Genes and Development Group, Biomedical Sciences, George Square, The University of Edinburgh, Edinburgh EH8 9XD, UK
* Author for correspondence (e-mail: t.pratt{at}ed.ac.uk)
Accepted 27 April 2004
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SUMMARY |
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Key words: Eye, BF1, Optic chiasm, Retinal ganglion cell axons, Ipsilateral, Contralateral, Ephb, Ephrin B, Tract tracing, Mouse
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Introduction |
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The behaviour of a growth cone at a specific point along its route is
defined by its interaction with the cells and molecules it encounters. In the
mouse, RGC axons exit the retina and start to navigate along the optic stalk
(forming the optic nerve) at embryonic day 11 (E11)
(Colello and Guillery, 1990).
By E15.5, the chiasm has acquired its mature configuration although
progressively more axons navigate this route during subsequent development
(Marcus et al., 1995
). At the
optic chiasm RGC growth cones respond to cues generated by a specialised
population of midline cells (Wizenmann et
al., 1993
; Sretavan and
Reichardt, 1993
; Marcus et
al., 1995
). Their importance is shown by the failure of optic
tract formation in mice with an immunoablated chiasm
(Sretavan et al., 1995
) and
the development of only ipsilateral projections in Pax2-/-
or Vax1-/- mice with agenesis of the chiasm
(Torres et al., 1996
;
Hallonet et al., 1999
).
Contralaterally projecting RGCs (the majority in mice) are distributed all
over the retinal surface. Ipsilaterally projecting RGCs are concentrated in
ventrotemporal retina and their axons are repelled at the optic chiasm
(Sretavan and Reichardt, 1993
;
Herrera et al., 2003
;
Williams et al., 2003
).
Several transcription factors are regionally expressed in the developing
visual system and are poised to regulate the development of its structures and
axonal connections (Torres et al.,
1996; Halonet et al., 1999;
Barbieri et al., 2002
;
Mui et al., 2002
;
Wang et al., 2002
;
de Melo et al., 2003
;
Herrera et al., 2003
). The
winged helix transcription factor Foxg1 is strongly expressed in the
developing nasal retina and optic stalk, optic chiasm, telencephalon and
superior colliculus (Xuan et al.,
1995
; Huh et al.,
1999
; Marcus et al.,
1999
). The most obvious anatomical defects in embryos homozygous
for a targeted deletion of Foxg1 are abnormally shaped eyes and a
hypoplastic telencephalon (Xuan et al.,
1995
; Huh et al.,
1999
). We have shown by injecting tract-tracers into the eyes of
these mutants that the retina projects axons to an optic tract growing over
the surface of the thalamus (Pratt et al.,
2002
).
To test the potential for Foxg1 to influence axon navigation, we used
carbocyanine dyes to trace retinal axons in mice carrying a targeted deletion
of Foxg1 (Xuan et al.,
1995; Hebert and McConnell,
2000
). We found that mice lacking Foxg1 have an increased
ipsilateral projection arising from both nasal and temporal retina. To
characterise this defect, we (1) used lacZ reporter transgenes to
identify potential sites at which Foxg1 expression might influence the
navigation of retinofugal axons; and (2) examined several molecular markers
expressed in the normal retina and optic chiasm, namely the transcription
factors Foxg1 and Nkx2.2, the receptor tyrosine kinase Ephb2 and its ligand
ephrin B2, and the stage-specific embryonic antigen 1 (SSEA-1; Fut4 - Mouse
Genome Informatics) (Marcus et al.,
1999
; Barbieri et al.,
2002
; Williams et al.,
2003
) in Foxg1-/- embryos.
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Materials and methods |
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PCR genotyping Foxg1 alleles
Foxg1+ was detected with primers Foxg1ORFFor
5'-CTG ACG CTC AAT GGC ATC TA-3' and Foxg1ORFRev 5'-TTT GAG
TCA ACA CGG AGC TG-3' which give a product of 438 bp;
Foxg1lacZ allele was detected with primers
Foxg1_5'UTRF 5'-GCT GGA CAT GGG AGA TAG GA-3' and
Foxg1lacZ_ORFR 5'-GAC AGT ATC GGC CTC AGG AA-3' which
give a 550 bp product; Foxg1Cre allele was detected with
primers NLSCreF 5'-CAT TTG GGC CAG CTA AAC AT-3'; NLSCreR
5'-ATT CTC CCA CCG TCA GTAC G-3', which give a 307 bp product. For
all primers, cycling conditions were 96°C for 2 minutes followed by
[96°C for 30 seconds, 58.5°C for 30 seconds and 72°C for 30
seconds] for 35 cycles.
lacZ staining
E14.5 Foxg1lacZ/+ and Foxg1lacZ/Cre
embryonic heads were dissected and fixed for 1 hour at 4°C in 4%
paraformaldehyde, 0.02% NP40, 0.01% sodium deoxycholate, 5 mM EGTA, 2 mM
MgCl2 in phosphate-buffered saline (PBS). In some cases, heads were
equilibrated in 30% sucrose/PBS and sectioned (10 µm) on a cryostat.
Tissues were rinsed several times in wash buffer (2 mM MgCl2, 0.02%
NP40, 0.01% sodium deoxycholate in PBS), transferred to staining solution
(wash buffer supplemented with 5 mM potassium ferricyanide, 5 mM potassium
ferrocyanide and 1 mg/ml X-gal), and stained overnight (cryostat sections on
slides) or for 2 days with agitation (wholemounts) at 37°C in darkness.
Staining was stopped with 20 mM EDTA in PBS. Some wholemounts were sectioned
with a vibratome (200 µm) or processed to wax, sectioned (10 µm) and
mounted. Cryostat sections were counterstained with Nuclear Fast Red.
Tract tracing
Embryos (E13.5) or heads (E15.5) were fixed at 4°C in 4%
paraformaldehyde in PBS overnight. For retrograde labelling, the back of the
head and tissues overlying the thalamus were removed, and small DiI or DiA
crystals (Molecular Probes) were placed in a line over the dorsal thalamus on
one side to ensure unilateral labelling of the optic tract. For anterograde
labelling, the lens was removed and the optic cup packed with larger clumps of
crystals. Embryos were returned to 4% paraformaldehyde in PBS in the dark at
room temperature for about one month to allow tracers to diffuse along axons.
Vibratome sections (200 µm) were cleared by sinking in 1:1 glycerol: PBS
containing the nuclear counterstain TOPRO3 (0.2 µM, Molecular Probes) and
then 9:1 glycerol:PBS. Sections were stored at 4°C. Images were acquired
using an epifluorescence microscope and digital camera (Leica Microsystems,
Germany) or a TCS NT confocal microscope (Leica Microsystems, Germany). For
quantification DiI-labelled RGCs were assigned to either nasal or temporal
retina (Fig. 1B,C,J,K) in
serial horizontal sections (for examples of sections used, see
Fig. 4). In epifluorescence
(TRITC filter) images (Fig.
3A-J; Fig.
4D-F,K,L) DiI appears orange and TOPRO3 appears red and in
confocal images (Fig. 3K,L;
Fig. 4A-C,G-J,M-O) DiI appears
red, DiA is green and TOPRO3 appears blue.
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Results |
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We examined Foxg1 expression in Foxg1lacZ/+
embryos at E14.5, because this is the time at which RGC axons are navigating
the midline. Foxg1 expression was strongest in the nasal retina
(Fig. 1B,C); 51% of nasal cells
were lacZ+ in counts across several sections. By contrast,
only a few temporal cells were lacZ+ (2%) (visible at higher
magnifications in Fig. 1D).
These findings are consistent with previous results of in situ hybridisation
(Hatini et al., 1994). In the
centronasal retina, staining was particularly prominent in the inner layer
occupied by the RGCs (Fig. 1E).
Foxg1 was expressed in the anterior hypothalamus
(Fig. 1F) with a population of
expressing cells at the ventral midline overlying bundled axons at the optic
chiasm (Fig. 1G,H). Foxg1 was
also expressed by other structures encountered (or avoided) by RGC axons,
including the telencephalon, dorsal midline and superior colliculus
(Fig. 1A).
We examined lacZ expression in Foxg1lacZ/Cre
compound heterozygotes, which can produce no functional Foxg1 protein, so as
to identify cells in which the mutant gene was transcriptionally activated at
E14.5. As Foxg1lacZ/Cre cells had the same lacZ
gene dose as Foxg1lacZ/+ cells (one copy), lacZ
staining intensity could be directly compared between the two genotypes. The
Foxg1-/- eye is distorted
(Fig. 1J,K) with an abnormally
small lens and a medially elongated retina that extends towards the optic
chiasm. The nasal bias of Foxg1 expression in
Foxg1lacZ/+ embryos described above was also apparent in
Foxg1lacZ/Cre embryos (see whole mount in
Fig. 1N and sectioned material
in Fig. 1J-M). We counted 21%
lacZ+ cells in nasal retina compared with only 3%
lacZ+ cells in temporal retina. The reduced count nasally
reflects the absence of lacZ+ cells in outer layers of the
Foxg1lacZ/Cre lateral retina (compare
Fig. 1B,C with J,K). Previous
work at a younger age, E12.5, also showed lacZ expression in the eyes
of Foxg1lacZ/lacZ embryos confined mainly to the nasal
region of the mutant retina (Huh et al.,
1999). As in Foxg1lacZ/+ embryos,
lacZ staining was prominent in the inner layer of the nasal retina
(compare Fig. 1J-M with B-E).
These data show that the nasotemporal polarity of the retina is maintained in
Foxg1-/- mutants until at least E14.5; this conclusion is
further supported by data presented later.
In E14.5 Foxg1lacZ/Cre compound heterozygotes, the anterior hypothalamus continued to express Foxg1 around the ventral midline at the point contacted by the expanded mutant retina (Fig. 1N). As in Foxg1lacZ/+ embryos, stained cells were detected at the ventral midline (Fig. 1O,P) indicating that Foxg1 is not required for the maintenance of this population of cells at the optic chiasm. lacZ staining was also retained in the telencephalon, along the dorsal midline and in the superior colliculus (Fig. 1I).
Lineage tracing of retinal cells that have expressed Foxg1
The experiments described above show that Foxg1 is expressed by
many nasal RGCs at the time their growth cones are navigating the chiasm. The
few expressing cells we detected in the temporal retina were near to the
lateral lip (or ciliary margin) of the temporal retina where progenitor cells
proliferate (Perron et al.,
1998; Zuber et al.,
2003
). It was therefore possible that Foxg1-expressing progenitors
might populate the temporal retina with RGCs, the navigational responses of
which were influenced by transient expression of Foxg1 earlier in their
lineage. Crossing Foxg1Cre/+ mice with R26RS
reporter mice generates embryos in which a Cre recombinase-mediated
recombination event irreversibly enables lacZ expression from the
Rosa26 locus in cells that express Foxg1
(Mao et al., 1999
;
Hebert and McConnell, 2000
).
These cells and their descendants express lacZ protein, regardless of
whether the Foxg1 locus remains active. The lacZ expression
pattern provides a convenient means of visualising cells whose fate may have
been influenced by autonomous exposure to Foxg1.
As expected, the nasal retina of Foxg1Cre/+; R26RS
embryos exhibited uniform lacZ staining
(Fig. 2A-D). Strong staining
was also seen in lens, optic nerve and at the optic chiasm. In the temporal
retina of Foxg1Cre/+; R26RS embryos, lacZ stained
cells were found in radial stripes most of which were one or two cells wide
while others were wider (Fig.
2B-D; arrows indicate radial stripes in temporal retina). A
similar stripy distribution has been described before in chimeras and arises
because the retina is constructed from radially oriented clones of cells
(Reese et al., 1999).
Examination of serial sections though the eyes of six embryos did not reveal
any clear pattern in the size or density of these radial stripes, implying
that the exposure of their ancestors to Foxg1 did not predispose them to
occupy any particular part of the temporal retina. It was also apparent that
most temporal RGCs were located outside the lacZ-expressing clones.
These lacZ-expressing clones included cells outside the RGC layer
showing that ancestral Foxg1 expression was not restricted to RGCs.
We also detected mosaic activation of lacZ expression in the temporal
retinal pigment epithelium (a derivative of the optic cup, as is the retina)
suggesting that a subpopulation of these cells are derived from
Foxg1-expressing cells.
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Disruption to RGC axon navigation in embryos lacking Foxg1
The polarisation of Foxg1 expression in the retina suggests that
it could play an autonomous role in the navigation of nasal RGC growth cones
but is unlikely to play an autonomous role in the navigation of most temporal
RGC growth cones. Foxg1 may, however, influence the navigation of both nasal
and temporal RGC growth cones by regulating the properties of the optic
chiasm. To test these possibilities, we used DiI and DiA to trace the RGC
projections in embryos lacking Foxg1.
We examined RGC projections in wild type at E13.5 (Fig. 3A-C) and E15.5 (Fig. 3F-H), over which period the optic chiasm develops its mature configuration. Progressing through the brain from rostral to caudal, sections show the injection in the eye (Fig. 3A,F), the optic nerve approaching the optic chiasm along the ventral surface of the brain (Fig. 3B,G) and the optic tract growing dorsally over the thalamus (Fig. 3C,H). At both ages, the contralateral optic tract is more strongly labelled, indicating that the majority of RGC axons cross the midline at the optic chiasm (Fig. 3C,H). Double labelling with DiI injected into one eye and DiA into the other illustrates the X-shape formed by RGC axons at the chiasm (Fig. 3K). Labelling of the optic tract is predominantly from the contralateral eye, as shown by cross-sections of the optic tract as it grows dorsally over the thalamus (Fig. 3K').
In E13.5 and E15.5 Foxg1-/- embryos many of the general features of RGC axon navigation were similar to those in wild type. RGC axons from both eyes converged on the ventral surface of the hypothalamus where they were sorted into ipsilateral and contralateral optic tracts growing dorsally over the thalamus (Fig. 3D,E,I,J). In contrast to the wild-type, the ipsilateral tract in mutants appeared greatly increased [compare panels for wild type (Fig. 3C,H) with mutants (Fig. 3E,J); arrows indicate the ipsilateral tract]. Double labelling with DiI injected into one eye and DiA into the other indicated that, although RGC axons converged at the hypothalamic midline, the balance of midline crossing was shifted in the mutants. Although many RGC axons still crossed the midline (Fig. 3L), a large proportion of axons in the optic tracts was labelled from the ipsilateral eye (Fig. 3L').
To quantify the shift in the proportion of RGC axons projecting
ipsilaterally and to identify the sites of origin in the retina of the
aberrant ipsilateral projections, we retrogradely labelled RGCs with DiI or
DiA from the thalamus. The numbers of nasal and temporal RGCs projecting
ipsilaterally and contralaterally were counted in wild-type and mutant embryos
at E15.5. As gene expression studies (Huh
et al., 1999) (present study) indicate that, despite its medial
elongation, the mutant retina does not exhibit greatly disturbed polarity,
mutant eyes were divided into nasal and temporal halves using the same
anatomical criteria as for wild-type eyes.
Wild-type embryos showed the expected patterns of projection. Retrograde label filled the optic tracts, optic chiasm and optic nerves, and showed most axons originating contralaterally (Fig. 4A-H). Contralaterally projecting RGCs were distributed across nasal and temporal retina (Fig. 4F,G). The much smaller number of ipsilaterally projecting RGCs were more frequently found temporally (Fig. 4G; Table 1). The density of labelled RGCs was highest medially, towards the optic nerve head, and decreased sharply in more lateral retina (Fig. 4F,G). RGC cell bodies were orientated radially with their axons projecting towards the optic nerve head along the inner surface of the retina (Fig. 4H).
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As in the wild type, the majority of mutant RGCs are located in the inner layer of the retina and are radially orientated with their axons projecting along the inner surface of the retina (compare Fig. 4O with Fig. 4H), although the mutant RGCs do exhibit a broader radial spread. Again, reminiscent of the wild-type situation, mutant RGC axons fasciculate to form the mutant correlate of the optic nerve, which exits the elongated retina at its point of attachment to the ventral brain (seen in Fig. 4K) and approaches the optic chiasm (seen in a ventral adjacent section in Fig. 4L). As in the wild type, mutant retinal axons form an X-shaped chiasm (compare wild types in Fig. 3K, Fig. 4B,C,E with mutants in Fig. 3L, Fig. 4I,J,L), although their shapes are not identical. Given the distortions to the mutant eye it is remarkable that its retinal projection retains so many features in common with the wild type.
Our results in Foxg1-/- mutants indicate an increase in
the proportion of RGCs whose axons are repelled by the midline and join the
ipsilateral optic tract. As in wild type, significantly more ipsilateral
projections originated in temporal retina (2 test;
Table 1). This finding provides
further evidence for the retention of nasotemporal polarity, as was suggested
by the expression pattern of Foxg1 in mutants (see above).
Expression of Ephb2 and ephrin B2 in the developing retina
As well as being expressed in the developing retina itself, Foxg1 is
expressed in cells that surround and may influence the patterns of expression
of other genes in the retina as it develops
(Dou et al., 1999). It was
possible that in embryos lacking Foxg1 the patterning of the retina might have
been altered fundamentally and this might have had consequences for RGC axon
navigation. Results above suggested that the nasotemporal polarity of the
Foxg1-/- retina is similar to wild type. Here, we
addressed the nature of the other major axis of the mutant retina, i.e.
dorsoventral. This was important as most ipsilateral RGC projections normally
originate ventrally in the temporal retina
(Drager, 1985
) and so a
repatterning of Foxg1-/- retina, such that it adopts a
predominantly ventral character might have explained an increased ipsilateral
projection.
The receptor tyrosine kinase Ephb2 and its ligand ephrin B2 are expressed
in complementary dorsoventral gradients in the developing retina and their
distributions in the retina and chiasm have been related to RGC axon midline
crossing behaviour (Barbieri et al.,
2002; Williams et al.,
2003
). In wild-type embryos at E13.5 and E16.5, ephrin B2 exhibits
a dorsal[high] to ventral[low] gradient of expression in
the retina with the strongest expression dorsolaterally
(Fig. 5A,B). At E13.5, Ephb2 is
expressed throughout the retina in a ventral[high] to
dorsal[low] gradient (Fig.
5E). By E16.5 Ephb2 is more evenly distributed across the retina
(Fig. 5F), although levels
still appear to be slightly higher ventrolaterally. Ephb2 is also strongly
expressed in the optic nerve, consistent with expression of Ephb2 protein on
the RGC axons (Fig. 5E-F,I-J).
RGC axons continue to express Ephb2 protein as they approach the optic chiasm
and exit into the optic tract (Fig.
5I,J). By contrast, the optic nerve stains weakly for ephrin B2
protein (Fig. 5A,B).
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In the wild type the eye is roughly spherical and is attached to the
ventral surface of the diencephalon by the optic stalk, whereas the mutant eye
lacks an optic stalk and is attached directly to brain. Perhaps as a
mechanical consequence of this the mutant eye is elongated medially and
squashed dorsoventrally relative to the wild-type eye. To distinguish whether
this is only a change in shape or also involves a change in retinal volume, we
measured retinal area (mm2) in every 20th 10 µm horizontal
serial section through the eyes of wild-type and mutant embryos. As each
section was 0.01 mm thick, retinal volumes approximated to (retinal area
x 0.2) mm3. Average volumes obtained for wild-type (0.86
mm3) and mutant (0.88 mm3) eyes were very similar. The
proportion of the Foxg1-/- retina that is elongated
medially (marked `er' in Fig.
1K) probably corresponds to the central third of the wild-type
retina. Consistent with this idea, the medial expansion of the mutant retina
expressed levels of ephrin B2 and Ephb2 intermediate between the extremes of
the expression gradients shown in Fig.
5D,H (data not shown), as did the wild-type central retina.
These data indicate that, as in the nasotemporal axis, patterning of the retina along the dorsoventral axis is similar in Foxg1-/- and in wild-type embryos.
Expression of Nkx2.2, SSEA-1 and ephrin B2 at the developing chiasm
The presence of cells at the chiasm that turn on Foxg1 in both
Foxg1+/+ and Foxg1-/- embryos
(Fig. 1) prompted us to examine
the mutant chiasm in more detail. The expression patterns of the transcription
factor Nkx2.2 and the cell-surface molecule stage-specific embryonic antigen 1
(SSEA-1) define stages of normal chiasm development
(Marcus and Mason, 1995;
Marcus et al., 1999
). SSEA-1
is expressed by a population of early differentiating neurons that provides an
anatomical template to guide RGC axons at the developing chiasm
(Sretavan et al., 1994
;
Marcus and Mason, 1995
;
Sretavan et al., 1995
). We
speculated that changes in the distribution of neurons expressing SSEA-1 in
the mutant might perturb RGC axon midline crossing behaviour. However, we were
unable to detect any clear shift in the expression domains of SSEA-1 between
the wild-type (Fig. 7A,C,E) and
mutant (Fig. 7B,D,F) chiasm at
E13.5. The transcription factor Nkx2.2 defines a domain through which RGC
axons grow at the midline (Marcus et al.,
1999
), but we did not find evidence of differences between Nkx2.2
expression in the wild-type and mutant (compare
Fig. 7G with
7H). Ephrin B2 is expressed by
radial glial cells that form a palisade at the chiasm and control RGC axon
divergence (Marcus and Mason,
1995
; Marcus et al.,
1995
; Nakagawa et al.,
2000
; Williams et al.,
2003
). Although the shape of the tissues surrounding the optic
chiasm was slightly abnormal in Foxg1 mutants, expression of ephrin
B2 was observed along the ventral midline of the diencephalon in both
wild-type and mutant embryos at E13.5 and E16.5
(Fig. 6). These data show that
the mutant chiasm does not show major defects in the expression of at least
three molecules known to be important for its interaction with RGC axons.
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Discussion |
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Our results show that Foxg1 is required to ensure a normal ipsilateral projection from RGCs. The Foxg1-/- retina produces an increased ipsilateral projection, arising from RGCs located both nasally and temporally. Given the expression patterns discussed above, abnormal development of RGC axons from both nasal and temporal territories argues against a completely cell-autonomous role for Foxg1 in the control of RGC projections. Foxg1 may autonomously regulate the behaviour of nasal RGC growth cones at the optic chiasm, e.g. by influencing their expression of cell surface molecules, but this can not be the case for most temporal RGCs.
The eyes of mice lacking Foxg1 have an abnormal shape
(Xuan et al., 1995;
Huh et al., 1999
) (present
study). A striking feature of the Foxg1-/- phenotype is
the lack of an optic stalk connecting the retina to the brain. This could
result in the retina becoming stretched medially as the head grows. Consistent
with this, the overall volume and patterning of gene expression along its
nasotemporal and dorsoventral axes are similar to wild-type retina. This
argues against a fundamental repatterning of the retina in the absence of
Foxg1. Changes in Foxg1-/- retinal morphology might also
be secondary to defective development of the lens, which expresses Foxg1 early
in its formation and is smaller than normal in mutants (Figs
2,
5)
(Hatini et al., 1994
;
Xuan et al., 1995
;
Dou et al., 1999
;
Huh et al., 1999
). Previous
studies have shown that, although lens is not necessary to instruct the
differentiation of neuroretina, it does influence the morphology of the retina
either mechanically or molecularly
(Ashery-Padan et al.,
2000
).
Foxg1 can be added to the growing list of proteins with a dual role in
tissue morphogenesis and axon growth and morphology. Others on this list
include transcription factors such as Pax6
(Ericson et al., 1997;
Mastick et al., 1997
;
Pratt et al., 2000
;
Pratt et al., 2002
) and
secreted proteins of the Wnt (Hall et al.,
2000
; Yoshikawa et al.,
2003
), bone morphogenetic protein (BMP)
(Augsburger et al., 1999
) and
hedgehog (Charron et al., 2003
)
families. Enzymes involved in regulating the sulphation status of
extracellular heparan sulphate proteoglycans are implicated in Shh, Wnt and
BMP signalling, tissue morphogenesis
(Merry and Wilson, 2002
;
McLaughlin et al., 2003a
) and
RGC axon guidance (Walz et al.,
1997
; Irie et al.,
2002
).
Foxg1 may act at the optic chiasm to control RGC growth cone navigation
The simplest explanation of our finding an increased ipsilateral projection
in embryos lacking Foxg1 is that Foxg1 normally regulates the proportions of
crossed and uncrossed projections by a direct action on both temporal and
nasal axons at the chiasm itself. In the chick, Foxg1 has been shown to
autonomously regulate the retinotectal mapping of RGC axons
(Yuasa et al., 1996) and it is
possible that in the mouse Foxg1 expression by RGCs is involved in
retinotectal mapping rather than in regulating midline crossing at the optic
chiasm.
In the wild-type the diencephalon is surrounded on each side by a large
telencephalic vesicle. This physical support is missing in the mutant, which
has a hypoplastic telencephalon and, as a consequence, the diencephalon adopts
a more `open' conformation. Nevertheless, we found that many morphological and
molecular features of the wild-type chiasm were retained in the mutant. The
normal developing chiasm has been characterised in terms of the expression of
transcription factors and regulatory molecules whose expression domains
coincide with navigating RGC axons (Marcus
et al., 1999; Nakagawa et al.,
2000
; Williams et al.,
2003
). We did not detect any gross abnormalities in the
distribution of cells expressing the transcription factors Foxg1 or Nkx2.2 or
of cells expressing cell surface molecules SSEA-1 or ephrin B2 at the
developing chiasm. Despite its elongated shape, the mutant eye projects a
correlate of the optic nerve, which converges to form the optic chiasm. It is
extremely unlikely that anatomical alterations of the mutant eye or brain are
solely responsible for the alterations in the balance of ipsilateral and
contralateral projections. Taken together, these findings point to a more
specific defect causing the change in RGC axon midline crossing in
mutants.
The mechanisms that regulate RGC growth cone navigation at the normal optic
chiasm are not well understood, although it is known that Ephb and ephrin B
family members are likely to participate in regulating midline crossing of RGC
growth cones (Nakagawa et al.,
2000; Barbieri et al.,
2002
; Williams et al.,
2003
). We did not detect obvious alterations in the distributions
of Ephb2 and ephrin B2 protein in the mutant retina and optic chiasm but this
does not necessarily preclude a role for Foxg1 in the control of Ephb/ephrin B
signalling. There are several precedents for a particular receptor/ligand
interaction leading to either attraction or repulsion depending on other
factors. The presence of the extracellular matrix molecule laminin has been
shown to convert the attraction of RGC growth cones towards a source of netrin
to a repulsion of RGC growth cones away from a source of netrin
(Hopker et al., 1999
). Levels
of an intracellular guanylate cyclase control the response of neocortical
growth cones to a Sema3a gradient (Polleux
et al., 2000
). Although an analagous mechanism has not been
demonstrated for Ephb/ephrin B signalling it appears that Ephb-expressing
axons can be either attracted or repelled by ephrin B in different situations
(McLaughlin et al., 2003b
;
Hindges et al., 2002
).
Foxg1 may, therefore, regulate expression at the optic chiasm of one or more of a potentially very large number of modulators of signalling pathways activated by, for example, Ephb/ephrin B. The identification of transcriptional targets of Foxg1 will allow a systematic survey of these possibilities. The final choice made by each RGC growth cone whether to go ipsilateral or contralateral at the optic chiasm probably depends on the balance between attractive and repulsive signals at the chiasm and the nature of the response of each growth cone to those signals. Most probably loss of Foxg1 promotes ipsilateral projections at the optic chiasm by perturbing these balances.
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ACKNOWLEDGMENTS |
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