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The Journal of Neuroscience, October 1, 2002, 22(19):8523-8531
Disruption of Early Events in Thalamocortical Tract Formation in
Mice Lacking the Transcription Factors Pax6 or Foxg1
Thomas
Pratt1,
Jane C.
Quinn1,
T. Ian
Simpson1,
John D.
West2,
John O.
Mason1, and
David J.
Price1
1 Biomedical Sciences and 2 Department of
Reproductive and Developmental Sciences, Genes and Development Group,
University of Edinburgh, Edinburgh EH8 9XD, United Kingdom
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ABSTRACT |
Early events in the formation of the thalamocortical tract remain
poorly understood. Recent work has suggested that thalamocortical axons
follow a path pioneered by transient thalamic afferents originating
from the medial part of the ventral telencephalon. We studied the
development of these transient afferents and the thalamocortical tract
in mutant mice lacking transcription factors normally expressed in the
dorsal thalamus or ventral telencephalon. Pax6 is expressed in
the dorsal thalamus, but not in the medial part of the ventral
telencephalon, and the thalamocortical tract fails to form in
Pax6 / embryos. We
found that transient thalamic afferents from the ventral telencephalon
do not form in Pax6/
embryos; this may contribute to the failure of their thalamocortical development. The distribution of
Pax6/ cells in
Pax6/
Pax6+/+ chimeras supports conclusions
drawn from forebrain marker gene expression that Pax6 is not required
for the normal development of the medial part of the ventral
telencephalon but is required in the dorsal thalamus. Failure of the
transient afferent pathway to develop is therefore likely a cell
nonautonomous defect reflecting primary defects in the thalamus. We
then examined the formation of thalamic afferents and efferents in
Foxg1/ embryos, which
lack recognizable ventral telencephalic structures. In these embryos
thalamic efferents navigate correctly through the thalamus but fail to
turn laterally into the telencephalon, whereas other axons are able to
cross the diencephalic/telencephalic boundary. Our results support a
role for the ventral telencephalon in guiding the early development of
the thalamocortical tract and identify a new role for the transcription
factor Pax6 in regulating the ability of the thalamus to attract
ventral telencephalic afferents.
Key words:
thalamocortical tract; diencephalon; ventral
telencephalon; Pax6; Foxg1; transcription factor; axon guidance; chimera; optic tract; mouse; transient thalamic afferents; tract
tracing
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INTRODUCTION |
The thalamocortical tract supplies
the cerebral cortex with innervation from the dorsal thalamus. In the
mouse, thalamocortical axons emerge from the dorsal thalamus at around
embryonic day 13 (E13) (Braisted et al., 1999 ; Tuttle et al., 1999;
Auladell et al., 2000), approximately coincident with the
cessation of neurogenesis in this structure (Angevine, 1970). The axons
then navigate ventrally through the thalamus and make a sharp lateral turn, avoiding the hypothalamus and entering the ventral telencephalon to form the internal capsule. The first thalamic axons arrive in the
cerebral cortex around E15 and form synaptic connections during
subsequent development (Braisted et al., 1999; Tuttle et al., 1999;
Auladell et al., 2000). Thalamic growth cones perform several
maneuvers on their way to the cortex, and their behavior at each point
is defined by the properties of the growth cones and the cells and
secreted molecules that they encounter (Braisted et al., 1999,
2000; Garel et al., 1999; Tuttle et al., 1999; Pratt et al., 2000;
Skaliora et al., 2000).
A model to explain how the thalamocortical tract forms postulates a
role for transient axons projecting from cells in the medial part of
the ventral telencephalon into the dorsal thalamus. According to this
model, these early thalamic afferents guide thalamocortical efferents
into the ventral telencephalon (Metin and Godement, 1996; Tuttle et
al., 1999). This raises the possibility that although later in
development the dorsal thalamus is the source of thalamocortical axons,
one of its important early functions is to accept innervation from the
ventral telencephalon. In this study we investigated how disturbances
inflicted on these tissues by mutating the transcription factors
Pax6 or Foxg1 affect the formation of this
transient afferent tract and the subsequent trajectory of the
thalamocortical tract.
Pax6 is expressed dynamically in the diencephalon, the lateral part of
the ventral telencephalon, and the cerebral cortex during the formation
of the thalamocortical tract (Stoykova et al., 1996, 2000; Warren and
Price, 1997; Hirata et al., 2002). The thalamocortical tract does not
form in Pax6/ mouse (Pratt
et al., 2000; Hevner et al., 2002) and rat (Kawano et al., 1999)
embryos. We used tract tracing in
Pax6/ embryos,
immunohistochemistry for Mash1 expression, and analysis of the
distribution of Pax6/
cells in Pax6/
Pax6+/+ chimeras to test the hypothesis
that the failure of the thalamocortical tract to form is preceded by a
failure of the dorsal thalamus to receive transient afferents from the
medial part of the ventral telencephalon.
Foxg1 (formerly BF-1) is expressed in the
telencephalon (with highest levels of expression ventrally), in the
nasal retina of the eye, and not in the diencephalon. The phenotype of
Foxg1/ embryos reflects
Foxg1 expression with the telencephalon reduced in size, the
eye distorted, and the diencephalon appearing normal. Judging from the
expression domains of forebrain marker genes (Foxg1,
Pax6, Emx2, and Dlx2), the
Foxg1/ telencephalon lacks
recognizable ventral structures but possesses a structural correlate of
the cerebral cortex, and the diencephalon is normal (Xuan et al., 1995;
Dou et al., 1999; Huh et al., 1999). We examined the trajectories of
thalamic and retinal axons in Foxg1/ embryos to further
test the hypothesis that a normal ventral telencephalon is required to
guide thalamocortical axons laterally toward the cerebral cortex.
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MATERIALS AND METHODS |
Pax6 and Foxg1 alleles. Both
Pax6 alleles (Pax6Sey and
Pax6Sey-Neu1) used in this study are
predicted to cause loss of Pax6 function (Favor et al., 1988; Hill et
al., 1991; Quinn et al., 1996).
Pax6Sey/Sey,
Pax6Sey/Sey-Neu1, and
Pax6Sey-Neu1/Sey-Neu1 embryos have been
reported to have the same phenotypic abnormalities (Quinn et al., 1996)
and are denoted Pax6/. Replacement of
the coding sequences of Foxg1 with LacZ coding sequences generated the Foxg1tm1M allele,
which causes loss of Foxg1 function (Xuan et al., 1995; Dou et
al., 1999, Huh et al., 1999). The
Foxg1tm1(cre)skm allele used in this study
was generated by targeted replacement of Foxg1 coding
sequences with Cre recombinase coding sequences [using a
targeting vector otherwise identical to that used by Xuan et al.
(1995)] (Hebert and McConnell, 2000). The anatomical defects reported
for Foxg1tm1M/tm1M embryos were
recapitulated in the Foxg1
tm1(cre)skm/tm1(cre)skm embryos used in this
study, and these are denoted
Foxg1/.
Animals for tract tracing.
Pax6/ and
Foxg1/ embryos were
obtained from Pax6Sey/+ × Pax6Sey/+ and Foxg1
tm1(cre)skm/+ × Foxg1
tm1(cre)skm/+ timed matings and were identified
by anatomical features described previously (Hill et al., 1991; Xuan et
al., 1995; Quinn et al., 1997; Huh et al., 1999). The day of finding a
vaginal plug was designated E0.5. Control embryos were obtained from
wild-type timed matings or nonhomozygous littermates. The numbers of
embryos examined in this study were as follows: injections into dorsal thalamus: E12.5: control, n = 5;
Pax6/, n = 6; Foxg1/,
n = 5; E13.5: control, n = 3;
Foxg1/, n = 3; E14.5: control, n = 3;
Pax6/, n = 3; Foxg1/,
n = 3; E15.5: control, n = 3;
Pax6/,
n = 3;
Foxg1/, n = 2; injections into E15.5 telencephalon: control, n = 9; Foxg/,
n = 9; injections into E15.5 optic cup: control,
n = 3;
Foxg1/, n = 3. For each embryo both left and right sides of the brain (or both
eyes) were injected with dioctadecyltetramethylindocarbocyanine perchlorate (DiI) (Molecular Probes, Eugene, OR).
DiI labeling and imaging. Whole embryos (E12.5, E13.5) or
heads (E14.5, E15.5) were fixed overnight at 4°C in 4%
paraformaldehyde (PFA) in PBS. For injections into the dorsal thalamus,
a coronal cut was made in the head to reveal the caudal end of the
thalamus. For injections into the optic cup, the lens of the eye was
removed to reveal the surface of the retina. For injections into the
telencephalon, cuts were made in the head to reveal the surface of the
telencephalon. DiI was applied to exposed telencephalon or thalamus by
stabbing several times with a pulled glass pipette coated with
microscopic DiI crystals. In the case of the eyes, the optic cup was
packed with larger clumps of DiI crystals. Embryos were returned to 4% PFA in PBS in the dark at room temperature for ~1 month to allow DiI
to diffuse along axons. Coronal vibratome sections (200 µm) were
counterstained with the nuclear dye TOPRO3 (0.2 µM; Molecular Probes) and cleared by serial
transfer through 1:1 glycerol/PBS and 9:1 glycerol/PBS at room
temperature until sections sank. Sections were stored at 4°C. Images
were acquired using either an epifluorescence microscope equipped with
a TRITC filter set and digital camera (Leica Microsystems, Hiedelberg,
Germany) or a TCS NT confocal microscope (Leica Microsystems).
In epifluorescence images, DiI appears orange and TOPRO3 appears red,
and in confocal images DiI appears red and TOPRO3 appears blue.
Mash1 immunohistochemistry.
Pax6+/+ and
Pax6Sey/Sey embryos were produced from
Pax6Sey/+ heterozygous timed matings and
fixed overnight in 4% PFA/PBS before processing to wax blocks. Wax
sections were cut at 10 µm and mounted on
poly-L-lysine (Sigma)-coated glass slides.
Sections were rehydrated through alcohols to PBS. Antigen unmasking was achieved by microwaving in 10 mM sodium citrate
buffer, pH 6.0. Sections were blocked with 10% normal goat serum in
0.1% Triton X-100/PBS. Primary antibody (anti-MASH1, 1:100; BD
PharMingen) in blocking solution (20% normal goat serum in 0.1%
Triton X-100/PBS) was applied overnight. Secondary antibody
(biotin-conjugated goat anti-mouse, 1:200 in blocking solution; Dako)
was then applied for 1 hr, before final visualization using
streptavidin-conjugated Alexaflour 488 (1:200; Molecular Probes) at
room temperature in the dark. Sections were mounted using 10%
Vectashield (Vector Laboratories) in 1:1 glycerol/PBS, and images were
captured using confocal microscopy.
Production of chimeras. Chimeras were produced as described
in Quinn et al. (1996). In brief, eight-cell embryos were obtained from
the parental cross Pax6Sey-Neu1/+,
Gpi1b/b female × Pax6+/Sey,
Gpi1b/b, Tg/Tg male, where Tg
denotes the presence of the reiterated  -globin transgene
TgN(Hbb-b1)83Clo (Lo, 1986). Pax6+/+,
Pax6Sey-Neu1/+,
Pax6+/Sey, and
Pax6Sey-Neu1/Sey embryos were produced,
all of which were Gpi1b/b and contained a
single copy of the -globin transgene
(Tg+). Donor embryos for aggregation were
obtained from inbred BALB/c mice, which were
Pax6+/+,
Gpi1a/a, and negative for the -globin
transgene (Tg). Embryos were collected
from superovulated females at 2.5 d after coitum and aggregated
according to West and Flockhart (1994). Aggregated embryos were
cultured overnight, transferred to recipient pseudopregnant
F1 homozygous albino and
Gpi1c/c females
(Pax6+/+,
Gpi1c/c,
Tg), and allowed to develop to
E12.5.
Tail and forelimb samples were taken for colorimetric analysis of GPI1
isotype contribution to give a global proportion of chimerism for each
embryo (West and Flockhart, 1994). In all embryos, all cells derived
from the eight-cell wild-type embryo were
Gpi1a/a, and all cells derived from the
Pax6Sey-Neu1/+ × Pax6+/Sey cross were
Gpi1b/b. A mean of tail and forelimb GPI1B
proportion was determined for each embryo. The genotype of each chimera
was determined by PCR and restriction digest analysis of genomic DNA as
described in Quinn et al. (1996). The use of two mutant Pax6 alleles,
PaxSey (Hill et al., 1991) and
Pax6Sey-Neu1 (Favor et al., 1988), allowed
identification of chimeras homozygous for the Pax6 mutations
(Pax6SeyNeu-1/Sey) and those heterozygous
(Pax6Sey-Neu1/+ and
Pax6+/Sey) or wild type
(Pax6+/+) at the Pax6 locus.
Identification of cells derived from the progeny of the
Pax6Sey-Neu1/+ × Pax6+/Sey crosses was achieved by DNA-DNA
in situ hybridization using a digoxygenin (DIG)-labeled
probe to the reiterated -globin transgene (Keighren and West, 1993;
Quinn et al., 1996). In brief, the head of each embryo was dissected
and fixed in alcohol/acetic acid (3:1, v/v). Samples were processed,
embedded in wax blocks, sectioned at 7 µm, and mounted on
3-aminopropyltriethoxysilane (Sigma)-coated slides. Sections were
hybridized with DIG-labeled DNA probe, detected with peroxidase-labeled
antibody, and visualized by diaminobenzidine staining. Slides were
counterstained with hematoxylin and eosin and examined using
bright-field and phase-contrast light microscopy.
Analysis of chimeras. Two balanced chimeras (those in which
the ratio of cells derived from the
Pax6Sey-Neu1/+ × Pax6+/Sey crosses to those from the
Pax6+/+×
Pax6+/+ crosses was ~1:1 as determined
by GPI1B analysis) from the
Pax6Sey-Neu1/Sey
Pax6+/+
(Pax6/
Pax6+/+) and
Pax6+/+
Pax6+/+ genotype groups were selected for
detailed analysis. Regions examined were the medial part of the ventral
telencephalon and the dorsal thalamus. In each chimera, the number of
Tg+ signals and individual cell nuclei in
a 250 × 250 µm grid area were counted in two nonconsecutive
sections for both the left and right sides of the brain (see Fig. 3 for
examples of sections used for analysis). A count of
Tg+ signal detected to nuclei counted does
not give a true estimate of the proportion of
Tg+ cells in a tissue. This is because of
tissue-specific differences in the efficiency of detecting
Tg+ signals and nuclei, caused by
variations in nuclear morphologies and packing densities (Quinn et al.,
1996). To correct for this effect we performed identical counts in
nonchimeric embryos, in which all cells contained the -globin
transgene, to generate tissue-specific correction factors for those
regions examined. These tissue-specific correction factors were as
follows: dorsal thalamus, 1.07; medial ventral telencephalon, 1.16. A
hybridization index was then calculated for each tissue [hybridization
index = (Tg+ signals/nuclei)/tissue
specific correction factor]. Because the corrected hybridization index
gives a true proportion of Tg+ cells in a
particular tissue, this can be compared directly with the global
contribution of Tg+ cells as determined by
GPI1 genotyping.
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RESULTS |
The ventral telencephalon does not project to the thalamus in
Pax6/ embryos
In control embryos at E12.5, DiI injections in the dorsal thalamus
labeled a tract coursing through the thalamus in a dorsoventral plane
and turning laterally into the ventral telencephalon. A population of
retrogradely labeled cell bodies in the ventral telencephalon marked
the lateral limit of DiI diffusion at this stage (Fig.
1a-d), indicating
that thalamic afferents precede thalamocortical efferents. Ventral
telencephalic cells projecting to the dorsal thalamus can be identified
only by retrograde DiI labeling from the dorsal thalamus, and the
number of cells labeled by injections that fill only a part of the
dorsal thalamus will always be less than the total number actually
projecting. This under-representation is likely to be compounded by the
transient nature of the projections, which may mean that at a given
time not all of the cells fated to project will be retrogradely labeled from the dorsal thalamus. Examination of our own material and previously published data (Tuttle et al., 1999) shows that retrogradely labeled cells can be scattered over a wide area in the medial part of
the ventral telencephalon or densely clustered (Fig.
1d), supporting the notion that a large proportion of
ventral telencephalic cells do project to the dorsal thalamus. By
E14.5 the thalamocortical axons had traversed the ventral telencephalon
(Fig. 1e-g). They had turned dorsally into the
cerebral cortex by E15.5 (Fig. 1h).
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Figure 1.
Labeling of thalamic afferents and efferents in
control embryos by DiI placement in the dorsal thalamus at E12.5
(a-d), E14.5
(e-g), and E15.5
(h). a, A caudal E12.5 section
showing the injection site and axons growing laterally at the
diencephalic/telencephalic boundary (white box).
b, A higher magnification of the area
boxed in a showing retrogradely labeled cell
bodies marking the lateral limit of DiI diffusion. c, A
more rostral E12.5 section with higher magnification
(d) of the area boxed in
c showing retrogradely labeled cell bodies in the medial
part of the ventral telencephalon. e-g,
Caudal to rostral series of sections showing injection site and the
trajectory of the thalamocortical tract at E14.5. Arrows
mark the lateral limit of the tract in each section. h,
By E15.5 the tract has reached the cerebral cortex (marked with
arrow). All sections were cut in the coronal plane.
e, Eye; cc, cerebral cortex;
dt, dorsal thalamus; ht, hypothalamus;
vtel, ventral telencephalon. Scale bars:
a, c, e-h,
500 µm; b, d, 50 µm.
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No labeled cells or axons were detected in the ventral telencephalon of
E12.5-14.5 Pax6/ embryos
after DiI injections into the dorsal thalamus (Fig.
2a,f-h) (serial sections from nine embryos were examined). At E14.5, we detected axons descending through the thalamus, but these did not
penetrate telencephalic tissues (Fig. 2f-i).
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Figure 2.
a,
f-i, Labeling in
Pax6/ embryos after
DiI placement in the dorsal thalamus at E12.5 (a)
and E14.5 (f-i).
a, An E12.5 rostral section, at a level similar to that
in Figure 1c, showing that no retrogradely labeled cells
were seen in the ventral telencephalon. Boxed area
indicates region of ventral telencephalon shown in
b-e. b-e,
Mash1 immunohistochemistry in E12.5 Pax6+/+
(c) and Pax6/
(e) ventral telencephalon. In both cases cells
expressing Mash1 protein (green) are distributed
in the ventricular zone (filled yellow arrows)
and not in the central region. b, d,
Phase-contrast images corresponding to fluorescent images in
c and e. E14.5 caudal
(f) and rostral (g)
sections showing injection site and the absence of DiI labeling in the
ventral telencephalon. h, Thalamic axons descending
through the thalamus (indicated by arrow); boxed
area in h is shown at higher magnification in
i illustrating an axon tipped with a growth cone
(arrow in i). dt*,
Pax6/ correlate of the
Pax6+/+ dorsal thalamus;
ht, hypothalamus; vtel, ventral
telencephalon. Pax6/
embryos lack eyes altogether. All sections were cut in the coronal
plane. Scale bars: a,
f-h, 500 µm;
b-e, 50 µm; i, 10 µm.
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Mash1-expressing cells occupy their normal positions in the medial
part of the Pax6/ ventral telencephalon
Previous studies have shown that Pax6 is not expressed
in the medial part of the ventral telencephalon (Stoykova et al., 1996; Hirata et al., 2002). In
Pax6/ embryos, the mRNA
expression domains of genes including Nkx2.1 and
Netrin1, which normally include the location of cells
projecting to the dorsal thalamus (Tuttle et al., 1999), are not
altered (Pratt et al., 2000; Stoykova et al., 2000). Expression of
Mash1 mRNA, the function of which is required for the
formation of these thalamic afferents (Tuttle et al., 1999), also
appears unaffected in
Pax6/ embryos (Stoykova et
al., 2000). Despite these findings, it remained possible that Pax6 is
expressed by early progenitors that give rise to ventral telencephalic
cells projecting to the thalamus. One consequence of this might be a
defect of Mash1 protein expression, and we tested this using Mash1
immunohistochemistry. Cells expressing Mash1 protein occupy their
normal positions in the ventricular zone of the ventral telencephalon
of E12.5 Pax6/ embryos
(compare expression pattern of Mash1 protein in E12.5 Pax6+/+ ventral telencephalon in Fig.
2c with that in
Pax6/ ventral
telencephalon in Fig. 2e). We did not detect any defects in
the pattern of Mash1 protein expression in
Pax6/ embryos throughout
the rostrocaudal axis of the medial ventral telencephalon. This
result implies that loss of Pax6 does not impair the ability of the
medial part of the ventral telencephalon to develop molecular features
associated with and essential for the formation of the transient tract
to the dorsal thalamus. The primary defect seen in our DiI tracing
experiments is therefore unlikely to be attributable to the
disappearance of these cells.
Pax6/ cells contribute normally to the
medial part of the ventral telencephalon but are under-represented and
segregate from Pax6+/+ cells in the dorsal
thalamus of Pax6/
Pax6+/+ chimeras
It has recently become apparent that the developing telencephalon
is a dynamic structure, with newly born cells participating in numerous
migratory streams that carry them from their birthplace to their final
destination (Wilson and Rubenstein, 2000). Although Pax6 is not
expressed in the medial part of the ventral telencephalon, it is
expressed more laterally (Stoykova et al., 1996, 2000; Hirata et al.,
2002). It is conceivable that cells born in Pax6-expressing territory
might migrate to the medial part of the ventral telencephalon before
projecting transiently to the dorsal thalamus. To address this issue we
turned to a Pax6/
Pax6+/+ chimera assay that directly tests
whether Pax6 is required autonomously by cells to occupy their correct
positions at a given developmental stage. The principle of this
approach is as follows. If there is an absolute requirement for Pax6
for a tissue to develop, then Pax6/ cells will not
contribute to that tissue in
Pax6/
Pax6+/+ chimeras, as occurs in the lens of
the eye and in the nasal epithelium (Quinn et al., 1996).
Alternatively, the requirement may be more subtle, as is seen in the
distal part of the optic cup (Collinson et al., 2000), and
Pax6/ cells either may
make a reduced contribution to the tissue or be distributed abnormally
within it. Pax6/ cells
would make a normal contribution if Pax6 is not required cell
autonomously for the development of the tissue. Previous studies (Quinn
et al., 1996; Collinson et al., 2000; J. C. Quinn and J. D. West, unpublished observations) have shown that the contribution of
Tg+ cells to a particular tissue of a
Pax6+/+,Tg+
Pax6+/+ chimera approximates the global
Tg+ contribution, although the two are
seldom identical (reflecting stochastic events during tissue
construction); large deviations from the global
Tg+ contribution in
Pax6/,Tg+
Pax6+/+ chimeras are likely to be a
consequence of the mutation. To provide a measure of inherent variation
between individual tissues in our chimeras, we performed counts in
several tissues in the control (Pax6+/+,Tg+
Pax6+/+) balanced chimeras and measured
the difference in each case from the global percentage chimerism and
found an SD of 7.5%. A
Pax6/ contribution to a
Pax6/
Pax6+/+ chimera of >15% (±2 SDs) above
or below the global contribution measured by GPI isoform composition
would therefore be evidence that
Pax6/ cells did not
contribute normally to that tissue, indicating a cell autonomous
requirement for Pax6 in that tissue.
We detect no abnormal distribution of
Pax6/,Tg+
cells in the medial part of the ventral telencephalon of
Pax6/,Tg+
Pax6+/+ chimeras.
Tg+,Pax6+/+
and
Tg+,Pax6/
cells were equally well mixed with unlabeled cells in
Pax6+/+,Tg+
Pax6+/+ (Fig.
3b) and
Pax6/,Tg+
Pax6+/+ (Fig. 3c)
chimeras. Previous examination of
Pax6/
Pax6+/+ chimeras has shown that
Pax6/,Tg+
cells exhibit abnormal patterns of distribution in relation to their
wild-type counterparts in all forebrain tissues that express Pax6
(Quinn et al., 1996; Collinson et al., 2000) (data from diencephalon in
this study, see below; Quinn, unpublished observations). Furthermore, cells were able to contribute to the medial part of the ventral telencephalon regardless of their Pax6 genotype. There was
no significant reduction in
Pax6/ cell contribution to
this region in the chimeras examined. In both
Pax6/,Tg+
Pax6+/+ and
Pax6+/+,Tg+
Pax6+/+ chimeras, the
Tg+ contribution to the medial part of the
ventral telencephalon was similar to the global
Tg+ contribution values defined by GPI
isoform composition for each chimera (Fig.
4). Therefore the loss of Pax6 function
does not detectably impair the ability of cells to contribute to the
medial part of the ventral telencephalon, implying that there is no
cell-autonomous requirement for Pax6 for the development of this part
of the brain. Taken together, the failure to detect abnormal
distribution or reduced contribution of
Pax6/ cells in our
chimeras provides compelling evidence that Pax6 does not participate
directly in the genesis of the region of the ventral telencephalon that
projects a transient tract to the dorsal thalamus.
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Figure 3.
E12.5
Pax6/
Pax6+/+ chimeras show an autonomous
requirement for Pax6 in the dorsal thalamus but not in the medial part
of the ventral telencephalon. a, d, Low
magnification of horizontal sections of chimeras like those used to
examine the contribution of cells to the medial part of the ventral
telencephalon (a) and the dorsal thalamus
(d) (red boxes indicate location
and orientation of higher magnification fields). b,
c, e-g, High
magnification of portions of typical fields used for quantification:
c, f, g, in
Pax6/,Tg+
Pax6+/+ chimeras; b,
e, in
Pax6+/+,Tg+
Pax6+/+ chimeras. In
these images medial is to the right, and
rostral is at the top. The Tg signals appear as
brown spots in the nuclei (here stained
blue). In the medial part of the ventral telencephalon,
both
Pax6+/+,Tg+
(b) and
Pax6/,Tg+
(c) cells intermingle equally well with
Pax6+/+,
Tg cells. In the dorsal thalamus,
although
Pax6+/+,Tg+
(e) cells are well mixed with the
Pax6+/+,Tg
cells,
Pax6/,Tg+
(f, g) cells do not intermingle
with their wild-type counterparts and instead form stripes highly
enriched for Pax6/
cells (red arrows in f, g)
separated by regions consisting almost exclusively of
Pax6+/+ cells (red
arrowheads in f). In some cases the
larger congregations of
Pax6/ cells seem to
have caused the ventricular surface of the dorsal thalamus to buckle
inward (red arrow in g).
e, Eye; cc, cerebral cortex;
dt, dorsal thalamus; vtel, ventral
telencephalon. Scale bars: a, d, 500 µm; b, c,
e-g, 50 µm.
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Figure 4.
Histogram showing quantification of chimerism in
Pax6/
Pax6+/+ and
Pax6+/+
Pax6+/+ chimeras. Global chimerism and
chimerism for the medial part of the ventral telencephalon and the
dorsal thalamus are shown for each chimera, with the
numbers above each bar indicating the
percentages of Tg+ cells. Primary
Tg+ signal/nuclei counts for the medial ventral
telencephalon and the dorsal thalamus were as follows:
Pax6+/+,Tg+
Pax6+/+ chimeras: JC24, medial
vtel = 2168/3721, dt = 2613/4390; JC58: medial vtel = 2135/3325, dt = 2427/3465;
Pax6/,Tg+
Pax6+/+ chimeras: JC56, medial
vtel = 1412/3484, dt = 692/3179; JC61: medial vtel = 1513/3591, dt = 511/3633. These primary counts were divided by the
tissue-specific correction factors (1.16 for the medial ventral
telencephalon, 1.07 for the dorsal thalamus) to give the corrected
hybridization index, which gives a true estimate of the percentage of
Tg+ cells in the tissue for comparison with the
global Tg+ contribution (see Materials and Methods).
Large variation between tissue-specific and global
Tg+ contribution, indicating a requirement for Pax6,
is seen only in the dorsal thalamus of
Pax6/
Pax6+/+ chimeras (marked with *),
where Pax6/ cells are
also abnormally distributed (Fig. 3). vtel, Ventral
telencephalon.
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In contrast, the ability of cells to contribute to the dorsal thalamus
was affected dramatically by their Pax6 genotype.
Tg+,Pax6+/+
cells were evenly distributed in the dorsal thalamus of
Pax6+/+,Tg+
Pax6+/+ chimeras (Fig. 3e), but
Tg+,Pax6/
cells were segregated from Pax6+/+ cells
into radial stripes in
Pax6/,Tg+
Pax6+/+ chimeras (Fig.
3f,g). These stripes could be followed in a
dorsoventral direction through many sections and extended into the
ventral thalamus (data not shown). When
Tg+ signals and nuclei were counted in
25-µm-wide strips running perpendicular to the ventricular surface,
we observed that either nearly all or virtually none of the cells in
these stripes were Pax6/
(Fig. 3f,g), suggesting that stripes of mutant
cells form perpendicular to the ventricular surface. The stripes of
mutant cells in the dorsal thalamus varied considerably in width from
one or two cells to tens of cells (Fig. 3, compare f,
g). These stripes were not a consequence of clonal expansion
during normal development, because they were not seen in the dorsal
thalamus of Pax6+/+
Pax6+/+ chimeras (Fig. 3e).
Although the overall morphology of the dorsal thalamus in the balanced
Pax6/
Pax6+/+ chimeras that were examined did
not appear disturbed [and certainly did not recapitulate the
distortions seen in Pax6/
embryos (Stoykova et al., 1996; Warren and Price, 1997)], the ventricular surface formed a dramatic kink coinciding with particularly large patches of mutant cells (Fig. 3g). This indicated that
the normal expansion of the diencephalic wall was retarded where large clumps of Pax6/ cells were
present. Analysis of broad areas of the dorsal thalamus showed that, on
average, Pax6/ cells were
under-represented in the dorsal thalamus of
Pax6/
Pax6+/+ chimeras. Although
Pax6+/+ cells contributed to the dorsal
thalamus of Pax6+/+
Pax6+/+ chimeras at levels comparable to
the global contribution, the contribution of
Pax6/ cells to the dorsal
thalamus of Pax6/
Pax6+/+ chimeras was less than half the
global contribution (Fig. 4). Taken together, the abnormal distribution
and under-representation of
Pax6/ cells in
Pax6/
Pax6+/+ chimeras provide strong evidence
that Pax6 is required autonomously for cells to make a full
contribution to the dorsal thalamus and to participate normally in its development.
Thalamocortical axons do not turn laterally into the telencephalon
in Foxg1/ embryos
In E12.5 Foxg1/
embryos, axons labeled from the dorsal thalamus coursed dorsoventrally
through the ventral thalamus (Fig.
5a), but although a few axons
approached the telencephalic/diencephalic boundary, hardly any entered
the telencephalon and those that did appeared disorganized (Fig.
5a-c). The telencephalon of
Foxg1/ embryos is highly
abnormal, and no retrogradely labeled cell bodies were detected in any
telencephalic tissues (Fig. 5d). At neither E14.5 nor E15.5
were thalamocortical axons seen turning laterally into telencephalic
structures (Fig. 5e-h). At E14.5 and E15.5, the
tract penetrated farther than normal into the lateral hypothalamus
(Fig. 5f-h, compare with Fig.
1f-h).
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Figure 5.
DiI labeling in
Foxg1/ embryos at
E12.5 (a-d), E14.5
(e-g), and E15.5
(h-j).
a-h, DiI injections into dorsal
thalamus. a, A caudal E12.5 section showing the
injection site and absence of DiI labeling from telencephalic
structures (the Foxg1/
telencephalon lacks recognizable ventral structures so is marked
cc*). b, A higher magnification of the
top boxed area in a showing that the very
few axons which approach the telencephalon are disorganized.
c, A higher magnification of the bottom area
boxed in a showing that labeled axons grow
ventrally and medially rather than turning laterally into the
telencephalon. d, A more rostral E12.5 section showing
that no retrogradely labeled cells or axons can be detected in the
rostral telencephalon. e-g, Caudal to
rostral series of sections at E14.5 show the injection site and the
trajectory of thalamic efferents. These do not penetrate the
telencephalon; arrows mark lateral limit of the tract in
each section. h, By E15.5 the tract has not penetrated
the telencephalon but has continued ventrally within the thalamus
toward the hypothalamus (marked with arrow).
i, j, DiI injection into telencephalic
structures at E15.5. i, Although no substantial tract
leaves the telencephalon in
Foxg1/ embryos, higher
magnification of the area boxed in j
shows that a few disorganized axons are able to cross the
telencephalic/diencephalic boundary. All sections were cut in the
coronal plane. cc* and e* denote
Foxg1/ correlates of
Foxg1+/+ cerebral cortex and eye;
dt, dorsal thalamus; ht, hypothalamus.
Scale bars: a, d,
e-h, 500 µm; b,
c, 50 µm.
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Placement of DiI crystals in the telencephalon of
Foxg1/ embryos labeled a
few axons that had crossed the telencephalic/diencephalic boundary
(Fig. 5i,j), although these appeared disorganized
and did not extend to the dorsal thalamus. Similar injections in
control embryos labeled a much larger number of axons that followed the trajectory of the thalamocortical tract and terminated in the dorsal
thalamus (data not shown).
Most of the retinal axons navigate the thalamus appropriately in
Foxg1/ embryos but a subpopulation
deviate into telencephalic structures
The optic tract forms at about the same time as the
thalamocortical tract. Retinal axons navigating toward the dorsal
thalamus encounter diencephalic and telencephalic tissues (Mason and
Wang, 1997) that are implicated in the formation of the thalamocortical tract (Braisted et al., 1999; Tuttle et al., 1999, Pratt et al., 2000).
We were therefore interested to see how retinal axons responded to the
disrupted forebrain environment present in
Foxg1/ embryos. In E15.5
control embryos, DiI crystals placed in the optic cup labeled a tract
leaving the retina (Fig. 6a),
coursing over the ventral surface of the brain, and continuing dorsally and caudally to the lateral dorsal thalamus (Fig.
6b,c). These axons were not seen entering
telencephalic tissue (Fig. 6b-e). In E15.5
Foxg1/ embryos, DiI
crystals placed in the optic cup label a tract in which the majority of
axons followed the same trajectory as that seen in control embryos to
reach the dorsal thalamus (Fig. 6f-j). Unlike in
the wild type, a subpopulation of axons deviated into telencephalic
structures (Fig. 6g,i).
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Figure 6.
DiI labeling of axons from the optic cup
in Foxg1/
(f-j) and control
(a-e) embryos at E15.5.
a-e, In control embryos, a rostral to
caudal series shows the tract leaving the optic cup
(a), growing over the lateral surface of the
hypothalamus (b), and reaching the lateral aspect
of the dorsal thalamus (c). Boxed
areas in b and c are shown at
higher magnification in d and e.
d, Axons do not penetrate into telencephalic structures.
e, Axons form a smooth tract running in a dorsoventral
direction in the lateral dorsal thalamus.
f-j, In
Foxg1/ embryos, a
rostral to caudal series shows the tract leaving the optic cup
(f), growing over the lateral surface of the
hypothalamus (g) (unfilled yellow
arrow in g shows axons penetrating the
telencephalon), and reaching the lateral aspect of the dorsal thalamus
(h). Boxed areas in
g and h are shown at higher magnification
in i and j. i, Some axons
leave the main tract and penetrate into telencephalic structures
(unfilled yellow arrow). j, Axons
form a smooth tract running in a dorsoventral plane in the lateral
dorsal thalamus. The main tract is indicated by yellow
arrows in a-c and
f-h. Note that short labeled fibers,
marked with yellow arrowheads, sprouting medially from
the main tract into the diencephalon are seen in both control
(d, e) and
Foxg1/
(i, j) embryos. cc* and
e* denote
Foxg1/ correlates of
Foxg1+/+ cerebral cortex and eye;
dt, dorsal thalamus; ht, hypothalamus;
vtel, ventral telencephalon. All sections were cut in
the coronal plane. Scale bars: a-c,
f-h, 500 µm; d,
e, i, j, 50 µm.
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DISCUSSION |
Disruption to thalamic afferents in
Pax6/ embryos
Tract-tracing studies in several rodent species have identified a
transient population of thalamic afferents that originate in the medial
part of the ventral telencephalon before thalamocortical tract
formation (Metin and Godement, 1996; Molnar et al., 1998; Braisted et
al., 1999). The cell bodies of these projecting neurons map to ventral
telencephalic domains expressing Nkx2.1 (transcription factor) and Netrin1 (secreted protein) mRNAs (Tuttle et al.,
1999). In Mash1/ embryos,
these domains of gene expression are missing, the transient afferent
tract does not form, and the thalamocortical tract does not penetrate
the telencephalon. This led to the proposal that Mash1 is required in
the medial part of the ventral telencephalon for the projection of this
transient tract that guides thalamocortical afferents laterally into
the ventral telencephalon (Tuttle et al., 1999).
Our observation that the ventral telencephalon does not project to the
dorsal thalamus in Pax6/
embryos could be explained by a primary defect in the projecting cells,
in the environment through which their axons navigate, or both.
Available evidence strongly suggests that the medial part of the
ventral telencephalon develops normally in
Pax6/ embryos. First, Pax6
is not expressed in this region at the time when its thalamic
connections form (Stoykova et al., 1996, 2000; Hirata et al., 2002).
Second, the expression patterns of Mash1 mRNA and protein,
Nkx2.1 mRNA, and Netrin1 mRNA are not altered in
this region in Pax6/
embryos (Pratt et al., 2000; Stoykova et al., 2000; the present study).
Third, Pax6/ cells in
Pax6/
Pax6+/+ chimeras show no abnormal
distribution and contribute normally to this region. In the forebrain
of Pax6/
Pax6+/+ chimeras, abnormalities always
manifest as segregation between Pax6/ and
Pax6+/+ cells rather than complete
exclusion of Pax6/ cells,
even in areas with high persistent expression of Pax6 (Quinn et al.,
1996; Collinson et al., 2000) (data from diencephalon in this study;
Quinn, unpublished observations). We cannot rule out the formal
possibility of a total exclusion of a subpopulation of
Pax6/ cells that would
normally project to the thalamus if that subpopulation makes up <30%
of the region analyzed (because this translates to a chimerism within
15% of the global chimerism of a balanced chimera, placing it within
the limits of inherent variations between our control chimeric tissues;
see Results). This is, however, unlikely because (1) the proportion of
ventral telencephalic cells that project to the thalamus is likely to
be high (see Results) and (2) a cell autonomous requirement for Pax6 is
manifested as a segregation defect rather than exclusion in all other
regions of the brain examined. The development of the thalamus is
compromised in Pax6/
embryos. First, Pax6/
thalamic cells exhibit reduced proliferation (Warren and Price, 1997).
Second, the Pax6/ dorsal
thalamus exhibits abnormal differentiation that is manifested as
altered patterns of gene expression and projection of axons with
altered navigation properties (Pratt et al., 2000). Third, Pax6/ cells are abnormally
distributed and under-represented in the dorsal thalamus of
Pax6/
Pax6+/+ chimeras (the present study).
Taken together these findings provide compelling evidence that a
primary defect in Pax6/
thalamus is responsible for its failure to receive transient afferents
from the ventral telencephalon. The lack of this afferent tract may be
one of several factors contributing to the failure of subsequent
thalamocortical development in Pax6/
embryos (Kawano et al., 1999; Hevner at al., 2002). Other factors are
likely to include cell-autonomous defects of thalamic cells and axons
(Pratt et al., 2000) (chimera results of the present study).
Axons forming the tract of the post-optic commissure (TPOC) fail to
reach the dorsal thalamus in
Pax6/ embryos (Mastick et
al., 1997). A recent preliminary report shows that TPOC axons can be
rescued nonautonomously by transient expression of Pax6 within the
thalamus at E10.5 (Mastick, 2001). This result is interesting in the
context of this study because it suggests a more general Pax6-dependent
role for the thalamus in attracting afferent axons.
Abnormal thalamic development in
Pax6/ embryos
Previous work has demonstrated that proliferation is reduced in
the Pax6/ dorsal thalamus
at E10.5 (Warren and Price, 1997). The under-representation of
Pax6/ cells in the dorsal
thalamus of E12.5 Pax6/
Pax6+/+ chimeras suggests a
cell-autonomous proliferation defect that cannot be rescued by
surrounding Pax6+/+ cells. Kinks in the
ventricular zone coinciding with large areas consisting almost
exclusively of Pax6/ cells
could reflect mechanical distortion imposed on the tissue by a patch of
relatively slowly dividing mutant cells surrounded by more rapidly
proliferating wild-type epithelium. That the tissue responds to these
patches of mutant cells by forming a kink, rather than relieving the
tension by allowing Pax6/
and Pax6+/+ cells to mix laterally,
provides a measure of the strong forces opposing their mixing.
Misexpression of cell surface molecules or secreted proteins in the
Pax6/ thalamus is a likely
mechanism for its failure to receive transient innervation from the
ventral telencephalon. That
Pax6/ and
Pax6+/+ cells did not intermingle in the
thalamus of E12.5 Pax6/
Pax6+/+ chimeras suggests a role for Pax6
in influencing cell-cell interactions in the developing thalamus by
regulating the expression of cell surface molecules or secreted
signaling proteins. This hypothesis is appealing because, in various
developing systems, Pax6 directly or indirectly regulates the
expression of many genes controlling cell-cell contact and signaling.
These include genes encoding cell surface adhesion molecules L1,
R-cadherin, 1 integrin, and trkB (Stoykova et al., 1997; Meech et
al., 1999; Warren et al., 1999; Duncan et al., 2000) and secreted
proteins Wnt7b, SFRP-2, and Netrin1 (Kim et al., 2001; Vitalis et al.,
2001). We have previously made a preliminary report that, at E14.5,
Pax6/ and
Pax6+/+ thalamic cells exhibit different
abilities to adhere to and grow on slices of wild-type forebrain in
culture (Pratt et al., 2001), suggesting that the control of thalamic
cell surface properties by Pax6 spans the period of thalamocortical
tract formation.
Disruption of the ventral telencephalon prevents thalamocortical
axons innervating the cerebral cortex in
Foxg1/ embryos
The thalamocortical phenotype of
Mash1/ embryos provides
evidence for the importance of transient thalamic afferents from the ventral telencephalon in guiding thalamocortical axons (Tuttle et al.,
1999), but the thalamus itself is also disrupted, making it hard to
determine where the primary defects causing thalamocortical axon
misrouting lie. In these mutants, most of the axons stall at the border
between the dorsal and ventral thalamus (Tuttle et al., 1999), raising
the possibility that the ventral telencephalon might be required to
guide axons through the thalamus. To test this we examined
Foxg1/ embryos in which
development of the thalamus is normal but the telencephalon is severely
disrupted and has no recognizable ventral structures (Xuan et al.,
1995; Dou et al., 1999). Thalamic axons in
Foxg1/ embryos showed no
clear pathfinding defects within the thalamus, indicating that a normal
ventral telencephalon is not required for this segment of the
thalamocortical tract to form. Thalamic axons did not, however, turn
laterally into the telencephalon, indicating that this turn requires a
normal ventral telencephalon. There is no evidence for a general
physical barrier to axons at the diencephalic telencephalic boundary in
Foxg1/ embryos because our
injections into the Foxg1/
telencephalon or eye (see below) identified axons crossing this boundary. A striking feature of the lateral turn of thalamocortical axons into the ventral telencephalon is their avoidance of the hypothalamus. In vitro studies show that thalamocortical
axons are repelled by molecules secreted by the hypothalamus (Braisted et al., 1999) and do not penetrate explants of hypothalamus (Pratt et
al., 2000). Our observation that dorsal thalamic efferents in
Foxg1/ embryos penetrate
farther ventrally than normal into the lateral part of the hypothalamus
suggests that repulsion by the hypothalamus in vivo is not
sufficient to propel thalamocortical axons laterally or to completely
repel invasion by thalamocortical axons diverted from their normal course.
Optic tract formation in Foxg1/
embryos
The optic tract grows from the retina to the dorsal thalamus and
encounters tissues involved in guiding the thalamocortical tract (Mason
and Wang, 1997). In Foxg1/
embryos we labeled many axons that navigated from the retina to the
lateral aspect of the dorsal thalamus along a similar trajectory to
that seen in control embryos, indicating that diencephalic tissues are
able to supply appropriate navigation cues to retinal axons in the
absence of Foxg1. This result supports our premise, based on the
absence of Foxg1 expression from the diencephalon and
appropriate expression of diencephalic marker genes in
Foxg1/ embryos, that the
Foxg1/ diencephalon is
normal (Xuan et al., 1995; Dou et al., 1999). We also observed a subset
of axons that projected into the telencephalon, a feature never seen in
control embryos. So although thalamocortical axons (which normally
penetrate the telencephalon) are excluded in the
Foxg1/ mutant, some
retinal axons (which are normally excluded from the ventral
telencephalon) are able to penetrate in the
Foxg1/ mutant. It is
tempting to speculate that the loss of Foxg1 from the developing
telencephalon results in the simultaneous loss of attractive cues for
thalamic axons and repulsive cues for some (but not all) retinal axons.
Foxg1 is expressed in the nasal portion of the retina
(Hatini et al., 1994; Xuan et al., 1995; Huh et al., 1999), so it is
also possible that the pathfinding errors in the
Foxg1/ optic tract reflect
an autonomous requirement for Foxg1 in a subset of retinal axons. These
issues deserve examination in a future study.
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FOOTNOTES |
Received Feb. 11, 2002; revised June 17, 2002; accepted July 9, 2002.
This work was supported by the Medical Research Council, Biotechnology
Biological Sciences Research Council, and Wellcome Trust
(065035). We thank Jean Hebert and Susan McConnell for the Foxg1tm1(cre)skm mice, Robert Hill for helping lay
the foundations for the chimera work, and Katy Gillies, Margaret
Keighren, and Jean Flockhart for expert technical assistance.
Correspondence should be addressed to Dr. David J. Price, Genes and
Development Group, Biomedical Sciences, University of Edinburgh, Hugh
Robson Building, George Square, Edinburgh EH8 9XD, UK. E-mail:
dprice{at}ed.ac.uk.
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TOP
ABSTRACT
INTRODUCTION
