak was detectable. In the same mutant embryos, considerable
fractions of E13.5 and E15.0 born cells are located marginally to the
smooth intermediate E12.0 peak. However, both E13.5 and E15.0 peaks are
displaced toward the ventricular side of the x-axis, and
the E13.5 and E15.0 populations are largely intermingled. In
Reln/ mice
(k), all E12.0 born cells are clustered
underneath the pia. All E13.5 and E15.0 born neurons lie deep to E12.0
born cells and are abundantly intermingled. Scale bar, 100 µm.
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In E19.0 normal embryos, E12.0 born neurons were clustered at two main
radial levels. Approximately one-fourth of them, including presumptive
Cajal-Retzius cells, lay underneath the pia mater; the rest was loosely
clustered at the level of the SP. E13.5 and E15.0 born cells were
located between the two main clustering levels of E12 born cells, and
the E15 cohort lay prevalently superficial to the E13.5 one (Fig.
4a,c,e,i). In brief, as
described previously, the neocortex is built-up biphasically and the CP
is laid down according to the so-called "inside-out" rule
(Marin-Padilla, 1978, 1998, and references therein; Bayer and Altmann,
1991).
In E19.0 Emx2/ embryos, the majority
of E12 born cells lay around the putative SP level (Fig. 4b,
j), and their presumptive SP laminar identity was confirmed
by colocalizing CaR with BrdU in a subset of them (data not shown);
almost no E12 born cell were detectable at the marginal edge of the
cortical wall (Fig. 4b,h, j). In the
same embryos, both E13.5 and E15.0 born cells were scattered throughout
the entire neocortical wall. A fraction of E13.5 and E15.0 cohorts lay
marginally to the SP E12.0 cohort; however, the average distance
between E13.5 and E15.0 born cells and the VZ was reduced, E13.5 and
E15.0 populations were much more intermingled, and the
inside-out rule was hardly followed (Fig. 4d,
f, j).
This picture is somehow reminiscent of the reeler mutant
cerebral cortex (Falconer, 1951; Caviness et al., 1988).
However, a major difference takes place between Emx2
knock-out and reeler mice. In reeler mutants, CP
neurons are not able to penetrate the preplate, which does not get
split and gives rise to the so-called superplate (Fig. 4k).
In Emx2/ embryos, CP neurons partially
retain this ability so that a subplate can be distinguished from the
subpial layer (Fig. 4b, j).
Radial glia and migrating neurons in the mutant cortical plate
In normal mouse embryos, regularly spaced radial glial bundles
span from the ventricular to the marginal edge of the cortical wall
since the very beginning of its development, and radially elongated
neurons climb along the surface of these bundles during their
translocation from the VZ up to the CP (Rakic et al., 1974; Caviness et
al., 1988). At approximately E16.5, the average diameter of glial
bundles varies dramatically around the SP (Fig.
5a) as a consequence of the
defasciculation process that normally occurs to radial glia at the
level of the CP, between E15 and E17 in the mouse. This phenomenon
consists in the separation between the 3-10 single cell fibers forming
each primary glial fascicle and is associated with the penetration of
each primary fascicle by radially migrating neurons (Gadisseaux and
Evrard, 1985; Caviness et al., 1988). Subtle morphological
abnormalities are displayed by radial glia of late gestation
Emx2/ fetuses. In these animals,
glial fascicles are, in general, less regularly organized (Fig.
5a,b), and it is possible to find thick bundles,
separated by wider interspaces (Fig. 5, compare
e,d), crossing the CP and reaching the
MZ. These dysmorphologies are somehow reminiscent of the classical
defasciculation defects displayed by radial glia in reeler
embryos (Caviness et al., 1998) (Fig. 5c,f) and are suggested to
underlie migration defects specific of these mutants (Caviness et al.,
1988). It is conceivable that the absence of the REELIN protein is the
common cause of late radial glia abnormalities in both
reeler and Emx2/ mice.
View larger version (112K):
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|
Figure 5.
Radial distribution of RC2 and MAP2 in
the neocortex of E16-E16.5 wild-type,
Emx2/, and
Reln/ embryos. a-f,
Radial glia in E16.5 wild-type (a),
Emx2/ (b),
and Reln/ (c)
embryos, stained by the monoclonal -RC2 antibody, rostral neocortex,
frontal sections. In c, d, and
e, there are magnifications of boxed
areas in a, b, and c,
respectively. In the normal cortex, the thick labeled glial bundles,
which ascend from the ventricular toward the marginal zone, undergo a
sudden reduction in their size at the level of the subplate
(a); in the cortical plate, they are regularly
arranged and separated by relatively narrow interspaces
(d, arrowheads). In
Emx2/ embryos, thick glial bundles ascend
from the ventricular zone and enter the cortical plate; they are
separated by wider interspaces (e, arrowheads)
and do not undergo any appreciable reduction in width, while crossing
the cortical plate (b, e). Similar distribution
of RC2 can be seen in Reln/ mutants
(c). In these animals, the glial bundles cross
the entire cerebral wall and reach, still thick, the marginal layer
commonly termed superplate (c); relatively large
interspaces are detectable among them, underneath the superplate
(f, arrowheads).
g-l, Distribution of neuron-specific MAP2
immunoreactivity on frontal sections through rostral telencephalons of
E16.0 wild-type (g),
Emx2/(h), and
Reln/ (i) embryos; in
j, k, and l, there are
magnifications of the boxed areas of g,
h, and i, respectively. In the case of
wild-type animals, cortical plate neurons are prevalently fusiform and
tightly clustered in a palisade-like structure; the marginal zone, less
cellularized, displays intense -MAP2 immunoreactivity, possibly
associated with terminal harborizations of cortical plate neurons
apical dendrites (j). In
Emx2/ embryos, the cortical
plate neurons appear more rounded and loosely distributed, and the
marginal zone is more irregularly shaped (k). All
of that is somehow reminiscent of what can be seen in the
Reln/ neocortex at the level of the
poorly laminated, broad layer replacing cortical plate and transitional
field, as well as in the overlying superplate
(l). CP, Cortical plate;
MZ, marginal zone; SbP, subplate;
SpP, superplate; TF, transitional field;
VZ, ventricular zone. Scale bar, 100 µm.
|
|
reeler-like abnormalities are also detectable in CP neurons
of Emx2/ embryos by -MAP2
immunostaining (Fig. 5g-l). At E16, the neocortical plate of mutant mice contains prevalently rounded neurons (Fig. 5k) instead of fusiform cells populating the wild-type plate
(Fig. 5j). In addition, these neurons are loosely clustered
so that Emx2/ CP lacks the tight
and palisade-like architecture characterizing the normal plate (Fig.
5j,k).
| |
DISCUSSION |
Impairment of Reelin expression in the neocortical
marginal zone
We have shown that CaR and Reln mRNA, normally
coexpressed at high levels by neocortical Cajal-Retzius neurons of late
gestation mouse embryos (Alcantara et al., 1998), are not detectable in the neocortical MZ of Emx2 knock outs. It is conceivable
that Reln- and CaR-expressing neurons affected in mutant
embryos correspond to the intensely -EMX2--CaR-immunoreactive
cells we described previously in the same area in wild-type embryos
(Mallamaci et al., 1998). At the moment, however, it is hard to say why
these two markers disappeared. It has been found that, in the rat, a large number of late CR cells are born at around a stage approximately corresponding to mouse E12 (Meyer and Fairèn, 1996). Given that and given the almost complete absence of E12 born cells in the MZ of
late gestation mutant embryos (Fig. 4h), one could conclude that our failure to detect CaR and Reln mRNA in mutant mice
could reflect the proper absence of the neurons normally expressing these markers. However, alternative explanations can be raised. For
example, it is possible that neurons normally fated to express Reln, not properly differentiated and still surviving, were
ectopically placed and/or heterochronically born. The ectopic
hypothesis would be in keeping with the wider radial distribution
displayed by E12 BrdU pulse-labeled cells in E19 knock-out embryos
compared with wild-type ones (Fig. 4i, j). About
heterochronic birth, it has to be mentioned that numerous subpial
neurons can be actually found by hematoxylin and cresyl violet staining
(data not shown) in the neocortex of E19 mutant embryos and that, in
the same animals, a small but substantive number of E13 (data not
shown) and E13.5 (Fig. 4d) born cells settle just beneath
the pia mater.
It is noteworthy that the impairment of Reln and CaR
expression is restricted to mid-late phases of corticogenesis. No
apparent differences in neocortical expressions of either
Reln mRNA (Fig. 2a,b) and CaR (data
not shown), in fact, could be detected at E11.5 between wild-type and
knock-out mice. What is the meaning of that? One could hypothesize that
the same CR neurons do not require the products of Emx2 at
the very beginning of their life and only subsequently become dependent
on them for surviving and/or retaining their proper differentiation
state. However, the overt increase of the absolute total number of
Reln-expressing cells taking place in wild-type animals
between E11.5 and E15 (data not shown; Alcantara et al., 1998), as well
as E10.5-E19 (data not shown) and E12-E19 (Fig.
4g,h) birthdating-survival data, suggest that at
least two different populations of Reln-expressing cells do
exist, which can be operationally distinguished on the basis of their
dependence on Emx2 function. We have an early transient population, prevalently generated before E11.0 and not dependent on the
Emx2 function, and a late, still detectable at approximately birth, population, prevalently generated after E11.5 and dependent on
the Emx2 function for crucial and still unknown steps of its development. Actually, two main neuronal populations were also described in the neocortical MZ of the rat. The first, early and transient, is generated in the neocortical VZ; the second, later born
and longer surviving, has been proposed to arise in the basal retrobulbar region and to reach the neocortical MZ by subpial tangential migration. These two rat populations nicely correspond, for
birthdating and survival profiles, to our MZ mouse populations (Meyer
and Fairèn, 1996; Meyer et al., 1998); however, we think that it
is premature to claim that a true homology takes place. Further
experimental work, including DiI tracing tests, will hopefully make
this point clear and will provide us with valuable suggestions about
the key role(s) played by Emx2 in the development of the mouse secondary population.
Axially restricted defects in the subplate
We have shown that, in Emx2 mutant mice, the
development of specific SP neuronal populations expressing CaR and EMX1
is impaired. The fact that these populations are affected, despite the
absence of any expression of Emx2 in normal SP cells and the
main restriction of EMX2 to the VZ (Gulisano et al., 1996; Mallamaci et
al., 1998), suggests that this phenotype is attributable to the
lack of critical functions played by Emx2 in SP neurons
precursors, when they still lie in the VZ. Emx2 could
normally either allow these progenitors to come out of the
mitotic cycle at the proper time or play a crucial role in preparing
the appropriate survival-differentiation programs of their postmitotic descendants.
These defects are not ubiquitous but are restricted to caudal and
medial SP. Interestingly, in the cortical VZ, Emx2 displays a graded expression with caudomedial maximum and rostrolateral minimum
(Mallamaci et al., 1998). Is there a link between axial restrictions in
SP phenotypes and this expression gradient? Two simple explanations can
be conceived. First, it is possible that Emx2 cooperates in
an additive way with other genes, characterized by complementary
expression patterns (e.g., Pax 6) (Walther and Gruss,
1991), in modulating the expression of a downstream effector necessary
to prime the development of the affected SP neuronal population. In
mutant embryos, the sum of Emx2 and, for example, Pax6 activities on this target should fall under the
critical threshold near the posterior and medial edges of the cortical VZ in which normally Emx2 is more intensely expressed.
Alternatively, it is possible that neurons belonging to an affected SP
population and displaying different axial locations, even if sharing a
given marker, are basically not homogeneous. At different locations along both main tangential axes, different doses of EMX2 might be
necessary to their VZ forerunners for initiating their specific and
different morphogenetic programs. If some functional interchangeability takes place between EMX2 and EMX1, it is conceivable that, in more
rostral or lateral neuroblasts, the absence of EMX2 can be rescued by
EMX1 protein, whereas this would not be possible in more caudal or
medial neuroblasts because of the very high, unrescuable levels of EMX2
activity these cells require.
Lack of Reln signaling and
migratory abnormalities
We found that radial migration of CP neurons is specifically
impaired in Emx2 mutant mice. It is conceivable that
intrinsic, cell-autonomous anomalies occurring in
Emx2/ migrating neurons could
take a part in causing that; testing this hypothesis will be the aim of
further experimental work. At the moment, however, a precise
causal relationship between time-restricted impairment of
Reln signaling from neocortical MZ and migratory phenotype
occurring to Emx2/ mice can be already hypothesized.
MZ cells normally are the only effectors of the Reln
function in the developing cerebral cortex, from at least E11.5 up to perinatal stages (D'Arcangelo et al., 1995, 1997; Hirotsune et al.,
1995; Ogawa et al., 1995; D'Arcangelo and Curran, 1998) (Fig. 2e-g,k). It has been shown that mice
constitutively lacking the Reln function display a severe
neocortical migratory phenotype, including two main features. First, CP
neurons do not penetrate the preplate, which is not split into MZ and
SP and gives rise to the so-called superplate; second, during their
radial migration, late born CP neurons do not overcome early born CP
neurons and remain largely intermingled with them so that the classical
inside-out rule is not followed (Caviness et al., 1988) (Fig.
4i).
As shown, Emx2/ mutants display a
specific time and space-restricted Reln functional
knock-out. In the neocortical MZ of
Emx2/ embryos, Reln mRNA
expression, apparently normal at E11.5, is reduced at E13.5 and
completely absent since E15.5. In the same mutants, early phases of
radial migration of CP neurons seem to be poorly affected, and late
phases are impaired in a reeler-like way. Like the wild-type
preplate, the Emx2/ preplate is
penetrated by CP neurons so that an SP and an MZ can be distinguished;
however, in the same mutants, late born CP neurons largely fail to
overcome early born CP neurons (Fig. 4j) so that the
inside-out rule is, again, hardly followed. It is possible that
transient exposure to Reln signaling is sufficient to stably
confer neurons fated to give rise to the SP the property to be able to
be subsequently "overcome" by CP neurons, even at stages at which
no more expression of Reln will be detectable. On the
contrary, the reduction-absence of Reln products in the E13.5-E15.5 MZ would account for the inability of the majority of E13.5
born neurons to let E15 born ones to settle superficially to them. Late
absence of Reln products in the MZ would also account for
abnormal neuronal packaging profiles, as well as for late radial glia
dysmorphologies, occurring in Emx2/
mutants in a reeler-related way (Fig. 5).
Finally, the possible role of nonmarginal Reln-expressing
cells in the aethiology of migratory anomalies described above has to
be mentioned. Nonmarginal Reln-expressing neurons
normally appear in the outer CP at approximately birth (Schiffmann et
al., 1997; Alcantara et al., 1998; Rice et al., 1998) (Fig.
2k), and it is likely that they could sustain the radial
migration of last born neocortical neurons. It is possible that, in
mutant Emx2/ embryos, earlier
appearance of nonmarginal Reln-expressing neurons (Fig.
2h-j), as well as their radial misplacement (Fig.
2l), could take part in causing the migratory
abnormalities outlined above. Selective in vivo ablation of
CP Reln signaling, as well as appropriate tests on brain
slice cultures, will hopefully allow us to make this point clear.
| |
FOOTNOTES |
Received Feb. 9, 1999; revised Nov. 11, 1999; accepted Nov. 15, 1999.
This work was supported by grants from the European Community BIOTECH
and BIOMED Programmes, the Telethon-Italia Programme, the Italian
Association for Cancer Research (the AIRC), and the Armenise-Harvard
Foundation. We thank Pierre Gressens for the RC2 antibody, Giacomo
Consalez for reeler founder mice, Vania Broccoli and
Larry Wrabetz for comments and suggestions, Mario Azzini for
photographic assistance, and Giorgio Corte, who collaborated previously
with us in generating the anti-EMX1 antibody. A special thanks to
Alessandro Bulfone for his helpful criticism and encouragement. Animals
were handled as recommended by the Institutional Animal Care and Use
Committee of H. San Raffaele.
Drs. Mercurio and Muzio contributed equally to this work.
Correspondence should be addressed to Antonello Mallamaci, Staff
Scientist, Unit of Molecular Biology of Development, DIBIT, H. San
Raffaele, via Olgettina 60, 20132 Milano, Italy. E-mail: mallamaci.antonio{at}hsr.it.
Dr. Mercurio's present address: National Institute of Medical
Research, Medical Research Council, Mill Hill, The Ridgeway, NW7 1AA
London, U.K.
| |
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C.-H. Chan and H. H Yeh
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A. Stoykova, O. Hatano, P. Gruss, and M. Gotz
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K. L. Ligon, Y. Echelard, S. Assimacopoulos, P. S. Danielian, S. Kaing, E. A. Grove, A. P. McMahon, and D. H. Rowitch
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S. Garel, K. J. Huffman, and J. L. R. Rubenstein
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H. Super, J. A. Del Rio, A. Martinez, P. Perez-Sust, and E. Soriano
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TOP
ABSTRACT
INTRODUCTION
Gene
