1 Department of Human Anatomy and Genetics, University of Oxford, South Parks
Road, Oxford OX1 3QX, UK
2 Max-Planck Institute of Biophysical Chemistry, Department of Molecular Cell
Biology, 37077 Gottingen, Germany
Author for correspondence (e-mail:
zoltan.molnar{at}anat.ox.ac.uk)
Accepted 25 July 2002
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SUMMARY |
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Key words: Axon guidance, Patterning, Telencephalon, Semaphorins, Mouse, Pallial/subpallial boundary, Thalamocortical projections, Corticofugal axons, Mouse
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INTRODUCTION |
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Early-generated, largely transient neuronal populations, have been
described to extend pioneering axonal projections through these critical
boundaries, thus providing scaffolds or temporary targets, `guide-post cells',
for the developing axonal projections
(McConnell et al., 1989;
Mitrofanis and Guillery,
1993
). It has been suggested that the early outgrowth of TCAs from
the diencephalon might be governed by pioneering projections from cells within
the internal capsule (Métin and
Godement, 1996
), while the crossing of the PSPB by the TCAs might
be dependent on selective fasciculation with these corticofugal projections
(Molnár and Blakemore,
1995
). Recent studies in homeobox gene mutants seem to support
these suggestions. In Mash1 (Ascl1 Mouse Genome
Informatics) homozygous mice, the internal capsule cells with thalamic
projections are missing and TCAs fail to enter the internal capsule
(Tuttle et al., 1999
). In
Emx2 homozygous mice, the early internal capsule projections take an
aberrant ventral route at the diencephalon-telencephalon junction, and some of
the TCAs follow them as they traverse that region
(López-Bendito et al.,
2002
). In Tbr1, Gbx2 and Pax6 mutants, both TCAs
and CFAs become derailed at the PSPB
(Miyashita-Lin et al., 1999
;
Hevner et al., 2001
;
Hevner et al., 2002
), but the
nature of these defects is not fully understood.
Pax6 has been found to have an essential role in brain
morphogenesis (Walter and Gruss,
1991; Schmahl et al.,
1993
; Stoykova et al.,
1996
; Stoykova et al.,
1997
; Caric et al.,
1997
; Warren et al.,
1999
), and has been implicated in early axonal pathfinding
(Mastick et al., 1997
;
Kawano et al., 1999
;
Pratt et al., 2000
). A
Pax6-positive group of cells was observed to extend along the PSBP,
from the ventricular/subventricular zones (VZ/SVZ) towards the mantle of the
basal telencephalon (Stoykova et al.,
1997
; Fernandez et al.,
1998
; Puelles et al.,
2000
). Lack of Pax6 function in the
Pax6Sey/Sey mutant brains, results in a prominent
ventralization of the molecular characteristics of the region of the ventral
pallium that leads to severe morphological malformation in the basolateral
cortex (Stoykova et al., 2000
;
Toresson et al., 2000
;
Yun et al., 2001
).
Using a Pax6/lacZ knockout mouse
(St-Onge et al., 1997), we
studied the consequences of the lack of Pax6 on the TCA and CFA
pathfinding with special interest to the axonal navigation within the PSPB. We
found that in Pax6-deficient brains, both TCAs and CFAs showed severe
malformations of their pathfinding when reaching the PSPB domain and these
correlated with ultrastructural defects of this region and a failure of the
expression of axonal guidance molecules, such as the attractants Sema3c and
Sema5a.
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MATERIALS AND METHODS |
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Detection of Pax6 promoter activity
The expression pattern of Pax6 in the isolated brains from
heterozygous and homozygous embryos was analysed by wholemount-ß-gal
staining (Gossler and Zachgo,
1993; Stoykova et al., 1988). Stained brains were embedded in 4%
agarose and coronal sections were cut with a Vibroslicer (Leica, VT1000S) at
50 µm. Sections were mounted on slides in PBS/glycerol, coverslipped and
examined with a light microscope. In situ hybridization on wholemounts and in
sections was performed with Pax6 in situ probe (Walther and Gruss,
1991) as described elsewhere (Stoykova and
Gruss, 1994
).
Immunohistochemistry
E14.5 and E18.5 wild-type, heterozygote and homozygous brains were used
(see numbers at Table 1 for
each time point). After ß-galactosidase staining of the isolated brains,
tissue was embedded in 4% agarose and sectioned at 40- 100 µm coronally
with a Vibroslicer. Alternatively, after immersion of brains in 30% sucrose
solution overnight at 4°C, tissue was sectioned coronally at 20 µm with
a microtome. Immunohistochemistry was carried out with rat anti-L1
(Boehringer, 1:100) and mouse monoclonal anti-TAG1 (Developmental Studies
Hybridoma Bank, Iowa; 1:100) antibodies. Sections were incubated overnight
with primary antibodies at 4°C. Primary antibodies were detected by a
biotinylated secondary antibody (1:100) and the avidin-biotin complex (ABC,
dilution 1:100) method (Vector). The reaction was visualized using 0.05%
diaminobenzidine solution (Sigma). Sections were rinsed, dehydrated and
mounted in Eukitt mounting media. To study cytoarchitecture, sections were
treated with 0.5% Cresyl Violet solution. Sections were visualized by light
microscopy and documented with a Leica DC500 digital camera.
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Axonal tracing with carbocyanine dyes
Brains at E14.5, E15.5 and E18.5 were fixed overnight in 4%
paraformaldehyde (see Table 1).
To expose the dorsal thalamus, a coronal cut was made at the caudal edge of
the diencephalon. The zona limitans intrathalamica provided the landmark for
orientation. Single crystals of DiI (1, 1- dioctadecyl -3, 3,
3',3'-tetramethyl-indocarbocyanine perchlorate; Molecular Probes),
and of DiA [4-(4-(dihexadecylamino)styryl)-N-methylpyridium iodide; Molecular
Probes], were placed with a fine tungsten wire into the dorsal thalamus or
dorsal cortex under a dissecting microscope. Brains were incubated in 4%
paraformaldehyde at room temperature (RT) for 3 weeks. The brains were then
embedded in 4% agarose and 100 µm coronal Vibroslicer sections were cut.
Tissue was counterstained with 0.002% bisbenzimide. Sections were coverslipped
in 1:1 mixture of PB and glycerol and analysed with a fluorescent microscope
(Leica DMR) or a laser scanning confocal microscope (Leica TCS).
Electron microscopy
Four E15.5 mice and four E18.5 mice (two Pax6-/- and
two Pax6+/+ brains for each age) were used. Brains were
immersed overnight in ice-cold fixative containing 2% paraformaldehyde and 2%
glutaraldehyde in 0.1 M phosphate buffer (PB, pH 7.2-7.4). Tissue blocks were
dissected and washed in 0.1 M PB. Coronal 60 µm sections of the
telencephalon were cut with a Vibroslicer. After several washes in 0.1 M PB,
selected sections were treated with OsO4 (1% in 0.1 M PB),
block-stained with uranyl acetate, dehydrated in a graded series of ethanols
and flat-embedded on glass slides in Durcupan (Fluka) resin. Ultrathin
sections (60 to 80 nm) from selected areas of the telencephalon were picked up
on coated copper grids. The grids were counterstained with saturated aqueous
uranyl acetate followed by lead citrate. The sections were examined with a
Joel (JEM 1010) transmission electron microscope.
In situ hybridization
In situ hybridization experiments were performed using
35Sriboprobes on 10 µm frozen sections from E14.5 and E18.5
brains (see Table 1) according
to standard protocols described previously
(Stoykova et al., 2000). The
cDNA probes were for Sema3a, Sema3c, Sema5A, Sema5b, Sema6a and netrin 1
(gifts from A. Puschel and M. Tessier-Lavigne).
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RESULTS |
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Aberrant thalamocortical axonal projections in the
Pax6-/- forebrain
In order to study axonal pathway formation in the
Pax6-/-, we used carbocyanine dyes to trace TCAs in fixed
brains (see Table 1). DiI
crystal placement into dorsal thalamus from E14.5 to E18.5 in wild-type brains
labelled TCAs extending to the primitive internal capsule having a normal
fasciculation pattern (see Fig.
3A,B). This was also seen in heterozygous brains
(Fig. 2D). DiI crystal
placements in the dorsal thalamus revealed a group of cells within the
internal capsule, which were situated medial to the ß-galactosidase
stained stripe of cells at the PSPB (Fig.
2G-I). It is known that some cells of the internal capsule project
to dorsal thalamus (Métin and
Godement, 1996; Molnár
et al., 1998
). However, in the homozygous brains, only a few
backlabelled cells were seen at the ventral telencephalon along the predicted
trajectory of the TCA pathway (Fig.
3I,J), but many were observed located the hypothalamus
(Fig. 3F-H).
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|
As it has been suggested that TCA development is delayed in the lack of
Pax6 (Pratt et al.,
2000), we examined TCA development in the
Pax6-/- at E15.5 and E18.5. In the E15.5 brains, TCAs
projected from the dorsal thalamus to the hypothalamus
(Fig. 3E-G) and by E18.5, some
TCAs reached the ventral telencephalon
(Fig. 3K,L) from where they
ascended dorsally towards the ganglionic eminences, following an aberrant
trajectory. Although they could be followed towards the PSPB, they were not
able to cross it to enter into the cortex
(Fig. 4Q).
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Corticofugal axonal projections are disrupted in the Pax6
knockout forebrain
We then examined whether the ability of CFAs to navigate across the PSPB
was affected in the Pax6-/- brains. In wild-type and
heterozygotes mice at E14.5 and E15.5, CFAs extended through the intermediate
zone to reach the PSPB, displaying a normal fasciculation pattern
(Fig. 4A,B). In the
Pax6-/- brains, CFA tracing studies revealed several
abnormalities. At E15.5, the CFA pathway started to descend towards the PSPB,
but it was displaced laterally (Fig.
4C,D). At later stages, large numbers of axons were seen to course
ventrolaterally through the developing lateral pallium, extending towards the
base of the ventral pallium (the amygdaloid region)
(Fig. 4H). The majority of CFAs
were unable to change their trajectories to cross the PSPB
(Fig. 4J), nevertheless some
entered into the subpallium fasciculating abnormally in large bundles at more
caudal levels (Fig. 4K-M). We
observed more numerous backlabelled cells in the marginal zone distant from
the cortical crystal placement sites in the mutants, suggesting that they
developed long range projections (Fig.
4N). The callosal projections were present in the mutants, but
their fasciculation pattern was also abnormal
(Fig. 4O). Cortical placement
of DiI crystals in wild-type brains at E15.5 revealed continuous axonal
bundles (Fig. 4F), which
extended to groups of backlabelled cells in the dorsal thalamus (data not
shown) (see Molnár et al.,
1998). As carbocyanine dyes travel in both anterograde and
retrograde directions in fixed tissue, and thalamic fibres arrive to the
cortex by E15.5, the bundles labelled at E18.5 contained both sets of fibres.
In the Pax6-/- brains, however, we did not observe
backlabelled groups of cells in the dorsal thalamus after cortical crystal
placements, and we could not detect any interaction between CFAs and TCAs when
we used double labelling (Fig.
4Q,S,U). This confirmed our observation obtained with anterograde
tracing from the thalamus, and indicated that, although thalamic axons
approached the PSPB, they did not reach the cortex in the mutants.
L1 expression and detection of thalamocortical axons
To examine the gross pattern of TCA growth, we performed L1
immunohistochemistry. Interestingly, we observed that L1 expression was not
limited only to TCAs at stage E14.5 and E18.5 as previously thought
(Fukuda et al., 1997), but L1
immunoreactive fibres were present in dorsal cortex at E14.5
(Fig. 5A,B). At this stage,
TCAs normally extend only to the level of the PSPB and do not invade the
intermediate zone of the cortex (De Carlos
and O'Leary, 1992
; Erzurumulu and Jhaveri, 1992). Therefore, the
detected immunoreactivity for L1 in the cortex at this age cannot be due the
presence of TCAs, suggesting that the CFAs were also labelled as they extend
in the intermediate zone of the dorsal cortex towards the PSPB.
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TCAs and CFAs are known to reach the PSPB synchronously
(De Carlos and O'Leary, 1992)
and the two sets of axons intermingle
(Molnár et al., 1998
).
By E18.5 in wild-type brains, TCAs have reached to the cortex through the
intermediate zone (Catalano et al.,
1991
). L1 immunolabelling in wild-type sections
(Fig. 5E,I) revealed the gross
organization of the fascicles in the striatum, PSPB and in the intermediate
zone. In Pax6-/-, L1 immunoreactivity was detected on both
CFAs and TCAs at the two studied ages (Fig.
5C,G) and at E14.5, TCAs were seen emerging from the basal ventral
telencephalon and had turned dorsomedially towards the subventricular zone of
the ganglionic eminence, but did not cross the PSPB
(Fig. 5C,D). At E18.5, in
Pax6-/- brains, L1 immunolabelled fibres reached the PSPB,
but they could not be followed crossing this boundary
(Fig. 5F,G,J,K). The
L1-positive fibre trajectory of CFAs in the cortex was displaced
ventrolaterally and axons continued their path to the ventral pallium as if
they were unable to cross the PSPB (Fig.
5D). As described above with carbocyanine dyes
(Fig. 4Q), using
immunohistochemistry for L1 we described similar results which point out that
TCAs and CFAs failed to meet each other at the PSPB.
TAG1 expression and corticofugal axons
CFAs selectively express the cell adhesion molecule TAG1
(Denaxa et al., 2001). The
immunostaining for TAG1, however, seemed to reveal only the initial part of
this pathway, as TAG1 immunoreactivity was drastically reduced at the level of
PSPB (see López-Bendito et al.,
2002
). In the wild-type forebrain at E14.5, TAG1 immunoreactive
CFAs appeared to extend through the intermediate zone of the developing
pallium to reach the PSPB (Fig.
5M,N). At E18.5, TAG1 immunoreactivity was expressed primarily by
cells of the cortical plate and marginal zone in the hippocampus and corpus
callosum (not shown). In the Pax6-/- brains, TAG1
immunoreactive fibres followed aberrant trajectories, being displaced
laterally in thick bundles (Fig.
5O,P). In addition, TAG1 expression in cortical plate and marginal
zone was reduced, preserved mostly within the medial pallium. These findings
were consistent with the abnormalities observed in CFA development in
Pax6-/- with DiI tracing.
Abnormalities at the PSPB in the Pax6-/-
brain
We further examined the cellular organization of the PSPB domain in
relation to the ß-galactosidase staining in the developing forebrain of
E14.5 and E18.5 heterozygous and homozygous embryos. In E14.5
Pax6-/- brains, the telencephalic neuroepithelium at the
PSPB appeared broader and more irregular compared with normal, and consisted
of aggregated cells which were heavily stained with Cresyl Violet
(Fig. 6D-F). By E18.5, a
considerable cell mass was prominent, which often occluded most of the lateral
ventricle at rostral level (Fig.
5F,G). The extended region consisted of cells that were heavily
stained for Nissl but negative for ß-galactosidase stain. These cells
were densely packed together and appeared to be continuous with the cortical
ventricular zone. The thickened pallial germinal neuroepithelium of the E18.5
Pax6-/- was also ß-galactosidase negative
(Fig. 4G, Fig. 5F,G,K). In the
Pax6-/-, the ventricular cellular mass and the atypical
PSPB did not show immunoreactivity for the glial cell markers GFAP or
vimentin, and did not express calbindin or GABA, but were immunoreactive for
the neuronal marker, ß-tubulin class III
(Menezes and Luskin, 1994)
(data not shown), suggesting that they comprised undifferentiated neurones. In
Pax6-/- brains, ß-galactosidase staining of the
dorsal cortex continued to the ventral pallium at the lateral surface
(Fig. 4G), whereas in
heterozygotes the unstained lateral cortex occupied this zone and the
ß-galactosidase staining formed a narrow stripe linking stained cells of
the amygdala to the cortex (Fig.
4E). In addition, Nissl staining revealed that the cortical plate
was drastically reduced in the ventral-most lateral pallium in the
Pax6-/- brains (Fig.
6F).
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Ultrastructural characterization of the wild type and
Pax6-/- pallium and PSPB at E15.5 and E18.5
In order to characterize further the morphological abnormalities seen in
the Pax6-/- mouse, which cause severe disruption in the
development of CFAs and TCAs at the PSPB, we performed electron microscopic
analysis at that boundary in wild-type and homozygous brains at two different
stages, E15.5 and E18.5. In wild-type brains, heterogeneous cell populations
within the region of this boundary were observed. Axons crossing the PSPB were
seen at E15.5 as well at E18.5 (Fig.
7E,O). However in the Pax6-/- brains, the
cells within the PSBP domain appeared to be more densely packed than in
wild-type brains. This abnormality was more prominent at later stages
(Fig. 7H,S). Cells located in
this area were seen tightly attached to neighbouring cells
(Fig. 7I). In accordance with
previous data (Stoykova et al.,
1997) and our tracing experiments described above, a few axonal
tracts were observed at electron microscopy level traversing the PSPB in the
Pax6-/- at both ages
(Fig. 7H). At the lateral
pallium of the Pax6-/- brains, bundles of axons were
observed extending superficially (Fig.
7J), possibly corresponding to the CFAs that in the mutant were
seen to follow an aberrant trajectory along this area (see
Fig. 4J,K).
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Altered expression of axonal guidance molecules in the
Pax6-/- pallium
In an attempt to look for possible molecular determinants for the observed
aberrant growth of the CFAs and TCAs in the Pax6-/- brain,
we examined the expression of several semaphorins (Sema3a, Sema3c, Sema5a,
Sema5b and Sema6a) and netrin 1, which have previously been
implicated in thalamocortical and/or corticofugal pathfinding
(Bagnard et al., 1998;
Métin et al., 1997
). A
prominent defect in the expression pattern of Sema3c and
Sema5b was detected within the embryonic pallium in
Pax6-/- (Fig.
8). Sema3c is assumed to act as attractive axon guidance
signal secreted by the cells of the SVZ
(Bagnard et al., 1998
). In
agreement with Bagnard et al. (Bagnard et
al., 1998
), the expression of Sema3c in E14.5 wild-type
cortex was confined to the SVZ and intermediate zone of the entire pallium,
thus including the dorsal, lateral and ventral pallial domains
(Fig. 8A). A similar pattern
was seen in the heterozygous brains (Fig.
8B). However, in the Pax6-/- brains the
expression of Sema3c was completely abolished in the region of the
ventral and lateral pallium, while in the dorsal pallium the expression was
shifted into the most superficial zone of the cortical plate
(Fig. 8C). In sections of E18.5
wild-type brains, the signal was fainter, but still detectable all along the
extending CFAs towards the basolateral telencephalon (data not shown).
Similarly as at stage E14.5, the expression of Sema3c was not
detectable within the ventral and lateral pallium in the
Pax6-/- mice.
|
In wild-type brains, Sema5a had a strong expression in the VZ of
the rostral pallium with a lateral (high) to medial (low) expression gradient
(Fig. 8D,E). In the
Pax6-/- brains, the lower expression level in the medial
pallium was preserved, while the very prominent hybridization signal seen
within the VZ at the PSPB was not visible any more (compare
Fig. 8G with
Fig. 8E, arrows). At E14.5,
netrin 1 was expressed in the basal telencephalon including the region of the
primitive internal capsule (Métin
et al., 1997). In the Pax6-/- brains, the
netrin 1 expression is present and might even appear stronger and more
widespread than in wild-type (data not shown), extending to the region where
the aberrant thalamic axons enter to the ventral telencephalon. These results
suggest that the altered expression pattern of Sema3c, Sema5b and
perhaps netrin 1 in the Pax6 mutant may be responsible for the
altered growth of the CFAs and TCAs.
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DISCUSSION |
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|
Aberrant TCA fibre growth is associated with disturbed CFA
projections in the Pax6-/- mice
We have previously suggested in our `handshake hypothesis', that axons from
the thalamus and from the early-born cortical preplate cells meet and
intermingle in the basal telencephalon, so that thalamic axons grow over the
scaffold of preplate axons and become `captured' for the waiting period in the
subplate (Blakemore and Molnár,
1990; Molnár and
Blakemore, 1995
). The use of the Pax6-/- mice
(St-Onge et al., 1997
) allowed
us to examine the relationship between Pax6 expression at the PSBP
and the cortical connectivity in normal and in Pax6 loss-of-function
conditions. In agreement with previous results
(Kawano et al., 1999
;
Pratt et al., 2000
;
Hevner et al., 2002
), we found
that the lack of PAX6 resulted in a failure of TCA growth into the
telencephalon, which is accompanied by a misrouting of the majority of the
TCAs into the hypothalamus. Remarkably, DiI labelling of the mutant cortex
revealed that, in addition to TCAs, the CFAs also follow an aberrant
trajectory with an abnormal fasciculation pattern. Although perinatally
(E18.5) the thalamic axons reach the PSPB, they are not able to cross this
region. Double DiI/DiA labelling experiments clearly indicated that no
interactions occur between TCAs and CFAs within the internal capsule, and
these two sets of axons remained separated by the PSPB. These observations
were further supported by the L1 and TAG1 immunohistochemistry results. The
aberrant development of these tracts may be partly explained by abnormal
axonal fasciculation because CFAs and TCAs would be unable to interact and use
each other as a scaffold to extend towards their targets. Accordingly, mice
with mutations in transcription factor genes expressed in the pallium
(Tbr1) and dorsal thalamus (Gbx2), show TCA and CFA
pathfinding errors in the region of the internal capsule
(Miyashita-Lin et al., 1999
;
Hevner et al., 2001
;
Hevner et al., 2002
). Tracing
experiments in these knockout mice have shown that lack of early CFA is
associated with abnormal development of TCAs. It is conceivable therefore that
the development of both fibre systems depends on the same factors requiring
the intact morphology of the PSPB and/or interactions between the two fibre
systems.
How does Pax6 affect TCA and CFA pathfinding?
The lack of Pax6 in the Pax6Sey/Sey causes a
complex phenotype reflected in defects of the molecular regionalization and
boundary formation in developing telencephalon
(Stoykova et al., 1996;
Stoykova et al., 1997
;
Stoykova et al., 2000
;
Warren and Price, 1997
;
Toresson et al., 2000
;
Yun et al., 2001
), axonal
pathfinding (Mastick et al.,
1997
; Pratt et al.,
2000
), adhesive properties
(Stoykova et al., 1997
;
Meech et al., 1999
), cortical
progenitor proliferation (Warren et al.,
1999
), cell migration (Caric et
al., 1997
; Brunjes et al.,
1998
; Chapouton et al.,
1999
), radial glia differentiation
(Götz et al., 1998
) and
neurogenesis (Heins et al.,
2002
). This phenotype is established early in embryonic
development, and becomes progressively more complex at later stages, which
seriously hampers the determination of the primary effect of Pax6 in
the developing brain.
The choreography and timing of developmental events seem to be important
for the normal development of telencephalic connectivity. We identified
several defects of TCA development in the Pax6-/- brains.
It is conceivable that changes in the location of forebrain domains, and their
boundaries, may displace transient `guide-post' cells and their pioneering
projections, which are thought to be crucial for TCA guidance
(Métin and Godement,
1996; Molnár et al.,
1998
). Cells with similar function have been described within the
internal capsule and ventral thalamus in numerous species
(Molnár, 1998
). In lack
of Pax6 in the Pax6Sey/Sey, the differentiation
of the ventral thalamic nuclei is completely abolished
(Stoykova et al., 1996
;
Warren and Price, 1997
)
(Fig. 1H), including the
reticular nucleus, which has an essential role in the control of the
thalamocortical circuits (Mitrofanis and
Guillery, 1993
). We found that only a few thalamic projecting
cells were labelled within the PSBP after a thalamic placement of the DiI
crystals and these cells were displaced towards the hypothalamus. In the
developing telencephalon, Pax6 expression is confined to the germinal
neuroepithelium (VZ/SVZ) with a rostrolateral (high) to caudomedial (low)
gradient, with a most prominent expression within the lateral and ventral
pallium (Stoykova et al.,
1997
; Smith-Fernandez et al.,
1998
; Puelles et al.,
2000
). In the Pax6-/- at the pallial VZ, there
is an intensive ectopic expression of ventral markers in the lateral and
dorsal pallium, which seems to include direct transcriptional regulation
between other genes, e.g. Pax6 and Ngn2; Pax6 and
Gbx2 (Stoykova et al.,
2000
; Toresson et al.,
2000
; Yun et al.,
2001
). These pattern abnormalities seem to affect the progenitor
identity of the ventrolateral telencephalon, leading to a failure of the
lateral cortical plate formation and a dysgenesis of basolateral structures,
claustrum, endopiriform nucleus and piriform cortex
(Stoykova et al., 2000
). It is
conceivable therefore that the described defects in the generation and/or
functional properties of the TCAs, guide-post cells within the internal
capsule, ventral thalamus and perhaps hypothalamus might be a further
consequence of the ventralization of the telencephalic neuroepithelium at the
PSBP border in Pax6-/- mice.
Semaphorins are thought to be involved in cortical and thalamic axon
guidance (Puschel, 1999;
Skaliora et al., 1998
). In the
Pax6-/- brains, the expression of Sema3c was
completely abolished from the SVZ and IZ of the lateral and ventral pallium,
thus including the region where the CFA defects were observed. In the dorsal
pallium, however, the expression was shifted into the most superficial zone of
the cortical plate. Sema3c (previously known as SemE) is assumed to
act as attractive axon guidance signal secreted mostly by the cells of the SVZ
(Bagnard et al., 1998
). Thus,
the abnormal expression pattern of Sema3c in the
Pax6-/- brain correlates well with both, the CFA
pathfinding defects within the PSBP and with the aberrant orientation of the
cortical axons towards the cortical surface and might be responsible for
them.
It has been suggested that secreted chemoattractants, e.g. netrin 1, might
influence the growth of navigating axons, especially when they change their
direction of growth (Métin et al.,
1997; Braisted et al.,
2000
; Tessier-Lavigne and
Goodman, 1996
). As reported in this work, the expression of netrin
1 in the basal telencephalon of the Pax6-/- embryos
(including the region of the internal capsule) was present and appeared wider
and stronger, when compared with the wild-type brain, which might additionally
hamper the proper growth of the early TCA axons through the altered
environment in the mutant. This possible difference in netrin 1 expression
pattern shall need further quantitative analysis.
Previous evidence indicated that the expanded VZ/SVZ in the
Pax6Sey/Sey pallium is a consequence of accumulation of
only partially differentiated (Stoykova et
al., 2000; Heinz et al., 2002) or prematurely differentiated
(Warren et al., 1999
)
neuroblasts. Furthermore, in this mutant, the adhesiveness of pallial cells is
increased, accompanied by a severe downregulation of the expression of the
homophilic cell-adhesion molecule R-cadherin and of tenascin (Stoykova et al.,
1977; Warren et al., 1999
). We
have described similar abnormalities at the PSPB in the
Pax6-/-. The densely packed adhering cells especially
apparent at the anterior part of this boundary might not be penetrable for
TCAs or CFA, thus making an additional obstacle for the TCAs and CFA to
traverse the IC, the gateway between thalamus and cortex.
Are axonal pathfinding and cortical regionalization defects
related?
There is a continuing debate as to how the developing pallium influences
TCA development, and in turn whether TCAs affect development of the cortex. It
has been demonstrated that there is a shift in the areal identity in the
cerebral cortex of Emx2 and Pax6Sey/Sey mutant
mice, which is matched with altered distribution of TCAs in the
Emx2-/- brains (Bishop
et al., 2000; Mallamaci et
al., 2000
). In the Pax6-/- brains, no thalamic
projections reach the cortex, suggesting that the shift observed in cortical
representation in the Pax6-/- cortex might be in fact
independent from TCA targeting and do not rely on the presence or absence of
thalamic projections. Our tracing studies demonstrated that in both
Emx2-/-
(López-Bendito et al.,
2002
) and Pax6-/- mice (present study) a large
proportion or the entire early TCA population were misrouted at the border
between the diencephalon and telencephalon. In addition, in the
Pax6-/- an additional defect prevented the TCAs from
invading the cortex through the PSPB. Although, both of these early TCA
targeting abnormalities are associated with the altered cortical Emx2
and Pax6 expression patterns they are related to earlier guidance
defects, most probably independent from the cortical alterations.
In summary, we have found that axonal pathfinding and fasciculation of both CFA and TCA are dependent upon the normal expression of Pax6 during early forebrain development. The pathfinding defects may be secondary to displacement and dispersion of guidepost cell populations within the ventral thalamus and internal capsule and to abnormal barrier formation at the PSPB as a result of defects in molecular patterning. These cellular changes are also associated with altered expression of proteins (including Sema3c, Sema5a and netrin 1) required for normal axonal guidance. Our study suggests that Pax6 has numerous roles in thalamocortical guidance, and these seem to be independent from the cortical regionalization.
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ACKNOWLEDGMENTS |
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Footnotes |
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