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The Journal of Neuroscience, December 15, 1999, 19(24):10877-10885
Graded and Areal Expression Patterns of Regulatory Genes and
Cadherins in Embryonic Neocortex Independent of Thalamocortical
Input
Yasushi
Nakagawa1,
Jane
E.
Johnson2, and
Dennis D. M.
O'Leary1
1 Molecular Neurobiology Laboratory, The Salk
Institute, La Jolla, California 92037, and 2 Center
for Basic Neuroscience, University of Texas Southwestern Medical
Center, Dallas, Texas 75235
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ABSTRACT |
The differentiation of areas of the mammalian neocortex has been
hypothesized to be controlled by intrinsic genetic programs and
extrinsic influences such as those mediated by thalamocortical afferents (TCAs). To address the interplay between these intrinsic and
extrinsic mechanisms in the process of arealization, we have analyzed
the requirement of TCAs in establishing or maintaining graded or areal
patterns of gene expression in the developing mouse neocortex. We
describe the differential expression of Lhx2, SCIP, and Emx1, representatives of three
different classes of transcription factors, and the type II classical
cadherins Cad6, Cad8, and
Cad11, which are expressed in graded or areal patterns, as well as layer-specific patterns, in the cortical plate. The differential expression of Lhx2, SCIP,
Emx1, and Cad8 in the cortical plate is
not evident until after TCAs reach the cortex, whereas Cad6 and Cad11 show subtle graded
patterns of expression before the arrival of TCAs, which later become
stronger. We find that these genes exhibit normal-appearing graded or
areal expression patterns in Mash-1 mutant mice that
fail to develop a TCA projection. These findings show that TCAs are not
required for the establishment or maintenance of the graded and areal
expression patterns of these genes and strongly suggest that their
regulation is intrinsic to the developing neocortex.
Key words:
cerebral cortex; area specification; transcription
factors; cadherins; Lhx2; Emx1; SCIP
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INTRODUCTION |
The neocortex, a major region of the
cerebral cortex, is divided into functionally specialized areas
characterized by a unique architecture and distinct sets of input and
output projections. Areas gradually differentiate within the cortical
plate (CP), which initially does not exhibit the anatomical features
that later distinguish different areas. The mechanisms that control neocortical arealization have been a debated issue, focusing on the
roles of intrinsic mechanisms, such as differential gene regulation autonomous to the developing neocortex, versus extrinsic mechanisms, such as the influence of thalamocortical afferents (TCAs), the principal input to the neocortex (Rakic, 1988 ; O'Leary, 1989). A role
of TCAs in controlling specific features associated with arealization,
including differential gene expression ranging from graded to area
specific, and anatomical properties that characterize neocortical areas
would be suggested if these features become apparent in the CP after
TCAs reach the neocortex. A role of genetic regulation would be
suggested if genes are differentially expressed in graded or areal
patterns before TCAs arrive and before other area-specific properties appear.
The developing neocortex exhibits considerable plasticity in the
differentiation of area-specific properties, and TCAs seem to be a key
regulator of this plasticity. For example, studies of the rodent
primary somatosensory area have demonstrated a critical role of TCAs in
the differentiation of barrels, a functional grouping unique to this
area (for review, see Woolsey, 1990; Schlaggar and O'Leary,
1993). Transplant experiments have shown that pieces of embryonic
neocortex grafted heterotopically to a different neocortical area can
acquire the area-specific architecture and connections characteristic
of the new area (O'Leary and Stanfield, 1989; Schlaggar and O'Leary,
1991). In addition, reductions in TCAs arising from the lateral
geniculate nucleus have been correlated with a corresponding reduction
in the extent of the neocortex that differentiates the architecture
characteristic of primary visual cortex, the target area of that
thalamic nucleus (Dehay et al., 1989, 1991; Rakic et al., 1991). The
influence of TCAs on areal plasticity, and by inference on normal
arealization, could be caused in part by its control of differential
gene expression in the developing CP.
Recent reports have described the differential expression of several
EphA receptor tyrosine kinases and their ephrin-A ligands in the CP
before the arrival of TCAs. In embryonic monkeys, the receptors
EphA3, A4, A6, and A7 are
expressed in graded or areal patterns before TCAs reach the cortex
(Donoghue and Rakic, 1999). On the other hand, the receptor
EphA5 and the ligands ephrin-A2, A3,
and A5, which are graded or areal at later stages, are
either not expressed when TCAs arrive or their expression is uniform. In rodents, EphA5 and ephrin-A5 also exhibit
substantial differences in their expression patterns before and after
TCAs arrive in the cortex (Zhang et al., 1997; Mackarehtschian et al.,
1999).
Although both intrinsic and extrinsic mechanisms contribute to the
process of arealization (O'Leary et al., 1994; Chenn et al., 1997;
Levitt et al., 1997), little is known about how these mechanisms
cooperate to establish area-specific properties. The purpose of this
study was to assess the potential interplay between these mechanisms by
examining the role of TCAs in influencing differential gene expression
in the CP. We focused on genes that are differentially expressed
tangentially across the developing CP and encode either nuclear
proteins that regulate gene expression or cell-surface proteins that
mediate cell-cell interactions. We wished to identify genes whose
expression becomes graded or areal in the CP either before or after
TCAs reach the cortex. We then assessed the role of TCAs in
establishing and maintaining differential gene expression patterns by
examining them in mice deficient for the basic helix-loop-helix
transcription factor gene Mash-1 (Guillemot et al., 1993),
which fail to develop a TCA projection (Tuttle et al., 1999).
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MATERIALS AND METHODS |
Animals. Embryos and postnatal pups obtained from
timed pregnant ICR mice (Harlan Sprague Dawley, Indianapolis,
IN) were used for all analyses except those examining the role
of TCAs, which used Mash-1 / mice (Guillemot et al.,
1993) and their wild-type littermates outcrossed into the CD1
strain. The day of insemination is designated embryonic day 0.5 (E0.5). The day of birth is designated postnatal day 0 (P0).
Maintenance and genotyping of the Mash-1 mutant embryos were
done as described previously (Guillemot et al., 1993; Tuttle et al.,
1999). Analysis of expression patterns was done blinded to genotype
(although the null mutant brains can be distinguished by their smaller
olfactory bulbs). Three to four brains per genotype were analyzed by
in situ hybridization.
In situ hybridization. In situ
hybridization and counterstaining on 20 µm cryostat sections were
done according to the methods of Tuttle et al. (1999). The following
digoxigenin-labeled RNA probes were used: Lhx2 (897-1482 of
mouse Lhx2; GenBank accession number AF124734; a gift from
S. Bertuzzi) (Xu et al., 1993); SCIP (rat full-length clone;
a gift from G. Lemke) (Monuki et al., 1989); Emx1 (mouse
full-length clone) (R. H. Dyck, J. Richards, J. J. A. Contos, C. Akazawa, J. Chun, D. D. M. O'Leary, unpublished observations); Cad6 (mouse full-length clone; a gift
from S. Mah and C. Kintner) (Inoue et al., 1997); Cad8
[241-1481 of mouse Cad8; GenBank accession number X95600;
obtained by reverse transcription (RT)-PCR] (Korematsu and
Redies, 1997); and Cad11 (1278-2121 of mouse
Cad11; GenBank accession number D31963; obtained by RT-PCR)
(Kimura et al., 1995). Hybridization using the rat SCIP
probe identified the same cell populations in rat and mouse tissues.
Whole-mount in situ hybridization was based on the method of
Wilkinson (1993), with the following modifications: brain were
pretreated with 10 µg/ml proteinase K for 30 min, and the
prehybridization, hybridization, and posthybridization washes were done
at 70°C. In addition, we used 3 µg/ml digoxygenin-labeled probes
for hybridization. Although the whole-mount analysis is useful to
survey the global gene expression patterns, we found that expression in
deeper layers of the cortical wall is often not detected, presumably
because of the poor penetration of riboprobes; for example, layer 5 expression of Cad8 along the whole rostrocaudal axis of the
neocortex at E18.5 is not detected with our whole-mount protocols.
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RESULTS |
For this analysis, we performed in situ hybridization
using digoxigenin-labeled riboprobes on E10.5-P2 mouse cerebral
cortex. TCAs pass from the internal capsule into the neocortex at
E14.5; by E15.5 they have spread across much of the neocortex and have begun to extend branches toward the CP (Bicknese et al., 1994). We use
the term "areal" to describe restricted tangential patterns of gene
expression and do not intend to imply that these patterns directly
relate to specific areas.
Graded expression of Lhx2, SCIP, and
Emx1 in developing neocortex
Lhx2 encodes an LIM-homeodomain transcription
factor postulated to control cortical neuron differentiation (Xu et
al., 1993). Lhx2 has been shown recently to be expressed in
mouse neocortex in both proliferating and nonproliferating cells
(Retaux et al., 1999) and to be involved in the proliferation of
cortical neuroepithelial cells (Porter et al., 1997). However, the
differential expression of Lhx2 along the tangential extent
of the neocortex has not been described.
Lhx2 is expressed in the dorsal telencephalic wall as early
as E10.5, but its expression is not graded (data not shown). In contrast, at E12.5, Lhx2 expression is graded in
high-medial-to-low-lateral (Fig.
1A) and
high-caudal-to-low-rostral (data not shown) patterns. Expression in the
preplate (PP) does not decline rostrally as much as that in the
ventricular zone (VZ), resulting in much higher expression in the PP
than in the VZ at rostral levels (data not shown).
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Figure 1.
Graded expression of regulatory genes in the
E10.5-E15.5 mouse neocortex. Coronal (A-C,
E-K) and sagittal (D) sections of
mouse forebrain show the expression of Lhx2
(A-D), SCIP
(E-G), and Emx1
(H-K). A, Lhx2
shows a graded pattern in the dorsal telencephalon at E12.5 with higher
expression medially than laterally (arrowheads).
B, At E14.5, Lhx2 expression is high in
the VZ and SVZ and graded with a high-medial-to-low-lateral
pattern but is very low in the CP with no obviously graded patterns
along the tangential axes. C, D, At E15.5,
Lhx2 expression exhibits a dramatic increase in the
upper CP of the caudolateral neocortex (arrowheads),
strongly graded in high-lateral-to-low-medial (B)
and high-caudal-to-low-rostral (C) patterns.
E, At E12.5, SCIP expression is detected
in the PP of the rostral cortex (arrowheads). SCIP is
expressed in a more ventral region, which appears to be the lateral
ganglionic eminence (E, arrows). F, G, At
E14.5, SCIP expression is limited to the IZ and is
graded in a high-lateral-to-low-medial pattern (F;
arrowheads) that is still present at E15.5
(G). At E15.5, it is also detected in the upper
CP (G; arrowheads), but the caudolateral
part of the neocortex, where Lhx2 is highly expressed,
shows much weaker expression (G; arrow).
H, Emx1 is in a slightly graded pattern
as early as E10.5, with higher expression more medially than laterally
(arrowheads). I, The same graded
expression is detected at E12.5 (arrowheads).
J, At E14.5, Emx1 is expressed in the
VZ/SVZ and IZ of the neocortex in a slightly graded,
high-medial-to-low-lateral pattern, whereas the level of expression in
the CP is very low. K, At E15.5, Emx1 is
most highly expressed in the upper CP of the caudolateral neocortex
(arrowheads). B and J are
from adjacent sections, and F is caudal to them.
C and K are from adjacent sections, and
G is rostral to them. A is slightly
caudal to I. In this and all subsequent figures, dorsal
is to the top and midline is to the right
in coronal sections, and dorsal is to the top and caudal
is to the right in sagittal sections, except for
H, which is a coronal section showing both sides of the
telencephalon. Scale bar, 100 µm.
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At E14.5, we find that Lhx2 expression is high in the VZ and
subventricular zone (SVZ) but very low in the CP (Fig.
1B). Within the VZ/SVZ, Lhx2
expression is graded in a high-medial-to-low-lateral, as well as a
high-caudal-to-low-rostral (data not shown), pattern, whereas graded
expression is not detected in the CP (Fig. 1B). At
E15.5, Lhx2 expression is increased substantially in the
upper CP of the caudolateral neocortex and is strongly graded in
high-lateral-to-low-medial and high-caudal-to-low-rostral patterns
(Fig. 1C,D). The graded expressions in the VZ/SVZ at E15.5
are the same as those at E14.5. Interestingly, the strongly graded
Lhx2 expression in the CP is in a countergradient along the
mediolateral axis compared with its expression in the VZ and SVZ (Fig.
1B,C). At both E18.5 (see Fig.
4A-C) and P2 (see Fig. 3A),
Lhx2 continues to be differentially expressed, with higher
expression laterally, in the putative auditory cortex, than medially,
in the putative visual cortex. The transition in expression level along
this axis shows a relatively abrupt decline. Even more medially,
expression is again slightly higher than that in the putative visual
area (see Fig. 3A).
The graded differential expression of Lhx2 occurs in a
layer-specific manner. At P2, Lhx2 is most highly expressed
in layers 2/3, 5, and 6 (see Fig. 3H,H'). Expression by the
deep layer neurons is evident as early as E15.5, at which time the CP
has strong expression and is mainly populated by future layer 5 and 6 neurons (Caviness, 1982; Frantz et al., 1994). The upper layer
expression is already present at E18.5 (see Fig. 4A),
which approximately matches the time when the future layer 2/3 neurons
start to reach the CP (Caviness, 1982; Frantz et al., 1994).
SCIP, which encodes a POU domain-containing
transcription factor (He et al., 1989; Monuki et al., 1989; Suzuki et
al., 1990), is expressed in a layer-specific manner in the developing
rat neocortex (Frantz et al., 1994), but differential tangential
expression has not been reported. At E10.5, SCIP is not
expressed in the dorsal telencephalic wall. At E12.5, it is expressed
in the PP of the rostral part of the dorsal telencephalon (Fig.
1E), as described previously (Frantz et al., 1994).
At E14.5, we find that SCIP expression is mostly limited to
the IZ and is graded in a high-lateral-to-low-medial pattern (Fig.
1F). At E15.5, SCIP expression is also detected
in the upper CP throughout most of the neocortex (Fig. 1G),
but in a graded pattern opposite to that observed at E14.5 in the IZ,
with much lower levels in the caudolateral part of the CP (Fig.
1G). Thus, SCIP expression in the CP is
complementary to that of Lhx2. At P2, the graded pattern of
SCIP expression is similar to that at E15.5 (see Fig.
3B) and in addition has a clear layer specificity (see Fig.
3I,I'; high in layer 2/3 and layer 5) as reported previously
(Frantz et al., 1994). In addition, a medial part of the caudal
neocortex, where Lhx2 expression is at a slightly higher
level than in the putative visual area, exhibits lower SCIP
expression, reinforcing the conclusion that graded patterns of
SCIP and Lhx2 expression in the CP are
complimentary (see Fig. 3A,B).
Emx1 is a homeodomain transcription factor expressed in
embryonic and postnatal mouse neocortex, in both proliferating cells and postmitotic neurons (Simeone et al., 1992; Gulisano et al., 1996).
A graded distribution of Emx1 transcripts has not been described, but immunostaining has revealed a graded pattern with higher
expression caudolaterally and lower expression rostromedially in
postnatal mouse neocortex (Briata et al., 1996). We find that Emx1 expression in the neocortex, both embryonic and
postnatal, is graded. At both E10.5 and E12.5, Emx1
expression in the dorsal telencephalic wall is slightly higher medially
than laterally (Fig. 1H,I). A
high-caudal-to-low-rostral-graded expression pattern is also found at
E12.5 (data not shown). At E14.5, Emx1 is expressed highly
in the VZ/SVZ and IZ and is still slightly graded in a high-medial-to-low-lateral pattern, whereas the expression in the CP is
very low, and graded expression is not detected (Fig. 1J). At E15.5, however, Emx1 is expressed
most highly in caudolateral neocortex in a pattern similar to that in
Lhx2 (Fig. 1K); this pattern is still
present at P2 (see Fig. 3C). Emx1 expression is
high in the putative auditory area, declines medial to it in the
putative visual area, but appears to increase further medially (see
Fig. 3C). The layer specificity of Emx1 is not as
evident as that of Lhx2 or SCIP, but its
expression appears to be higher in layers 2/3, 4, and 6 than in layer 5 (see Fig. 3J,J'), which is consistent with the results of
Gulisano et al. (1996).
In summary, Lhx2, SCIP, and Emx1 are
expressed in graded patterns in the developing neocortex, including the
CP, with different degrees of layer specificity. Because the expression
of these genes in the CP becomes differential only after TCAs arrive in the cortex (Bicknese et al., 1994), TCAs could play a role in controlling the establishment and maintenance of these differential expression patterns. In addition, the finding that Lhx2 and
SCIP expression in the CP is in countergradients to their
expression in the VZ/SVZ and IZ, respectively, makes them particularly
promising candidates to be potentially regulated by TCAs.
Temporal differences in the onset of areal expression
of cadherins
Cad6, Cad8, and Cad11 have been reported to
be expressed in area-specific manners in P2 mouse cortex by whole-mount
in situ hybridization (Suzuki et al., 1997), and
Cad6 expression has been reported to be graded in the CP at
E14.5 (Inoue et al., 1998). We have examined the expression of
Cad6, Cad8, and Cad11 at embryonic and postnatal
ages, using both cryosections and whole-mount brains, to determine when
they take on their graded and areal patterns of expression.
In agreement with Inoue et al. (1998), we find that Cad6
expression is graded in a high-lateral-to-low-medial pattern in the CP
as early as E14.5 (Fig.
2A). However, in
addition, we find that Cad6 is also expressed in the VZ and
SVZ at E14.5 in a similarly graded manner to that in the CP (Fig.
2A). The expression in the VZ is detected at E12.5,
but no expression is evident at E10.5 (data not shown). Cad6
expression in the CP appears more clearly graded at E15.5, because of
an apparent increase in Cad6 expression in the lateral
neocortex, whereas expression in the VZ/SVZ appears to be substantially
diminished (Fig. 2B,C). The graded expression in the
CP is still present at P2. Caudally, the putative auditory area
expresses the highest level of the Cad6 transcript, with the
highest expression in layers 2/3 and 5 (Fig.
3D,K,K'). The expression level
in these layers declines medially (Fig. 3D); although even
more medially in the putative visual area, a detectable level of
Cad6 is found, but in contrast to that in the auditory area,
expression is mainly in layer 4.
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Figure 2.
Graded expression of cadherin genes in the E14.5
and E15.5 mouse neocortex. Coronal sections of mouse forebrain show the
expression of Cad6 (A-C) and
Cad8 (D-F), and sagittal sections
show that of Cad11 (G, H).
A, At E14.5, expression of Cad6 is
already graded in a high-lateral-to-low-medial pattern both in the CP
(arrowheads) and in the VZ/SVZ (arrows).
B, C, At E15.5, this graded expression becomes more
pronounced in the CP (B, C; arrowheads),
but the expression declines in the VZ/SVZ, especially at rostral levels
(B). The medial part of the neocortex expresses a
very low level of Cad6 (B, C;
arrows). D, Cad8
expression is detected only in the intermediate zone at E14.5 and is
not graded (arrowheads). E, F, But at
E15.5, it is graded in a high-medial-to-low-lateral pattern in the
upper CP (arrowheads for high-medial expression).
Rostrocaudal differences in expression are not clear for either
Cad6 or Cad8 (data not shown).
G, Cad11 expression in the CP is slightly
graded in a high-caudal-to-low-rostral pattern at E14.5
(arrowheads). H, At E15.5, this graded
expression becomes more evident (arrowheads).
A and D as well as C and
F are from adjacent sections. E is
slightly caudal to B. Scale bar, 100 µm.
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Figure 3.
Differential expression of regulatory genes and
cadherins in the P2 mouse neocortex. A-L, Coronal
sections of mouse forebrain show graded expression and areal patterns
(A-G) and layer specificity
(H-L) of the expression of
Lhx2 (A, H), SCIP
(B, I), Emx1 (C,
J), Cad6 (D, K),
Cad8 (E, F, L; E is
rostral to F), and Cad11
(G). H'-L', These panels show
4,6-diamidino-2-phenylindole counterstaining of
H-L, respectively. The boxes in
A-D and F show the approximate locations
of panels of a higher magnification, H-L. The graded
patterns of gene expression that are observed at E15.5 are maintained
at P2. A, H, Lhx2 is highly expressed in
layers 2/3, 5, and 6 of the caudolateral neocortex, corresponding to
the putative auditory area (A; box,
H), whereas its expression level is much lower in
the more medial, putative visual area (A;
single arrow); expression increases again
more medially (A; double
arrows). B, I, SCIP
expression is mainly detected in layers 2/3 and layer 5 (I) and is lowest in the auditory area
(B; single arrow) and much
higher in the visual area (B; box) a
graded expression pattern opposite to that of Lhx2. The
area indicated by double arrows (B),
where Lhx2 expression is slightly higher than that in
the putative visual area, exhibits a lower expression level of
SCIP compared with that in the visual area. C,
J, Emx1 is expressed in a tangential pattern
similar to that of Lhx2 (C;
box for the auditory area with higher expression,
single arrow for the visual area with
lower expression, and double arrows for
the area with slightly higher expression) and is higher in layers 2/3,
4, and 6 than in layer 5 (J). D,
K, Cad6 expression is graded in a
high-lateral-to-low-medial pattern (D) with the
highest level in layer 5 and the lower aspect in layers 2/3
(K); the putative auditory area shows a high
level of expression (D; box), whereas the
visual area shows much lower expression (D;
double arrows) with a different layer
specificity (mainly in layer 4). Expression between these areas is at
lower levels in all layers (D; single
arrow). E, F, L, Cad8 is
more highly expressed medially than laterally in layer 5 (E,
F; arrowheads) and, in addition, is expressed in
the upper layers of the putative motor (E;
arrow) and visual (F; box,
L) areas. G, Cad11 is
expressed higher in the putative visual (double
arrows) than in the auditory (single
arrow) area, without much layer preference. A-D,
F, and G are from adjacent sections. Scale bars:
A-G, 1 mm; H-L, H'-L',
100 µm. iz, Intermediate zone; mz,
marginal zone; sp, subplate.
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In contrast to the graded expression of Cad6,
Cad8 is more uniformly expressed at E12.5 and E14.5 along
both the mediolateral (Fig. 2D for E14.5) and
rostrocaudal (data not shown) axes, and its expression is most
pronounced in the intermediate zone at E14.5 (Fig.
2D) and in the PP at E12.5 (data not shown). However, at E15.5 Cad8 expression exhibits a graded pattern with a
higher level medially than laterally in the CP (Fig.
2E,F). At P2, Cad8 expression has a
graded pattern similar to that at E15.5 (Fig. 3E,F)
and is strong in layer 5 (Fig. 3L,L'). Interestingly, the laminar pattern of Cad8 expression also shows areal
differences. In most of the neocortex, Cad8 expression is
primarily limited to layer 5. However, in addition to the layer 5 expression, the putative motor (Fig. 3E) and visual (Fig.
3F) areas have higher levels of Cad8
expression in layers 2/3 and 4 (Fig. 3L,L'). This upper
layer expression is observed as early as E18.5 (Fig.
4G,H), which
approximately coincides with the arrival of upper layer neurons at the
CP (Caviness, 1982; Frantz et al., 1994).
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Figure 4.
Graded and/or areal distributions of regulatory
genes and cadherins are not altered in the E18.5 Mash-1
mutant neocortex. Coronal sections (A, D-H, J, A', D'-H',
J') and whole mounts (B, C, I, B', C', I') of
wild-type (A-J) and Mash-1 null
(A'-J') mutant forebrains show the comparison of
Lhx2 (A-C, A'-C'), SCIP
(D, D'), Emx1 (E, E'),
Cad6 (F, F'), Cad8
(G-I, G'-I'), and Cad11 (J,
J') expression between the two genotypes. The
Mash-1 mutant does not show any significant differences
from wild type in graded, areal, or laminar cortical expression
patterns of the genes examined. The whole-mount panels
show the left hemispheres; B and B' are
the caudolateral view with the olfactory bulbs on the
left, and C, I, C', and I'
are dorsal views with the midline to the right.
A-C, A'-C', Lhx2 is highly expressed in the
caudolateral neocortex in both genotypes (arrows).
D-F, J, D'-F', J', Tangentially graded patterns of
SCIP, Emx1, Cad6, and
Cad11 are similar between the wild type and the mutant
(arrows show areas with higher expression). G, H,
G', H', Cad8 expression in layer 5 is graded in a
high-medial-to-low-lateral pattern in both genotypes
(arrowheads), and expression in the upper layers of the
frontal (G, G'; arrows) and occipital
(H, H'; arrows) cortex is also unchanged
in the mutant. I, I', The whole-mount pictures show the
Cad8 expression in the frontal cortex, with the
arrowheads showing the caudal boundary of the expression
domains. In the Mash-1 mutant, the medial portion of
this expression domain extends further caudally than in the wild type.
Scale bars: sections, 500 µm; whole mounts, 1 mm.
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Cad11 expression is detected in the CP, but not in the
VZ/SVZ, at all ages examined (E14.5, E15.5, E18.5, and P2). Expression is not detected at E10.5 or E12.5 (data not shown). Cad11
exhibits a graded expression in a high-caudal-to-low-rostral pattern in the CP (Fig. 2G,H) as well as a
high-medial-to-low-lateral one (Fig. 3G), which are subtle
at E14.5 but pronounced by E15.5.
In conclusion, our findings reveal temporal differences in the onset of
the graded expression of Cad6, Cad8, and
Cad11. Because the areal pattern of expression of
Cad6 observed postnatally is reflected by its graded
expression as early as E14.5 in the CP, as well as in the proliferative
layers that give rise to it, the initial establishment of this
patterned expression appears to be independent of TCAs. The later onset
of the graded expressions of Cad8 and Cad11
suggests that TCAs could control the establishment of their expression
patterns or be involved in refining and maintaining the patterned
expression of all three cadherins.
Graded or areal patterns of gene expression in the neocortex of
Mash-1 mutant mice
We next examined whether TCAs are required to establish and/or
maintain the graded or areal gene expression patterns described above.
For this, we analyzed gene expression in mice deficient for
Mash-1 (Guillemot et al., 1993), which fail to develop a TCA projection (Tuttle et al., 1999). Because Mash-1 itself is
not expressed at detectable levels in the cortex (Torii et al., 1999; Tuttle et al., 1999), differences in the expression patterns between wild-type and mutant littermates would strongly imply a role of TCAs in
regulating these patterns. We performed these comparisons of gene
expression at E18.5 (which coincides with the day of birth), because
Mash-1 mutant mice die soon after birth (Guillemot et al.,
1993).
As shown in Figure 4, Mash-1 mutant mice do not exhibit any
significant differences from wild-type mice in the graded, areal, or
laminar cortical expression patterns of Lhx2 (Fig.
4A-C,A'-C'), SCIP (Fig.
4D,D'), Emx1 (Fig.
4E,E'), Cad6 (Fig.
4F,F'), or Cad11 (Fig.
4J,J'). In addition, Cad8 expression in
wild-type and mutant mice shows a similarly graded pattern in layer 5 with a higher level medially than laterally (Fig.
4G,H,G',H') and exhibits area-specific laminar differences
in expression, characterized by increased expression in the upper
layers of frontal (Fig. 4G,G') and occipital (Fig.
4H,H') cortex. We did note, however, that the shape
and extent of the Cad8 expression domain in rostral cortex
differ between wild-type (Fig. 4I) and mutant (Fig.
4I') brains. Specifically, we find that the medial
portion of this Cad8 expression domain extends further
caudally than normal. This change is also seen in sections and appears
to be mainly caused by an abnormal caudal extension of the upper layer
Cad8 expression typical of frontal cortex (data not shown).
These results show that the establishment and maintenance of the graded
or areal gene expression patterns described here, with the possible
exception of some features of Cad8 expression, do not
require TCAs.
| |
DISCUSSION |
The primary goal of our study was to determine the requirement of
TCAs for establishing and maintaining graded or areal patterns of gene
expression in the CP of the developing neocortex. As a prerequisite, we
have described several novel patterns of gene expression that may be
relevant to neocortical arealization. Among these, we have shown that
the regulatory genes Lhx2, SCIP, and Emx1 are expressed in graded patterns in the developing
neocortex, including the CP, have defined the onset of the areal
expression of Cad8 and Cad11, and have described
some unique features of Cad6 expression.
The differential gene expression patterns that we describe become
evident at different ages. Graded expression of Lhx2,
SCIP, Emx1, and Cad8 in the CP is not
detected until E15.5, by which time TCAs have already entered the
cortex (Bicknese et al., 1994). Therefore, the time of emergence of
these patterns is consistent with the hypothesis that they require TCAs
for their establishment. Cad6 and Cad11 show
slightly graded patterns of expression in the CP at E14.5, which are
more robust at E15.5. The earlier onset of these patterns suggests that
they do not require TCAs to be established but may nonetheless require
TCAs to be refined and maintained. However, we find that each of these
genes exhibits normal-appearing graded or areal expression patterns in
Mash-1 mutant mice that fail to develop a TCA projection
(Tuttle et al., 1999). These findings indicate that TCAs are not
required for the establishment or maintenance of the graded and areal
expression patterns of the genes analyzed here. The possible exception
is the relatively minor change in the rostral expression domain of Cad8, which shows a caudalward extension along its medial edge.
The graded patterns of gene expression that we observe in the CP are
layer specific and become apparent soon after the appropriate set of
neurons reaches the CP. The graded patterns observed in the CP are
unlikely to be maturation dependent, because they are maintained over a
long time period extending from the peak of cortical neurogenesis until
after neurogenesis has ceased and most, if not all, neurons have
reached the CP. In addition, the graded expression of Lhx2
and Emx1 would be difficult to explain on the basis solely
of gradients of maturation because along the medial-lateral axis the
oldest neurons express the highest levels, whereas along the
rostral-caudal axis the youngest neurons express the highest levels.
For the genes analyzed here, with the exception of Cad6, the
graded expression observed in the CP is not seen in the VZ/SVZ or as
the postmitotic neurons migrate through the IZ to the CP. Thus, these
expression patterns exhibited by CP neurons are not simply a
maintenance of the relative expression levels found in their progenitor
cells in the VZ/SVZ. This difference between the CP and VZ/SVZ in
expression patterns is particularly interesting for Lhx2 and
Emx1, because their graded pattern of expression in the CP
is the opposite of those in the VZ/SVZ. Likewise, the graded expression
of SCIP in the CP is the opposite of that in the IZ. Thus,
the regulation of these genes in the CP differs from that in the VZ/SVZ
and IZ.
Our findings also suggest that potentially different regulatory
mechanisms control the expression of the cadherin genes that we have
analyzed. For example, Cad6 is in similarly graded patterns in the VZ and the CP, and therefore its expression pattern is expected
to be determined early. In contrast, Cad8 expression appears
to be mediolaterally graded only in the CP at E15.5. Later, at E18.5,
this graded CP expression is found in layer 5 throughout the neocortex,
and layered onto it is Cad8 expression in the upper layers,
but only in restricted domains in rostral and occipital cortex. This
finding suggests that the graded and areal expression of
Cad8 is regulated by mechanisms that are linked to those
regulating the layer specificity of cortical cells.
As we were preparing to submit this paper, a study by Miyashita-Lin et
al. (1999) was published that addresses a similar issue, although they
used a different mutant mouse (deficient for the homeodomain
transcription factor gene Gbx2) and examined a different set
of genes (Id-2, a helix-loop-helix transcription factor;
Tbr-1, a T-box transcription factor; RZR- , an
orphan nuclear receptor; EphA7; and Cad6).
As in the Mash-1 mutants (Tuttle et al., 1999), TCAs also
fail to project to the neocortex in the Gbx2 mutants (Miyashita-Lin et al., 1999). They found that the graded or
differential gene expression patterns, as well as the layer-specific
patterns, observed at P0 appear normal in the Gbx2 mutants.
The study by Miyahsita-Lin et al. (1999) and our study complement one
another well, because the two groups analyzed different genes in
different mutants but obtained the similar finding that TCAs are not
required to establish or maintain the differential gene expression
patterns normally observed in the developing neocortex. When we
consider the diverse and large set of genes analyzed in the two studies together, they reinforce one another and strongly support the conclusion that much of differential gene expression in the embryonic neocortex is established by mechanisms intrinsic to the telencephalon.
However, despite the findings presented in our study and that of
Miyashita-Lin et al. (1999), it may be premature to conclude that all
differential gene expression in the developing neocortex is TCA
independent. Both studies are limited by the fact that the
Mash-1 and Gbx2 mutants die on the day of birth
(E18.5/P0); therefore it was not possible to assess effects that TCAs
may have on gene expression at later stages of development. This is a
fairly significant caveat because most aspects of area-specific architecture and connectivity emerge postnatally. In addition, a
"rerouting" of TCAs to inappropriate target areas of the neocortex, rather than removing TCAs, may provide a more revealing test of their
potential influences on cortical gene expression. A late influence of
TCAs has been described for maintaining the area-specific distribution
of the  1 subunit of the GABAA receptor
observed in the somatosensory area of P7 rats, which only begins to
emerge a day or two before birth (Paysan et al., 1997). Even the
relatively late removal of TCAs by ablation of the dorsal thalamus at
P0 results in the loss of expression of this receptor subunit in the
somatosensory area (Paysan et al., 1997). This influence of TCAs
appears to be activity independent (Penschuck et al., 1999). A
particularly intriguing example is the expression of the H-2Z1 transgene that is primarily restricted to layer 4 of the granular parts
of somatosensory cortex (Cohen-Tannoudji et al., 1994). Although the
identity of an endogenous gene regulated in this manner is not known,
findings obtained from cortical slice cultures and heterotopic cortical
transplantation suggest that the area-specific expression of the
transgene is specified early in embryonic cortical development, even
though the transgene itself is not expressed until P2 (Cohen-Tannoudji
et al., 1994; Gitton et al., 1999a). Curiously, although cortical
slices removed from embryonic mice before TCA ingrowth and cultured for
a long term will later express the transgene, transgene expression
in vivo is dramatically attenuated in mice with a neonatal
thalamic ablation. Thus, although the early area-specific determination
of the transgene expression is independent of extrinsic influences
including TCAs, in vivo expression of the transgene does
seem to require TCAs (Gitton et al., 1999b).
Similarly, in vivo and in vitro studies on the
regional expression of the limbic system-associated protein (LAMP;
which is preferential for limbic cortex) and latexin (which is found in the infragranular layers of lateral cortex, including the neocortex and
the archicortex) have provided evidence that the regional specification
of the cerebral cortex, as measured by the commitment to differential
gene expression, occurs early during corticogenesis, probably within
the ventricular zone (Barbe and Levitt, 1991; Arimatsu et al., 1992;
Ferri and Levitt, 1993), although it is not known whether the in
vivo expression of LAMP and latexin requires TCAs. Nevertheless,
it is not clear whether it is valid to extrapolate the mechanisms
controlling regionalization of the cerebral cortex to the process of
arealization of the neocortex.
The graded and areal expression of the genes analyzed here is likely
controlled by a combinatorial action of regulatory genes that are
differentially expressed at earlier stages in the dorsal telencephalic
neuroepithelium, which gives rise to the CP. Candidates include the
homeodomain gene Emx2 and the paired domain gene
Pax6 that are expressed at the onset of cortical
neurogenesis in countergradients along the rostrolateral-to-caudomedial
extent of the dorsal telencephalic neuroepithelium (Walther and Gruss,
1991; Gulisano et al., 1996; Dyck et al., 1997; Mallamaci et al.,
1998). These genes have been proposed to be involved in regulating the
expression of axon guidance molecules that control the area-specific
targeting of TCAs (O'Leary et al., 1994), as well as imparting areal
identities to cortical neurons reflected by their gene expression
profiles and the axonal connections that they subsequently form (Chenn
et al., 1997). In turn, the differential expression of these early
regulatory genes is likely controlled by patterning centers localized
to the telencephalon (for review, see Rubenstein and Beachy, 1998). A
better understanding of the control of differential gene expression patterns intrinsic to the neocortex, as well as other features related
to neocortical arealization, will require defining the action and
downstream targets of early-expressed regulatory genes such as
Emx2 and Pax6 and the even earlier patterning
mechanisms that establish their differential expression across the
dorsal telencephalic neuroepithelium.
| |
FOOTNOTES |
Received Aug. 23, 1999; revised Sept. 28, 1999; accepted Sept. 28, 1999.
This work was supported by National Institutes of Health Grants NS31558
(D.D.M.O) and NS32817 (J.E.J). Y.N. was supported by the Human Frontier
Science Program and the Uehara Memorial Foundation. We thank T. Savage
for genotyping Mash-1 mutant embryos, S. Bertuzzi, G. Lemke, S. Mah, and C. Kintner for providing cDNAs, K. Bishop, A. Butler, N. Dwyer, and L. Krubitzer for helpful comments on this
manuscript, and K. Yee for help in the blind analysis of
Mash-1 mutant embryos.
Correspondence should be addressed to Dr. Dennis D. M. O'Leary,
Molecular Neurobiology Laboratory, The Salk Institute, 10010 North
Torrey Pines Road, La Jolla, CA 92037. E-mail:
dennis_oleary{at}qm.salk.edu.
| |
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T. Shimogori and E. A. Grove
Fibroblast Growth Factor 8 Regulates Neocortical Guidance of Area-Specific Thalamic Innervation
J. Neurosci.,
July 13, 2005;
25(28):
6550 - 6560.
[Abstract]
[Full Text]
[PDF]
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T. T. Kroll and D. D. M. O'Leary
Ventralized dorsal telencephalic progenitors in Pax6 mutant mice generate GABA interneurons of a lateral ganglionic eminence fate
PNAS,
May 17, 2005;
102(20):
7374 - 7379.
[Abstract]
[Full Text]
[PDF]
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K.-i. Mizutani and T. Saito
Progenitors resume generating neurons after temporary inhibition of neurogenesis by Notch activation in the mammalian cerebral cortex
Development,
March 15, 2005;
132(6):
1295 - 1304.
[Abstract]
[Full Text]
[PDF]
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C. Zimmer, M.-C. Tiveron, R. Bodmer, and H. Cremer
Dynamics of Cux2 Expression Suggests that an Early Pool of SVZ Precursors is Fated to Become Upper Cortical Layer Neurons
Cereb Cortex,
December 1, 2004;
14(12):
1408 - 1420.
[Abstract]
[Full Text]
[PDF]
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K. J. Huffman, S. Garel, and J. L. R. Rubenstein
Fgf8 Regulates the Development of Intra-Neocortical Projections
J. Neurosci.,
October 13, 2004;
24(41):
8917 - 8923.
[Abstract]
[Full Text]
[PDF]
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N. Funatsu, T. Inoue, and S. Nakamura
Gene Expression Analysis of the Late Embryonic Mouse Cerebral Cortex Using DNA Microarray: Identification of Several Region- and Layer-specific Genes
Cereb Cortex,
September 1, 2004;
14(9):
1031 - 1044.
[Abstract]
[Full Text]
[PDF]
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A. Leingartner, L. J. Richards, R. H. Dyck, C. Akazawa, and D. D.M. O'Leary
Cloning and Cortical Expression of Rat Emx2 and Adenovirus-mediated Overexpression to Assess its Regulation of Area-specific Targeting of Thalamocortical Axons
Cereb Cortex,
June 1, 2003;
13(6):
648 - 660.
[Abstract]
[Full Text]
[PDF]
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Z. Molnar, S. Higashi, and G. Lopez-Bendito
Choreography of Early Thalamocortical Development
Cereb Cortex,
June 1, 2003;
13(6):
661 - 669.
[Abstract]
[Full Text]
[PDF]
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S. Garel, K. J. Huffman, and J. L. R. Rubenstein
Molecular regionalization of the neocortex is disrupted in Fgf8 hypomorphic mutants
Development,
May 1, 2003;
130(9):
1903 - 1914.
[Abstract]
[Full Text]
[PDF]
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S. Garel, K. Yun, R. Grosschedl, and J. L. R. Rubenstein
The early topography of thalamocortical projections is shifted in Ebf1 and Dlx1/2 mutant mice
Development,
March 14, 2003;
129(24):
5621 - 5634.
[Abstract]
[Full Text]
[PDF]
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A. Bellion, M. Wassef, and C. Metin
Early Differences in Axonal Outgrowth, Cell Migration and GABAergic Differentiation Properties between the Dorsal and Lateral Cortex
Cereb Cortex,
February 1, 2003;
13(2):
203 - 214.
[Abstract]
[Full Text]
[PDF]
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D. Uziel, S. Muhlfriedel, K. Zarbalis, W. Wurst, P. Levitt, and J. Bolz
Miswiring of Limbic Thalamocortical Projections in the Absence of Ephrin-A5
J. Neurosci.,
November 1, 2002;
22(21):
9352 - 9357.
[Abstract]
[Full Text]
[PDF]
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K. M. Bishop, J. L. R. Rubenstein, and D. D. M. O'Leary
Distinct Actions of Emx1, Emx2, and Pax6 in Regulating the Specification of Areas in the Developing Neocortex
J. Neurosci.,
September 1, 2002;
22(17):
7627 - 7638.
[Abstract]
[Full Text]
[PDF]
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D. M. Kahn and L. Krubitzer
Massive cross-modal cortical plasticity and the emergence of a new cortical area in developmentally blind mammals
PNAS,
August 20, 2002;
99(17):
11429 - 11434.
[Abstract]
[Full Text]
[PDF]
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C. Zhou, S. Y. Tsai, and M.-J. Tsai
COUP-TFI: an intrinsic factor for early regionalization of the neocortex
Genes & Dev.,
August 15, 2001;
15(16):
2054 - 2059.
[Abstract]
[Full Text]
[PDF]
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Y. Nakagawa and D. D. M. O'Leary
Combinatorial Expression Patterns of LIM-Homeodomain and Other Regulatory Genes Parcellate Developing Thalamus
J. Neurosci.,
April 15, 2001;
21(8):
2711 - 2725.
[Abstract]
[Full Text]
[PDF]
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S. Tole and E. A. Grove
Detailed Field Pattern Is Intrinsic to the Embryonic Mouse Hippocampus Early in Neurogenesis
J. Neurosci.,
March 1, 2001;
21(5):
1580 - 1589.
[Abstract]
[Full Text]
[PDF]
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Q. Liu, N. D. Dwyer, and D. D. M. O'Leary
Differential Expression of COUP-TFI, CHL1, and Two Novel Genes in Developing Neocortex Identified by Differential Display PCR
J. Neurosci.,
October 15, 2000;
20(20):
7682 - 7690.
[Abstract]
[Full Text]
[PDF]
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TOP
ABSTRACT
INTRODUCTION
