1 Division of Developmental Biology, Children's Hospital Research Foundation,
Cincinnati, OH 45229-3039, USA
2 Wallenberg Neuroscience Center, Division of Neurobiology, Lund University,
Solvegatan 17, BMC A11, S-221 84 Lund, Sweden
3 Howard Hughes Medical Institute, The Salk Institute, 10010 North Torrey Pines
Road, La Jolla, CA 92037, USA
* Author for correspondence (e-mail: kenneth.campbell{at}chmcc.org)
Accepted 12 December 2002
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SUMMARY |
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Key words: Acetylcholine esterase, Amygdala, DLX, GSH2, MASH1, Neurogenin 2, NR2E1, Pallium, Pallio-subpallial boundary, Subpallium, Tailless, Ventral pallium, Mouse
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INTRODUCTION |
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The dorsal and ventral portions of the telencephalon are separated by a
morphologically identifiable radial glial palisade
(Stoykova et al., 1997;
Götz et al., 1998
;
Chapouton et al., 1999
). The
location of this glial palisade marks the pallio-subpallial boundary and
coincides with the juxtaposition of cells expressing distinct developmental
control genes. Precursor cells on the subpallial side express the homeobox
gene Gsh2 and the bHLH gene Mash1 (Ascl1
Mouse Genome Informatics), whereas cells on pallial side express the paired
homeobox gene Pax6 as well as the bHLH genes neurogenin 1
(Ngn1) and Ngn2. Several of these developmental control
genes play important roles in the establishment and/or maintenance of this
boundary (Stoykova et al.,
1997
; Stoykova et al.,
2000
; Chapouton et al.,
1999
; Chapouton et al.,
2001
; Corbin et al.,
2000
; Toresson et al.,
2000
; Yun et al.,
2001
). For example, Gsh2 is required to repress pallial
gene expression in the subpallium and in its absence ventricular zone cells in
the lateral ganglionic eminence (LGE) are misspecified, resulting in a
truncation of the LGE-derived striatum
(Corbin et al., 2000
;
Toresson et al., 2000
;
Yun et al., 2001
). Conversely,
in Small eye (Sey) mutants, which contain a point mutation
in the Pax6 gene (Hill et al.,
1991
), the pallium is misspecified by ectopic ventral gene
expression along with restricted loss of pallial gene expression
(Stoykova et al., 1996
;
Stoykova et al., 2000
;
Toresson et al., 2000
;
Yun et al., 2001
;
Muzio et al., 2002
).
The above-mentioned genes are unlikely to be the only molecular players
involved in the establishment and/or maintenance of the pallio-subpallial
boundary. In the present study, we have examined a role for the orphan nuclear
receptor Tlx (also known as tailless; Nr2e1
Mouse Genome Informatics) (Yu et al.,
1994; Monaghan et al.,
1995
) in this process. Homozygous Tlx mutants have been
shown to exhibit altered telencephalic morphology as well as abnormally
aggressive behavior (Monaghan et al.,
1997
; Young et al.,
2002
). However, the underlying mechanisms that lead to these
defects have not been elucidated. We show here that Tlx is required
for correct dorsal-ventral patterning of the embryonic telencephalon and for
the normal differentiation of amygdalar structures in the ventrolateral
telencephalon. Moreover, our data demonstrate that Tlx and
Pax6 cooperate genetically to establish the pallio-subpallial
boundary.
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MATERIALS AND METHODS |
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Histological analysis
Embryos were fixed and sectioned on a cryostat as previously described
(Toresson et al., 2000). Adult
brains were removed from 3-week- to 8-month-old animals and fixed overnight by
immersion in 4% paraformaldehyde at 4°C before sinking in 30% sucrose and
sectioning at 30 µm on a cryostat. Immunohistochemistry was performed on
slide-mounted sections for the embryonic tissue, whereas the adult brain
sections were immunostained under free-floating conditions. The protocol was
as previously described (Olsson et al.,
1997
) with the modification that 0.3% H2O2
was used for 10-15 min instead of 3% H2O2. Primary
antibodies were used at the following concentrations: rabbit anti-DARPP-32
(1:1000, Chemicon); rabbit anti-distalless (i.e. pan DLX) (1:1000, provided by
G. Panganiban); rabbit anti-Er81 (1:5000, provided by S. Morton and T.
Jessell); rabbit anti-GSH2 (1:5000)
(Toresson et al., 2000
);
rabbit anti-ISL1/2 (1:500, provided by T. Edlund); rabbit anti-MASH1 (1:1000,
provided by J. Johnson); rabbit anti-MEIS2 (1:5000, provided by A. Buchberg);
rabbit anti-nestin (1:500, provided by R. McKay); rabbit anti-parvalbumin
(1:1000, provided by P. Emson); rabbit anti-PAX6 (1:200, Covance) and goat
anti-PAX6 (1:250, Santa Cruz). The secondary antibodies used were biotinylated
swine anti-rabbit antibodies (DAKO), FITC-conjugated anti-rabbit (Jackson
Immunoresearch) and Cy3-conjugated anti-goat antibodies (Jackson
Immunoresearch). The ABC kit (Vector labs) was used to visualize the reaction
product for the biotinylated antibodies with diaminobenzidine as the final
chromogen. Confocal microscopy was performed on a Zeiss LSM510 confocal
microscope.
For acetylcholine esterase (AChE) staining, adult brain sections were mounted on slides and dried at 37°C for 1 hour before placing in incubation medium (3 mM cupric sulfate, 10 mM glycine, 15 mM acetic acid, 35 mM sodium acetate, 0.08 mM tetraisopropyl pyrophosophoramide, 1.5 mM acetylthiocholine iodide, adjusted to pH 5.0) at 37°C for 4 hours. They were then developed in a 40 mM sodium sulphide solution (pH 7.5).
Non-radioactive in situ hybridization was performed as described in
Toresson et al. (Toresson et al.,
1999) using the following probes: Dbx1 (IMAGE clone
AA003371) (Yun et al., 2001
),
Sfrp2 (Kim et al.,
2001
), Ngn2 (Sommer
et al., 1996
) and Tlx
(Yu et al., 1994
;
Monaghan et al., 1995
).
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RESULTS |
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At E12.5, Tlx mutants appear morphologically normal, however, at
the molecular level they exhibit defects in the patterning of the lateral
telencephalon. The pallio-subpallial boundary is marked by the juxtaposition
of GSH2- and PAX6-expressing cells of the subpallial and pallial ventricular
zones, respectively (Fig.
2A-C). Confocal micrographs show only a minimal overlap (i.e. 2-3
cell diameters) of cells expressing these two proteins in the wild-type
telencephalon (Fig. 2C,D). In
Tlx mutants, GSH2-expressing cells are found dorsal to their normal
limit (i.e. up to and in the LGE-pallium angle,
Fig. 2E). In these mutants, the
overlap of GSH2 and PAX6 in ventricular zone cells is much broader than in the
wild types (Fig. 2E-H).
Interestingly, this broader overlap correlates with the altered organization
of PAX6-positive cells in the subventricular region. In wild types,
PAX6-positive cells are seen to emanate from the region where GSH2 and PAX6
overlap in the ventricular zone and to form a stream from the subventricular
zone (SVZ) out to the pial surface (Fig.
2B-D) (Puelles et al.,
1999). In Tlx mutants, however, the PAX6-positive cells
form a broader domain in the SVZ (Fig.
2H), which corresponds well with the wider overlap of GSH2 and
PAX6 expression. The dorsal shift of GSH2 expression in the Tlx
mutants is still observed at E14.5 (Fig.
3E), however, high-level PAX6 expression appears to have retracted
dorsally by this stage (Fig.
3H). Moreover, in the Tlx mutants, the position of the
PAX6-expressing cells in the SVZ region has shifted dorsally and there are
2.2-fold more of these cells (Fig.
3H) than in wild types (Fig.
3D; 514±89 versus 235±90 cells, respectively,
P<0.02, n=3).
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|
The bHLH genes Mash1 and Ngn2 also mark cells on the pallial and subpallial sides of the pallio-subpallial boundary, respectively. In addition to the dorsal expansion of GSH2 expression in Tlx mutants (Fig. 2E, Fig. 3E), MASH1-positive cells can also be found in a more dorsal position in the mutant telencephalon (Fig. 3F). Concomitant with this, Ngn2 expression in Tlx mutants is retracted from its normal ventral limit (Fig. 3G), as is the case for PAX6 (Fig. 3H). These alterations in gene expression are seen at both rostal and caudal levels. Taken together, these results demonstrate a dorsal shift in the expression limits of genes, which normally abut at the pallio-subpallial boundary.
Based on gene expression patterns, the pallium has recently been divided
into medial, dorsal, lateral and ventral portions
(Puelles et al., 1999;
Puelles et al., 2000
;
Yun et al., 2001
). The ventral
pallium is a small domain located immediately dorsal to the pallio-subpallial
boundary. This pallial region is normally marked, at least in part by the
expression of Dbx1, a homeobox gene
(Yun et al., 2001
), and
Sfrp2 (secreted frizzled related protein 2), which encodes a putative
Wnt inhibitor (Kim et al.,
2001
). In addition to the dorsal shift in the expression limits of
GSH2, MASH1, Ngn2 and PAX6 in the lateral telencephalon, the ventral
pallial region of the Tlx mutants lacks expression of both
Dbx1 (Fig. 4B) and
Sfrp2 (Fig. 4D). This
loss in expression is specific to the ventral pallium as both genes continue
to be expressed in the mutant diencephalon
(Fig. 4B and
Fig. 4D).
|
Unlike the case at E12.5, by E14.5 and onwards the Tlx mutant
forebrains do not appear normal in that there is a significant reduction in
the size of the LGE (see Figs
3,
4,
5). Despite this reduction, the
dorsal limit of the DLX-expressing SVZ appears to have shifted dorsally in
accordance with the observed shift of GSH2 and MASH1
(Fig. 5B). Notably, this is not
the case for the dorsal limit of Islet1 expression
(Fig. 5D). However, we observed
a dorsal expansion in the LGE SVZ-expression domain of the ETS transcription
factor Er81 (Fig. 5F). In the
wild type, Er81 is also expressed in the ventricular zone of the pallium,
including at least the ventral and lateral pallium
(Fig. 5E). Commensurate with
the dorsally expanded subpallial SVZ expression of Er81, the pallial
ventricular zone expression is greatly reduced in at least the ventral pallium
(Fig. 5F). We have recently
shown that Er81 and Islet1 mark separate dorsal and ventral progenitor pools
in the DLX-expressing SVZ of the LGE, respectively
(Stenman et al., 2003). These
findings indicate that the patterning defects around the pallio-subpallial
boundary in Tlx mutants result in the selective expansion of certain
dorsal LGE characteristics (i.e. DLX and Er81 expression) at the expense of
those normally marking the ventral pallium. Thus the data presented above show
that the loss of Tlx gene function results in a misspecification of
the ventral pallium (Fig.
6).
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|
In addition to gene expression patterns, the pallio-subpallial boundary is also marked by a palisade of radial glial fibers originating in the ventricular zone near the LGE-pallium angle and coursing to the pial surface. In the wild-type telencephalon, this glial palisade can be visualized by Nestin staining (Fig. 7A). Tlx mutants show fewer nestin-positive radial glial fibers in the region of the pallio-subpallial boundary. Moreover, these fibers do not appear to fasciculate to form the palisade (Fig. 7B). The calcium-binding protein parvalbumin also marks radial glia in the pallio-subpallial boundary (Fig. 7C). Again, the parvalbumin-positive radial glial fibers in the mutant telencephalon fail to fasciculate (Fig. 7D). Thus, in addition to regulating gene expression at the pallio-subpallial boundary, Tlx gene function is also required for the normal formation of the radial glial palisade. Indeed, the altered patterning, described above, may contribute significantly to the abnormal formation of this radial glial palisade.
|
Gene dosage of Tlx and Pax6 regulates the
patterning of the lateral telencephalon
Several studies have shown that Pax6 is required for the correct
patterning at the pallio-subpallial boundary
(Stoykova et al., 1996;
Stoykova et al., 2000
;
Toresson et al., 2000
;
Yun et al., 2001
) as well as
the formation of the radial glial palisade
(Stoykova et al., 1997
;
Götz et al., 1998
;
Chapouton et al., 1999
).
Homozygous Sey mutants show ectopic ventral (i.e. Gsh2,
Mash1 and Dlx) gene expression in the pallium and a loss of dorsal (i.e.
Ngn1 and Ngn2) gene expression in the corresponding domain.
Moreover, the ventral pallium markers Dbx1
(Yun et al., 2001
) and
Sfrp2 (Kim et al.,
2001
; Muzio et al.,
2002
) are both lost in the Sey/Sey mutants. The fact that
Tlx mutants exhibit a similar, but much less severe, phenotype to
Sey homozygotes could be because of Pax6 directly or
indirectly regulating Tlx expression. This does not seem to be the
case, however, because Tlx is expressed in Sey/Sey mutants
both at E12.5 and E14.5 (data not shown). Furthermore, the data presented
above demonstrate that Tlx is not a general regulator of
Pax6 expression.
Tlx and Pax6 may, therefore, co-operate genetically to establish the pallio-subpallial boundary. To analyze this we generated a series of compound Sey and Tlx alleles at E14.5 and E16.5. Mice heterozygous for either the Sey or Tlx mutations do not show alterations in gene expression at the pallio-subpallial boundary with respect to any of the markers we have employed here (data not shown). However, in Tlx+/;Sey/+ compound heterozygotes, a few scattered GSH2- and MASH1-positive cells are seen in the region of the ventral pallium (data not shown). Furthermore, the expression of Sfrp2 is reduced, and at some levels missing, in Tlx+/;Sey/+ compound heterozygotes (data not shown). The double heterozygous phenotype, however, is much less severe than that observed in Tlx homozygous mutants (described above). We also analyzed Tlx/;Sey/+ mutants and in all cases the limit of GSH2 (Fig. 8D), MASH1 (Fig. 8E) and DLX (data not shown) expression extends further dorsally into the pallium than in Tlx homozygous mutants (Fig. 8A,B). Furthermore, Ngn2 is downregulated in the pallial domain containing ectopic GSH2 and MASH1 cells (Fig. 8F). These findings demonstrate that removal of one allele of Pax6 on the Tlx/ mutant background results in a significant dorsal shift in the expression limits of GSH2, MASH1, DLX and Ngn2, as compared to the Tlx/ mutants alone. It is interesting to note that the patterning defects in the Sey/Sey mutants (Fig. 8G-I) are more severe than those observed in Tlx/;Sey/+ mutants. Moreover, the patterning defects observed in Tlx+/; Sey/Sey (data not shown) and Tlx/;Sey/Sey mutants (Fig. 8J-L) were not noticeably different from those seen in the Sey/Sey mutants. Thus Pax6 appears to be required for correct patterning in broader portions of the pallium (i.e. lateral and dorsal pallium) than Tlx. Both, however, are important for the correct patterning of gene expression around the pallio-subpallial boundary. Moreover, our findings show that correct gene dosages of both Tlx and Pax6 are required to properly establish this boundary (Fig. 9).
|
|
Altered amygdalar development in Tlx mutants
Tlx mutants have smaller than normal brains, which exhibit gross
morphological defects in numerous telencephalic regions, including the
amygdalar region (Monaghan et al.,
1997). However, specific defects in the amygdala of these mutants
have not, as yet, been described. Previous studies have suggested that the
basolateral amygdala derives from the ventral pallium
(Fernandez et al., 1998
;
Puelles et al., 1999
;
Puelles et al., 2000
). Given
the molecular misspecification of the ventral pallial region of Tlx
mutants (described above), alterations in the development of this amygdalar
nucleus would be predicted. We analyzed the amygdalar region in perinatal
animals, however, because Tlx mutants are viable, we were also able
to analyze this region in mature brains (i.e. three weeks to eight months
old).
In wild-type animals, the basolateral amygdala is marked by the expression of Er81 from late-embryonic stages into adulthood (Fig. 10A). Tlx mutant brains stained for Er81 reveal little evidence of a normal basolateral nucleus in either perinatal or mature brains (Fig. 10B). In addition to marking the basolateral nucleus, acteylcholine esterase (AChE) staining also reveals the lateral nucleus and the central nucleus of the amygdala (Fig. 10C). In the Tlx mutants, some AChE staining is found in the presumptive region of the basolateral and lateral amygdala (Fig. 10D), however, the amount of staining is drastically reduced as compared to the wild type. Despite this, the size of the central nucleus appears rather similar to that in wild types (Fig. 10C,D). Staining for the phosphoprotein DARRP-32 outlines the lateral and basolateral amygdala in wild types, revealing the `teardrop'-shaped nuclei (Fig. 10E). This morphology is not apparent in Tlx mutants (Fig. 10F). DARPP-32 marks the interstitial nucleus of the amygdala (Fig. 1E), as does MEIS2 (Fig. 10G). This nucleus also seems to be present in Tlx mutants and although its morphology is changed, it appears to be somewhat similar in size to that in wild types (Fig. 10G,H). Thus the alterations in the Tlx mutant amygdala seem to be rather specific to the basolateral and lateral amygdala.
|
In order to strengthen the correlation between the misspecification of the
ventral pallium and the alterations in the development of the basolateral and
lateral amygdalar nuclei, we have performed fate-mapping studies that, by
exclusion, support a pallial origin for these nuclei. Using a Dlx5/6
enhancer (Zerucha et al.,
2000) to drive cre recombinase in the subpallial SVZ
(Stenman et al., 2003
) of
gtROSA reporter mice (Mao et al.,
1999
), we have found that the lateral and basolateral amygdala
contain very few cells originating in the subpallium
(Fig. 10I). In fact, few, if
any, of the subpallium-derived cells in the basolateral amygdala express Er81
(Fig. 10J). Interestingly, the
normal expression of Er81 in the ventricular zone of the ventral pallium is
lost in Tlx mutants (Fig.
5F). Thus, this data, together with the data presented above,
indicate that the Er81-expressing cells in the basolateral amygdala are
probably derived from the ventral pallium and may represent the glutamatergic
projection neurons characteristic of this nucleus
(Swanson and Petrovich, 1998
).
The small population of subpallium-derived cells
(Fig. 10J) might represent the
GABAergic interneuron population in this nucleus (e.g.
Smith et al., 2000
). In
addition, our data show that the central and medial nuclei of the amygdala are
largely derived from the subpallium (Fig.
10I). In summary, the present findings suggest that the patterning
defects around the pallio-subpallial boundary in Tlx mutants have
severe consequences for the development of basolateral and lateral nuclei in
the amygdalar complex.
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DISCUSSION |
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A stream of PAX6-positive cells, which emanate from the ventricular zone
and course downward towards the pial surface, is found at the
pallio-subpallial boundary (Puelles et
al., 1999). Interestingly, the point at which these cells emerge
from the ventricular zone seems to correlate with a slight overlap of GSH2-
and PAX6-expressing cells (see Fig.
2), which is also the case in the embryonic chick telencephalon
(von Frowein et al., 2002
).
Gsh2 appears to be important for the development of this stream of
cells because the number of PAX6-positive cells is reduced (but not missing)
in Gsh2 mutants (Toresson et al.,
2000
). Although the PAX6-positive stream of cells has been
suggested to belong to the subpallium
(Puelles et al., 1999
;
Puelles et al., 2000
), it may,
in fact, represent a population of transitional cells between the pallial and
subpallial compartments. Unlike the case in Gsh2 mutants, the
subventricular domain of PAX6-positive cells appears to be broader in
Tlx mutants as compared to that in wild types. This correlates well,
at least at early stages, with the increased overlap of the GSH2- and
PAX6-expression domains in the Tlx mutant ventricular zone.
In Tlx mutants, the dorsal shift in the expression limits of genes
that abut at the pallio-subpallial boundary, is accompanied by a loss of
Dbx1 and Sfrp2 expression (i.e. ventral pallial identity).
It is unclear, however, whether this indicates a direct role for Tlx
in the development of the ventral pallium or if the effect is indirect.
Because Tlx is not required for the diencephalic expression of
Dbx1 and Sfrp2, a direct role for this gene in the
regulation of these ventral pallial markers seems unlikely. Alternatively, the
dorsal expansion of GSH2 and MASH1 in the mutants might suggest a role for
Tlx in the repression of these factors within the ventral pallium.
The loss of ventral pallial identity in the mutants could therefore be because
of the ectopic expression of subpallial genes. In support of this, the ventral
pallium marker Dbx1 is up-regulated in the LGE of homozygous
Gsh2 mutants (Yun et al.,
2001), suggesting a role for Gsh2 in the repression of
Dbx1. Homozygous Sey mutants (which display ectopic
Gsh2 gene expression in the pallium) also exhibit a loss of ventral
pallial identity (Kim et al.,
2001
; Yun et al.,
2001
; Muzio et al.,
2002
). However, Sey/Sey mutants display more
severe patterning defects, including both the lateral and the dorsal pallium
as well (Toresson et al.,
2000
; Yun et al.,
2001
).
The origins of different amygdalar nuclei has previously been unclear.
Although it has been suggested that its nuclear components derive from both
the dorsal and ventral halves of the embryonic telencephalon
(Swanson and Petrovich, 1998).
In particular, the basolateral amygdala and the lateral amygdala have been
suggested to derive from the ventral pallium and the lateral pallium,
respectively (Fernandez et al.,
1998
; Puelles et al.,
1999
; Puelles et al.,
2000
). However no direct evidence for this has been provided thus
far. A recent study (Gorski et al.,
2002
) has fate mapped the Emx1-expression region of the
dorsal telencephalon. This study showed that, in addition to the neocortex and
hippocampus, many structures in the ventrolateral cortical region are also
derived from the Emx1-expression domain, including both the
basolateral and lateral amygdala. A defining feature of the ventral pallium is
the lack of Emx1 gene expression, along with the expression of
Pax6, Tbr1, Dbx1 and Sfrp2
(Puelles et al., 1999
;
Yun et al., 2001
;
Kim et al., 2001
). Thus the
Emx1-expressing pallial regions should not contribute neurons to the
basolateral and lateral amygdala. However, it is possible that low levels of
Emx1 are normally expressed in the ventral pallium. This expression
might not be easily detected by in situ hybridization but could drive
sufficient levels of cre recombinase to mark cells derived from the
ventral pallium (i.e. neurons in the basolateral and lateral amygdala). Our
data support a ventral pallial origin for both of these amygdalar nuclei
because the patterning defects observed in the Tlx mutants appear to
be restricted to the ventral pallium. However, it is difficult to determine
whether a portion of the lateral pallium is also affected in these mutants
because of a lack of specific markers for this pallial region. These amygdalar
nuclei are largely generated between E11 and E14 in the mouse
(McConnell and Angevine,
1983
), which correlates well with the timing of the observed
patterning defects in Tlx mutants. Moreover, our fate-mapping
studies, using the subpallial Dlx5/6 expression domain, demonstrate a
largely pallial origin for the basolateral and lateral amygdala because only a
few cells are labeled in these nuclei. In contrast, the central and medial
amygdalar nuclei do appear to derive from subpallial sources. Taken together,
these fate-mapping studies provide convincing evidence for both pallial and
subpallial contributions to the amygdalar complex.
Homozygous Tlx mutants can survive after birth and have been
reported to display abnormally aggressive behavior
(Monaghan et al., 1997;
Young et al., 2002
). Because
the lateral and basolateral amygdala are both thought to be involved in the
regulation of fear rather than aggression
(Oakes and Coover, 1997
;
Nader et al., 2001
), it is
unlikely that the amygdalar defects in these mutants are responsible for the
aggressive behavior. It should be noted, however, that other morphological
defects are present in the Tlx mutant forebrain
(Monaghan et al., 1997
), which
may contribute more or less to their aggressive behavior. Notably, the LGE in
Tlx mutants is considerably more reduced in size than other
telencephalic structures such as the cortex. This reduction in the size of the
LGE is unlikely to contribute significantly to the observed defects at the
pallio-subpallial boundary in the Tlx mutants because the opposite
phenotype would be predicted, at least with respect to the gene expression.
Indeed, it is probable that a smaller LGE will result in a ventral shift of
the expression limits of genes that abut at the pallio-subpallial boundary. We
are currently investigating the mechanisms that underlie the reduced LGE size
in the Tlx mutants, which may include either, or a combination of,
cell death, lack of proliferation or a patterning defect.
Tlx and Pax6 interactions
As mentioned above, the pallial phenotype of homozygous Tlx and
Sey mutants share several similarities, specifically the alteration
in gene expression around the pallio-subpallial boundary as well as altered
development of the radial glial palisade. This motivated us to further examine
the relationship between these two genes in the process of telencephalic
dorsal-ventral patterning. Our findings show that the correct gene dosage of
both Tlx and Pax6 is crucial for the establishment of the
pallio-subpallial boundary. Although the loss of one allele of Pax6
on the homozygous Tlx mutant background results in a significant
worsening of the pallial phenotype, removal of either one or both of the
Tlx alleles on the homozygous Sey background does not
further exacerbate the phenotype as compared to Sey/Sey mutants
alone. Therefore Tlx is required to augment Pax6 gene
function in the ventral-most portions of the pallium and thereby to correctly
position the pallio-subpallial boundary. This genetic interaction with
Pax6 provides an explanation for why Tlx, despite its broad
expression pattern, is crucial for the establishment of the pallio-subpallial
boundary. It seems that Tlx is not the only gene that is expressed
across the pallio-subpallial boundary and regulates correct gene expression at
this boundary. The zinc finger gene Gli3, which is expressed on both
sides of the pallio-subpallial boundary, is also required in this process
(Tole et al., 2000;
Rallu et al., 2002
). As is the
case with Tlx, this is not through the direct regulation of
Pax6 expression.
The fact that Tlx and Pax6 interact genetically in the
establishment of telencephalic dorsal-ventral identity suggests that their
protein products might do so through direct molecular interactions. PAX6 is
known to physically interact with the HMG box protein SOX2 in the regulation
of eye development (Kamachi et al.,
2001). Sox2 is expressed in the telencephalic ventricular
zone in regions overlapping with Pax6 expression
(Zappone et al., 2000
),
suggesting that similar interactions may be involved in telencephalic
patterning. No interacting partners for TLX have, as yet, been identified in
vertebrates, not even retinoid X receptors (RXRs), which are known to interact
with many orphan nuclear receptors (for a review, see
Blumberg and Evans, 1998
).
Furthermore, we have not been able to detect a physical interaction between
TLX and PAX6 (unpublished data). It seems therefore that these two genes
regulate telencephalic patterning through independent but convergent pathways.
The convergence of Tlx and Pax6 to pattern the
pallio-subpallial boundary could be mediated through the regulation of common
gene targets. Pax6 has been implicated in the regulation of
Ngn2 expression in the pallium
(Stoykova et al., 2000
;
Toresson et al., 2000
;
Yun et al., 2001
). Recently,
an enhancer, which is capable of driving Ngn2 expression in the
pallium, including the ventral pallium, has been identified
(Scardigli et al., 2001
). This
enhancer was shown to require Pax6 gene function for its correct
expression. Interestingly, this enhancer element contains a putative TLX
binding site (J. S., K. C. and F. Guillemot, unpublished data), suggesting
that TLX as well as PAX6 may be involved in directly regulating Ngn2
gene expression in the ventral pallial region. Such a regulation may explain,
at least in part, the Tlx mutant phenotype, because
Neurogenins have previously been shown to negatively regulate
subpallial (e.g. Mash1 and Dlx genes) gene expression
(Fode et al., 2000
).
Interestingly, co-regulation of a common gene by TLX and PAX6 has been shown
to occur in eye development. The paired homeobox genes Pax6 and
Pax2 are expressed in the developing retina and optic stalk,
respectively. These factors regulate the development of these two eye regions,
in part, through direct mutual repression (i.e. PAX6 represses Pax2
gene expression in the retina and vice versa)
(Schwarz et al., 2000
).
Moreover, the Pax2 promoter contains a functional TLX binding site,
which, when bound by TLX, results in the repression of promoter activity
(Yu et al., 2000
). It is
interesting to note that Tlx is expressed in both the retina and the
optic stalk (Yu et al., 1994
;
Monaghan et al., 1995
;
Yu et al., 2000
), and yet it
is proposed to participate in patterning the retina-optic stalk transition
(Yu et al., 2000
). This is
similar to the data presented here in which Tlx is expressed on both
sides of the pallio-subpallial boundary but is involved in the establishment
of this boundary. The present results indicate that this function is dependent
on a genetic interaction with the pallial-enriched Pax6 gene.
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ACKNOWLEDGMENTS |
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