Nina Ireland Laboratory of Developmental Neurobiology, Department of Psychiatry, University of California, San Francisco, CA 94143-0984, USA
* Author for correspondence (e-mail: jlrr{at}cgl.ucsf.edu)
Accepted 31 January 2003
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SUMMARY |
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Key words: Fgf8, Neocortex, Regionalization, Topography, Thalamocortical axons
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INTRODUCTION |
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How is the regional organization of the neocortex established? This issue
has been and remains controversial
(O'Leary et al., 1994;
Levitt et al., 1997
;
Rubenstein and Rakic, 1999
;
Monuki and Walsh, 2001
;
Pallas, 2001
;
Ragsdale and Grove, 2001
;
Ruiz i Altaba et al., 2001
;
Sur and Leamey, 2001
;
O'Leary and Nakagawa, 2002
).
It has been proposed that regionalization is induced by extrinsic cues, in
particular by incoming thalamic axons, which convey positional and functional
specification (the `protocortex' model)
(O'Leary, 1989
). Conversely,
it has been suggested that intrinsic regional differences are established
within the neuroepithelium by molecular determinants that regulate neocortical
areal specification, including the targeting of thalamic axons (the `protomap'
model) (Rakic, 1988
). Although
thalamic inputs have been implicated in regulating aspects of area-specific
properties (O'Leary et al.,
1994
; Paysan et al.,
1997
; Gitton et al.,
1999a
; Dehay et al.,
2001
; Gurevich et al.,
2001
; Monuki and Walsh,
2001
; Pallas,
2001
; Ragsdale and Grove,
2001
; Sur and Leamey,
2001
; O'Leary and Nakagawa,
2002
), several studies in mice have recently demonstrated that
mechanisms intrinsic to the telencephalon play a major role in the
regionalization of the neocortex.
Transplantation and in vitro experiments using the transgenic mouse line
H-2Z1, which expresses ß-galactosidase in the primary
somatosensory area, have shown that positional information is already present
at early stages of neocortical development
(Cohen-Tannoudji et al., 1994;
Gitton et al., 1999b
).
Furthermore, multiple genes whose expression prefigures neocortical areal
domains or boundaries have been identified
(Bulfone et al., 1995
;
Korematsu and Redies, 1997
;
Suzuki et al., 1997
;
Inoue et al., 1998
;
Nothias et al., 1998
;
Donoghue and Rakic, 1999a
;
Donoghue and Rakic, 1999b
;
Mackarehtschian et al., 1999
;
Miyashita-Lin et al., 1999
;
Nakagawa et al., 1999
;
Rubenstein et al., 1999
;
Liu et al., 2000
;
Sestan et al., 2001
). The
expression of several of these genes is not perturbed by the lack of thalamic
input in Gbx2-/- and Mash1-/-
(Ascl1 Mouse Genome Informatics) mutant mice
(Miyashita-Lin et al., 1999
;
Nakagawa et al., 1999
).
Similarly, cortical explant assays have demonstrated that thalamic inputs are
not required for the induction of H-2Z1 transgene expression
(Gitton et al., 1999a
). Thus,
these experiments show that positional information is present within the
neocortex at early stages of development, and that later steps in neocortical
molecular regionalization do not require thalamic innervation.
Several early positional information determinants have recently been
identified: Emx2 and Pax6, which encode homeobox
transcription factors; and COUP-TFI (Nr2f1 Mouse
Genome Informatics), which encodes an orphan nuclear receptor
(Wang et al., 1991;
Simeone et al., 1992
;
Stoykova and Gruss, 1994
).
Emx2 is expressed in a high caudodorsal to low rostroventral gradient
in the neuroepithelium of the cortical anlage, and Pax6 is expressed
in a complementary high rostroventral to low caudodorsal gradient
(Simeone et al., 1992
;
Stoykova and Gruss, 1994
;
Gulisano et al., 1996
;
Stoykova et al., 2000
;
Toresson et al., 2000
;
Yun et al., 2001
;
Muzio et al., 2002b
). Mice
carrying null alleles of Emx2 and Pax6 have a
rostral-to-caudal and caudal-to-rostral shift, respectively, in the expression
of neocortical regionalization markers during embryogenesis
(Muzio et al., 2002b
) and at
birth (Bishop et al., 2000
;
Mallamaci et al., 2000b
;
Bishop et al., 2003
).
COUP-TFI is expressed in a high caudoventral to low rostrodorsal
gradient in the neocortex (Jonk et al.,
1994
; Qiu et al.,
1994
; Liu et al.,
2000
) and COUP-TFI homozygous mutant mice show molecular
regionalization defects at birth, although Emx2 and Pax6
expression is not altered (Zhou et al.,
2001
). Furthermore, changes in the targeting of thalamic
projections, which mirror the neocortical molecular defects, have been
observed in Emx2 and COUP-TFI mutants
(Bishop et al., 2000
;
Mallamaci et al., 2000b
;
Zhou et al., 2001
). Thus,
Emx2, Pax6 and COUP-TFI control neocortical molecular
patterning and may regulate the specific targeting of thalamic axons.
What are the molecules that control the early gradients of transcription
factor expression within the telencephalon? Discrete sources of secreted
signaling molecules that influence early telencephalic patterning include FGFs
along the rostral and dorsal midline
(Crossley et al., 1996;
Shimamura and Rubenstein,
1997
; Crossley et al.,
2001
; Ragsdale and Grove,
2001
), BMPs and WNTs along the dorsal midline
(Furuta et al., 1997
;
Grove et al., 1998
;
Lee et al., 2000
;
Monuki and Walsh, 2001
), and
SHH along the rostroventral margin (Kohtz
et al., 1998
; Crossley et al.,
2001
; Ruiz i Altaba et al.,
2001
). For example, BMP and WNT signaling pathways positively
regulate Emx2 expression in the telencephalon
(Ohkubo et al., 2002
;
Theil et al., 2002
), while
ectopic FGF8 expression, which is generated either via bead implantation in
chicken embryos or via electroporation in mouse explants, downregulates
Emx2 expression (Crossley et al.,
2001
; Storm et al.,
2003
). Furthermore, in utero electroporation experiments in mice
have shown that early ectopic expression of Fgf8, or of a
dominant-negative form of FGF receptor 3 (Fgfr3), shifts molecular,
histological and functional aspects of regionalization in newborns and
postnatal mice (Fukuchi-Shimogori and
Grove, 2001
). These results showed that modifying FGF signaling
has a major impact on neocortical regionalization. However, the specific roles
of endogenous levels of FGF8 in the formation of gradients of transcription
factor expression, and in the establishment of thalamocortical connectivity,
remain to be elucidated.
In the present study, we have undertaken a genetic approach to investigate
the function of Fgf8 in neocortical regionalization. Because a
complete loss or severe reduction of Fgf8 levels blocks embryonic
development (Meyers et al.,
1998; Sun et al.,
1999
) or severely perturbs telencephalic growth
(Meyers et al., 1998
;
Reifers et al., 1998
;
Shanmugalingam et al., 2000
;
Storm et al., 2003
), we took
advantage of a hypomorphic allele that has been generated in mice by the
intronic insertion of a neo cassette (Fgf8neo)
(Meyers et al., 1998
). In
Fgf8neo/neo embryos, the insertion of the neo cassette
causes aberrant splicing of Fgf8 transcripts, reducing the amount of
mRNA encoding functional FGF8 protein to
40% of normal levels (G. Martin,
unpublished) (Meyers et al.,
1998
). Fgf8neo/neo embryos survive until birth, have a
hypoplastic midbrain and cerebellum, and appear to lack olfactory bulbs
(Meyers et al., 1998
).
However, these embryos have a cerebral cortex of apparently normal size
(Meyers et al., 1998
),
enabling the study of neocortical regionalization.
We show that a reduction of Fgf8 levels in Fgf8neo/neo embryos creates a rostral shift in the graded expression of transcription factors, including Emx2 and COUP-TFI, in cortical progenitors. Furthermore, this early caudalization of neuroepithelial molecular properties correlates with a reduction in the size of a rostral neocortical molecular domain and a rostral expansion of more caudal regions. Surprisingly, we find that these molecular changes do not affect the targeting of thalamic axons to different neocortical domains. Taken together, our results show that Fgf8 participates in regulating early gradients of cortical gene expression and in later neocortical molecular regionalization. Furthermore, our results raise the possibility that the initial targeting of thalamic axons may be partially independent of neocortical molecular regionalization.
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MATERIALS AND METHODS |
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In situ hybridization
Embryos were fixed overnight in 4% paraformaldehyde (PFA) at 4°C. In
situ hybridization was performed on 80-100 µm vibratome sections as
described previously (Garel et al.,
1999) with the following probes: cadherin 6 and cadherin 8
(Cdh6 and Cdh8) (a gift from M. Takeichi); COUP-TFI
(a gift from M. Tsai); Dbx1 (a gift from F. Ruddle); Emx2 (a
gift from A. Simeone); Epha7 (a gift from A. Wanaka); Fgf8
(G. Martin); Fgfr3 (a gift from D. Ornitz); Id2 (a gift from
M. Israel); Lef1 (a gift from R. Grosschedl); Otx2 (a gift
from A. Simeone); Pax6 (a gift from P. Gruss); RZRß (a
gift from M. Becker-Andre); and Wnt3a (a gift from A. McMahon).
Sections were mounted in glycerol and analyzed on a dissection microscope.
Axonal tracing
After overnight fixation in 4% PFA at 4°C, single crystals of the
fluorescent carbocyanide dye DiI (1,1'-dioctadecyl
3,3,3',3'-tetramethylindocarbocyanine perchlorate; Molecular
Probes) or DiA (4-4-dihexadecyl aminostyryl N-methyl-pyridinium iodide;
Molecular Probes) were placed in single or multiple locations in the neocortex
(Godement et al., 1987). After
3-7 weeks at room temperature in 4% PFA to allow dye diffusion, the samples
were embedded in 5% agarose and cut at 100 µm on a vibratome.
Counterstaining was performed using Hoechst (Aldrich Chemicals) and digital
images were taken using a Spot II camera on a fluorescent microscope or
dissection microscope.
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RESULTS |
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Molecular identity of the frontal neocortex is abnormal in
Fgf8neo/neo late embryos and neonates
We examined if the early molecular modifications in the cortical
neuroepithelium of Fgf8 hypomorphic embryos changed the regional
properties of the neonatal neocortex. Between E17.5 and birth,
Fgf8neo/neo brains have a variable external phenotype
(Meyers et al., 1998) that
generally fit into two categories: (1) a `mild' phenotype in which there is
hypoplasia of the olfactory bulb, midbrain and cerebellum; (2) a `severe'
phenotype, in which the olfactory bulbs are not morphologically detectable and
there is a large reduction of the midbrain and cerebellar structures. In both
`mild' and `severe' cases the size and external morphology of the cerebral
cortex appeared normal (Figs
5,6,7,8)
(Meyers et al., 1998
).
To study molecular regionalization of the neocortex at birth, we analyzed
the distribution of genes that are expressed in a laminae- and region-specific
pattern that correlates with functional subdivisions of the neocortex
(Suzuki et al., 1997;
Miyashita-Lin et al., 1999
;
Nakagawa et al., 1999
;
Rubenstein et al., 1999
;
Bishop et al., 2000
;
Bishop et al., 2003
).
Id2 (Idb2 Mouse Genome Informatics) expression in
layers 2/3 delimits a rostrodorsal domain in the orbitofrontal and frontal
neocortices (Id2 rostral-superficial, Id2rs),
which extends medially into the occipital neocortex (Id2
caudal-superficial, Id2cs), whereas Id2
expression in layer 5 is restricted to a complementary parietal domain
(Id2 layer 5, Id25)
(Fig. 5A,G)
(Rubenstein et al., 1999
). In
`mildly' affected Fgf8neo/neo embryos, we observed a pronounced
reduction in the rostral Id2rs domain, a rostral expansion
of Id25 and a slight rostral shift in
Id2cs (compare Fig.
5A,G with 5B,H). Similarly, the Cdh8 rostral superficial
expression domain (Cdh8 rostral-superficial,
Cdh8rs) is reduced
(Fig. 5C,D), whereas the domain
of high Cdh6 expression, which is normally restricted to the parietal
neocortex expands rostrally (Fig.
5E,F) (Nakagawa et al.,
1999
). In `severe' Fgf8neo/neo embryos, these shifts are
even more pronounced and in some cases Id2rs and
Cdh8rs expression is not detected (see
Fig. 8A,B; data not shown),
whereas high Cdh6 expression expands into the most rostrodorsal
neocortex (Fig. 8C,D). By
contrast, Epha7 expression in the caudal and rostral neocortex and
RZRß (Rorb Mouse Genome Informatics) expression
in layer 4 (Fig. 5I,K)
(Miyashita-Lin et al., 1999
;
Rubenstein et al., 1999
) does
not show a clear change (Fig.
5G,J and data not shown).
To better understand the relationship between the different shifts in expression that we observed in the rostral neocortex of Fgf8neo/neo neonates, we carefully compared the expression patterns of Id2 and Cdh6 in adjacent coronal sections (Fig. 6). In the wild-type rostral neocortex, Id2rs, Id25 and Cdh6 high expression define a boundary between two molecular domains: (1) a rostral and medial domain (including the presumptive prefrontal and motor neocortex) delimited by high Id2rs, low Id25 and low Cdh6 expression; (2) a complementary parietal domain (including the presumptive somatosensory cortex) delimited by low Id2rs, high Id25 and high Cdh6 (Fig. 6A-D'). In `mildly' affected Fgf8neo/neo embryos, the boundary of expression between these two molecular domains was still present but was displaced to a more rostral position (Fig. 6E-H').
Thus, in Fgf8neo/neo newborns, there is a reduction of a molecularly defined rostral domain and a complementary expansion of the adjacent parietal domain. This change in the relative size of molecular domains implies that a substantial part of the frontal and orbitofrontal neocortex of Fgf8neo/neo neonates has molecular properties characteristic of the parietal neocortex.
The organization of thalamocortical projections to the rostral
neocortex is not modified in Fgf8neo/neo embryos
In wild-type embryos, different thalamic nuclei form connections with
specific neocortical domains (Crandall and
Caviness, 1984; Miller et al.,
1993
; O'Leary et al.,
1994
; Molnar and Blakemore,
1995
; Levitt et al.,
1997
; Monuki and Walsh,
2001
; Pallas,
2001
; Ragsdale and Grove,
2001
; O'Leary and Nakagawa,
2002
). As previous analysis of Emx2-/- and
COUP-TFI-/- mice suggested that changes in patterning are
linked with changes in the specificity of thalamic connections
(Bishop et al., 2000
;
Mallamaci et al., 2000b
;
Zhou et al., 2001
), we
examined the organization of thalamocortical projections in
Fgf8neo/neo neonates. DiI crystals alone, or paired with DiA
crystals, were placed in occipital, parietal or frontal neocortices of
control, `mildly' and `severely' affected Fgf8neo/neoneonatal brains
to retrogradely label thalamic cells projecting towards these domains
(Fig. 7). We assessed the
severity of the rostral-to-caudal molecular shift in each of these brains by
performing in situ hybridization with Id2, Cdh6 and/or Cdh8
on the opposite hemisphere (Fig.
8).
Dye injections in control occipital, parietal and frontal neocortex labeled
cells in the dorsal lateral geniculate nucleus (dLGN), the ventroposterior
nucleus (VP) and a more medial domain including the ventromedial (VM) and
mediodorsal (MD) nuclei, respectively (Fig.
7A,A',C,C',E,E',G,G')
(Jones, 1985;
Molnar et al., 1998
).
Surprisingly, in both `mild' and `severe' Fgf8neo/neo embryos, dye
placement within the occipital (n=5) and parietal neocortex
(n=9) showed the same pattern of connectivity as in controls
(Fig. 7). However, we noted two
differences in caudal neocortical dye tracing experiments in
Fgf8neo/neo mutants. First, dye placement in the occipital neocortex
of four out of nine mutants, did not label axons even within the cortical
plate. Second, large dye injections in the caudal parietal cortex of
Fgf8neo/neo embryos sometimes labeled a few cells in the dLGN
(Fig. 7C-D') (two cases
out of five), although the vast majority of labeled cells were detected in VP,
as in controls (Fig.
7C-F').
As molecular changes are more robust in the rostral neocortex of Fgf8neo/neo neonates, we focused on the pattern of connectivity of this neocortical region. Dye injections within the rostral parietal and frontal neocortex of `mild' (n=5) and `severe' (n=3) Fgf8neo/neo neonates labeled cells and axons in similar thalamic domains as in controls (data not shown and Fig. 7G-H'). To test if this observation was due to the persistence of a small rostral molecular domain, we examined thalamocortical projections in `severe' Fgf8neo/neo newborns that lack detectable Cdh8rs expression and show a massive rostral expansion of high Cdh6 expression (Fig. 8A-D). Dye injections in the frontal and rostral parietal neocortex of such `severe' mutants labeled a similar distribution of thalamic regions as in controls (Fig. 8E-L). Thus, in Fgf8neo/neo neonates, although the frontal neocortex has molecular characteristics of more caudal neocortical regions, it lacks a detectable defect in its pattern of connections with the thalamus.
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DISCUSSION |
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Reduced Fgf8 levels in Fgf8neo/neo embryos allow
the study of neocortical regionalization
FGF8 has been implicated in a variety of processes including cell
proliferation, cell death, cell migration and tissue regionalization
(Crossley et al., 1996;
Lewandoski et al., 1997
;
Shimamura and Rubenstein,
1997
; Meyers et al.,
1998
; Reifers et al.,
1998
; Ye et al.,
1998
; Martinez et al.,
1999
; Sun et al.,
1999
; Trumpp et al.,
1999
; Lewandoski et al.,
2000
; Shanmugalingam et al.,
2000
; Wilson and Rubenstein,
2000
; Crossley et al.,
2001
; Martin,
2001
; Shinya et al.,
2001
; Kobayashi et al.,
2002
; Ornitz and Marie,
2002
). In utero electroporation experiments driving the ectopic
expression of Ffg8 or blocking FGF signaling in the telencephalon of
E11.5 mouse embryos have implicated FGF signaling in neocortical
regionalization (Fukuchi-Shimogori and
Grove, 2001
). As multiple Fgf genes are expressed in the
telencephalon (Maruoka et al.,
1998
; Bachler and Neubuser,
2001
; Shinya et al.,
2001
; Gimeno et al.,
2002
), genetic analyses that eliminate the function of a single
gene are required for the investigation of their specific functions(s) in
neocortical patterning. The use of hypomorphic Fgf8neo/neo mutants
(Meyers et al., 1998
) allowed
us to circumvent the effects of a complete loss or severe reductions in
Fgf8 expression on early telencephalic development
(Meyers et al., 1998
;
Reifers et al., 1998
;
Sun et al., 1999
;
Shanmugalingam et al., 2000
;
Shinya et al., 2001
;
Storm et al., 2003
) and to
study the effects of reduced Fgf8 levels on the regionalization of a
roughly normally sized neocortex (Figs
1,2,3,4)
(Meyers et al., 1998
). Our
results show that a reduction of endogenous Fgf8 levels changes the
molecular regional properties of neuroepithelial progenitors and postmitotic
neurons in the rostral neocortex (Figs
3,
5 and
6).
Fgf8 levels regulate Emx2 and COUP-TFI
expression gradients
The inactivation of Emx2, Pax6 and COUP-TFI has revealed
the key roles of these transcription factors in the molecular regionalization
of the neocortex (Bishop et al.,
2000; Mallamaci et al.,
2000b
; Zhou et al.,
2001
; Bishop et al.,
2003
; Muzio et al.,
2002b
). These genes have a graded expression within the cortical
neuroepithelium, suggesting that gradients of diffusible molecules may
regulate their expression patterns. Experiments in chicken embryos
(Crossley et al., 2001
) and in
mouse telencephalic explants (Storm et
al., 2003
) have shown that ectopic FGF8 in the telencephalon
downregulates Emx2 expression, suggesting a link between FGF
signaling and Emx2 in neocortical regionalization. Our study shows
that a hypomorphic Fgf8 mutation shifts rostrally the graded
expression of Emx2 and COUP-TFI, indicating that reduced
Fgf8 levels caudalize the rostrodorsal telencephalon. Furthermore, it
suggests a role for FGF8 in creating and/or maintaining a rostral
neuroepithelial domain where caudally expressed genes such as Emx2
and COUP-TFI are not present. This role may not be restricted to the
neocortex, because we observe a similar rostral expansion of
Dbx1expression (Fig.
3E,F), which is normally restricted to the caudal part of the
non-neocortical ventral pallium (Puelles
et al., 2000
; Yun et al.,
2001
).
At this point, we are uncertain about how FGF8 regulates gradients of
transcription factor expression in the embryonic cortex. Interestingly,
Fgfneo/neo embryos have a rostral expansion of FgfR3
expression. Thus, as at the midbrain/hindbrain boundary, FGF8 may regulate the
expression of one of its own receptors in the forebrain
(Sleptsova-Friedrich et al.,
2001). In addition, Emx2 inactivation has been shown to
reduce the expression domain of Fgfr3
(Muzio et al., 2002b
),
suggesting that Emx2 may modulate FGF-signaling in the neocortex.
Taken together, these observations raise the possibility of a negative
feedback loop between Fgf8, Emx2 and Fgfr3, which could
contribute to patterning the rostral neocortical neuroepithelium.
Fgf8 regulates rostral neocortical molecular
regionalization
Our study shows that regionalization markers of the cortical plate are
preferentially perturbed in the rostral neocortex in Fgf8neo/neo
neonates. These modifications, which are reminiscent of the ones induced by
reducing of FGF-signaling in the cortex
(Fukuchi-Shimogori and Grove,
2001), include the reduction of frontal expression of
Cdh8rs and Id2rs, and the expansion of
parietal Id25 and Cdh6 expression. Overall, the
changes in Id2, Cdh8 and Cdh6 expression in
Fgf8neo/neo mice are opposite to the ones observed in
Emx2-/- mice and share similarities with the ones observed
in the Pax6sey/sey phenotype
(Bishop et al., 2000
;
Mallamaci et al., 2000b
;
Bishop et al., 2003
;
Muzio et al., 2002b
). However,
in Fgf8neo/neo embryos, we have detected only subtle changes in
Pax6 expression, whereas a clear rostral shift in Emx2 and
COUP-TF1 expression was observed (Figs
2,3,4).
As Emx2 expression is also increased in Pax6sey/sey mutant
mice (Muzio et al., 2002b
),
our results suggest the possibility that the common molecular changes found in
Pax6sey/sey and Fgf8neo/neo embryos are both mediated by
Emx2 expansion into the rostrolateral neocortical
neuroepithelium.
Thalamic projections to the abnormally patterned frontal neocortex
are apparently normal
Neocortical areas are defined by their architecture, molecular identity and
connectivity and distinct neocortical areas are specifically interconnected
with different thalamic nuclei. Thus, understanding the mechanisms regulating
the early targeting of thalamic axons during embryogenesis and the
establishment of these connections is a key step in the study of neocortical
regionalization. In both Emx2-/- and
COUP-TFI-/- mutant mice, the occipital neocortex forms
aberrant embryonic thalamic connections with the ventroposterior nucleus,
instead of the dLGN (Bishop et al.,
2000; Mallamaci et al.,
2000b
; Zhou et al.,
2001
). Emx2-/- and
COUP-TFI-/- mice have reduced caudal molecular markers and
abnormal molecular regionalization, respectively
(Bishop et al., 2000
;
Mallamaci et al., 2000b
;
Zhou et al., 2001
;
Bishop et al., 2003
),
suggesting that early gradients of Emx2 and COUP-TFI
expression may regulate the expression of guidance cues within the neocortex
that direct the targeting of thalamic axons during embryonic development.
We show that in Fgf8neo/neo embryos, despite a rostral shift in Emx2 and COUP-TFI expression and a change in the molecular identity of the rostral neocortex, this rostral neocortical domain has a pattern of axonal connections with the dorsal thalamus that is indistinguishable from wild-type embryos. It is important to note that DiI and DiA injections may not allow the detection of subtle changes in connectivity, owing to the difficulty in controlling the size of the cortical region where axons are labeled, as well as the lack of morphological boundaries delineating cortical areas and thalamic nuclei in neonates. Thus, in `mildly' affected Fgf8neo/neo newborns, the potential rerouting of thalamic axons by the moderate shift in molecular cues might induce technically undetectable changes in connectivity. However, the pattern of thalamocortical projections appears normal even in `severely' affected mutants that lack the frontal Cdh8rs domain and have the extensive rostral expansion of Cdh6 expression (Fig. 8).
How could this apparent uncoupling between molecular regionalization and
thalamocortical connectivity in Fgf8neo/neo mutants occur? One
possibility is that the mutation might not extensively perturb the expression
of the molecules that control the early targeting of thalamic axons such as
Pax6 (Fig. 4C,D)
(Hevner et al., 2002;
Jones et al., 2002
). In such a
case, it would imply that the changes in gradients of Emx2 and
COUP-TFI cortical expression might not be sufficient to modify the
initial targeting of thalamic axons. This hypothesis is not supported by a
straightforward interpretation of the phenotypes observed in
Emx2-/- and COUP-TFI-/- mice
(Bishop et al., 2000
;
Mallamaci et al., 2000b
;
Zhou et al., 2001
;
Bishop et al., 2003
). However,
it is possible that the thalamocortical topographic defects in
Emx2-/- and COUP-TFI-/- mice may not
be entirely due to neocortical defects. For example, in both mutants thalamic
axons show pathfinding errors inside the basal ganglia
(Mallamaci et al., 2000b
;
Zhou et al., 2001
;
Lopez-Bendito et al., 2002
).
As COUP-TFI is widely expressed in the dorsal thalamus and basal
ganglia (Jonk et al., 1994
;
Qiu et al., 1994
;
Liu et al., 2000
), and
Emx2 is expressed at the boundary between the basal ganglia and the
diencephalon (Lopez-Bendito et al.,
2002
), these pathfinding defects may be due to abnormalities in
the dorsal thalamus and/or basal ganglia. Thus, early molecular
regionalization of the neocortex and the initial targeting of thalamic axons
during embryogenesis might be regulated by partially independent mechanisms.
Support for this hypothesis is provided by the study of
Ebf1-/- and Dlx1/2-/- embryos. In
these mice, which have basal ganglia defects, subsets of thalamic axons fail
to reach the cortex. Remarkably, the axons that do reach the neocortex have a
shifted topography in the absence of neocortical molecular defects
(Garel et al., 2002
). Thus,
the analysis of Fgf8neo/neo, Ebf1-/- and
Dlx1/2-/- mutants support the possibility that neocortical
molecular regionalization and neocortical targeting of thalamic axons are
partially independent during embryonic development.
Integration of molecular regionalization in neocortical
arealization
How might these early embryonic events affect the formation of postnatal
cortical areas? It is possible that if the Fgf8neo/neo mice lived
beyond the day of birth, the distribution of thalamocortical projections would
more closely match the molecular changes in the neocortex. This possibility is
consistent with several experimental results, including those observed after
ectopic expression of Fgf8 in the caudal neocortical anlage. This
treatment induces an astonishing mirror-image duplication of the somatosensory
barrel field, which is tightly linked to thalamic innervation and peripheral
sensory information (Fukuchi-Shimogori and
Grove, 2001). These observations are made several days after
birth, when thalamic axons have grown into the cortical plate and formed
collaterals (Crandall and Caviness,
1984
; Miller et al.,
1993
). In addition, several lines of evidence suggest that
important regulatory processes in thalamocortical connectivity may take place
shortly after birth in rodents. For example, cortical transplantation
experiments at birth have shown that thalamic axons can later be redirected
towards the ectopically grafted cortex
(Levitt et al., 1997
;
Frappe et al., 1999
;
Gaillard and Roger, 2000
;
Ragsdale and Grove, 2001
;
O'Leary and Nakagawa, 2002
).
Thus, in response to local cues within the neocortex, early projections may be
redirected or refined and collaterals specifically formed and targeted
(Levitt et al., 1997
;
Gao et al., 1998
;
Mann et al., 2002
). Therefore,
our study supports a model in which the initial targeting of thalamic axons
during embryogenesis is not strictly controlled by regionally expressed cues
in the neocortex. However, once thalamic axons reach the neocortex,
interactions between regional cortical cues and thalamic axons probably modify
the initial pattern of thalamic projections and promote the formation of
mature neocortical areas.
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ACKNOWLEDGMENTS |
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