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First published online 12 November 2008
doi: 10.1242/dev.023572
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Centre for Integrative Physiology, The University of Edinburgh, Hugh Robson Building, George Square, Edinburgh EH8 9XD, UK.
* Author for correspondence (e-mail: david.price{at}ed.ac.uk)
Accepted 10 October 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Foxg1, Retinal ganglion cell, Chiasm, Mouse
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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The optic chiasm begins forming around embryonic day 12 (E12) in mouse,
shortly after the initiation of RGC genesis. At first, RGCs in dorsocentral
retina project pioneering contralateral axons and a smaller number of
transient ipsilateral axons (Colello and
Guillery, 1990
; Guillery et
al., 1995; Insausti et al.,
1984; Marcus et al.,
1995; Mason and Sretavan,
1997). The peak phase of RGC genesis and RGC axon growth through
the chiasm occurs at E14-16, when the permanent ipsilateral projection forms
(Colello and Guillery, 1990).
Contralateral RGC axons arise from the entire retina, whereas permanent
ipsilateral RGC axons arise mainly from the ventrotemporal crescent (VTC), a
small region of peripheral ventrotemporal (VT) retina. The adult pattern of
decussation is established by birth with a 95-97% to 3-5% ratio of
contralateral to ipsilateral axons (Drager
and Olsen, 1980; Drager,
1985).
In vitro and in vivo experiments have demonstrated that
ipsilateral-contralateral divergence at the chiasm involves repulsive factors
acting at or around the chiasm (reviewed by
Erskine and Herrera, 2007).
During the peak period of retinal axon divergence, the mouse optic chiasm
expresses molecules inhibitory to axon growth, including ephrin B2
(Nakagawa et al., 2000;
Williams et al., 2003),
heparan sulphate proteoglycan modifying enzymes
(Pratt et al., 2006),
chondroitin sulphate proteoglycans (Chung
et al., 2000a; Chung et al.,
2000b; Tuttle et al.,
1998), CD44 (Sretavan et al.,
1994; Sretavan et al.,
1995), stage-specific embryonic antigen 1
(Marcus and Mason, 1995;
Sretavan et al., 1994)
(SSEA-1) and slit proteins (Erskine et al.,
2000; Niclou et al.,
2000; Plump et al.,
2002; Ringstedt et al.,
2000; Thompson et al.,
2006a; Thompson et al.,
2006b). Most of these reduce axon growth from all retinal regions,
rather than VT axons selectively. Ephrin B2, however, which is expressed by
midline radial glia, is necessary and sufficient for the repulsion of
EphB1-bearing VT axons into the ipsilateral optic tract
(Williams et al., 2003). By
contrast, contralateral axons do not express EphB1 during the peak phase of
ipsilateral projections and so their axons are not repelled by ephrin B2 at
the chiasm. To date, there is no evidence for an attractive factor in
developing mouse ventral diencephalon promoting growth of contralateral but
not ipsilateral axons across the chiasm.
Recent work has identified transcription factors that regulate the
expression of key RGC axon guidance molecules (reviewed by
Erskine and Herrera, 2007). A
notable example of an ipsilateral determinant is the zinc-finger transcription
factor Zic2, which is expressed by RGCs in the VTC, is sufficient for their
axons to project ipsilaterally and is thought to act via transcriptional
regulation of their EphB1 axon guidance receptor levels
(Herrera et al., 2003;
Williams et al., 2003;
Lee et al., 2008;
Garcia-Frigola et al., 2008).
A transcription factor implicated in promoting the contralateral projection of
RGC axons is Foxg1, a winged helix transcription factor expressed by nasal
RGCs, nasal optic stalk and presumptive optic chiasm
(Hatini et al., 1994;
Huh et al., 1999;
Pratt et al., 2004).
Foxg1-/- mouse embryos show a significant increase in the
number of ipsilateral projections (Pratt
et al., 2004); strikingly, they develop a major ipsilateral
projection from nasal retina.
Here, we have investigated the mechanism of action of Foxg1 in determining
the laterality of RGC axon projections. We found a significant increase in the
number of Zic2-expressing RGCs and ectopic Ephb1 expression in
Foxg1-/- DN retina. We used an in vitro assay to test
whether Foxg1 is required by nasal RGCs, by comparing axon growth from
Foxg1-expressing and Foxg1-/- dorsonasal (DN)
retinal explants co-cultured with dissociated chiasm cells from
Foxg1-expressing embryos. This co-culture approach has an established
track record in demonstrating differential responses of different types of
RGCs to chiasm cells: for example, wild-type VT retinal axons grow less well
than wild-type DN retinal axons on chiasm cells, reflecting the fact that many
VT retinal axons are repelled from the chiasm into the ipsilateral optic tract
in vivo (Herrera et al., 2003;
Herrera et al., 2004;
Marcus et al., 1995;
Marcus and Mason, 1995;
Marcus et al., 1996;
Wang et al., 1995). We found
that Foxg1 is required in DN retina for its axons to grow normally on chiasm
cells. We also tested whether Foxg1 is required by chiasm cells, by comparing
axon growth from Foxg1-expressing DN or VT retina co-cultured with
dissociated chiasm cells from either Foxg1-expressing or
Foxg1-/- embryos. These experiments indicated that Foxg1
is also required at the optic chiasm for its cells to support the normal
growth of retinal axons.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) enables
identification of cells in which the Foxg1 locus is transcriptionally
active. Co-cultures used pigmented mouse embryos from
Foxg1LacZ/+ (CBA) and Foxg1Cre/+
(Swiss Webster) matings. The Foxg1-coding sequences in the
Foxg1Cre allele are replaced by a Cre recombinase cassette
(Hebert and McConnell, 2000).
Foxg1LacZ and Foxg1Cre are predicted
null alleles (Hebert and McConnell,
2000; Xuan et al.,
1995).
PCR genotyping Foxg1 alleles
Foxg1LacZ/LacZ and Foxg1Cre/Cre
embryos were identified by their severely hypoplastic telencephalon and eye
deformities. Foxg1LacZ/+ embryos were distinguished from
Foxg1+/+ embryos by PCR of embryonic tails using primers
lacZ F2 (5'-TTG AAC TGC CTG AAC TAC CG-3') and
lacZ R2 (5'-CCT GAC TGG CGG TTA AAT TG-3'). Cycling
conditions were as previously described
(Pratt et al., 2004).
Histochemistry
Embryos were fixed in 4% paraformaldehyde (PFA) in phosphate-buffered
saline (PBS) for 2 hours. lacZ staining on cryostat sections (10
µm) or whole mounts and immunohistochemistry on cryostat sections (10
µm) or wax sections (10 µm) were performed as previously described
(Pratt et al., 2004). Primary
antibodies were: rabbit polyclonal Zic2 antibody (1/8,000) (Stephen Brown,
Columbia University, New York (Brown et
al., 2003); mouse monoclonal Brn3a antibody (1/300) (Chemicon
International). Fluorescent secondary detection used goat anti-rabbit IgG
AlexaFluor 488 and goat anti-mouse IgG AlexaFluor 546 antibodies (1/150;
Molecular Probes) and sections were counterstained using TO-PRO-3 (1/2,000;
Molecular Probes). Non-fluorescent detection of Zic2 or Brn3a was via a
diaminobenzidine (DAB) colour reaction (Zic2: Rabbit Envision+ kit, Dako
K4010; Brn3a: mouse Envision+ kit, Dako K4006).
For each eye, numbers of labelled cells were counted in six evenly spaced sections from dorsal to ventral: the dorsal- and ventral-most sections in which Brn3a-positive cells were visible were identified and the intervening four sections were then chosen by dividing up the distance between the dorsal- and ventral-most sections equally. One-way ANOVA was used to assess the significance of differences between groups and, where differences were significant (P<0.05), two-tailed Student's t-tests assuming equal variances were used to assess differences between pairs of groups.
Co-cultures
Retinal explants were cultured in collagen gels surrounded by dissociated
chiasm cells of the same age (E14.5). Co-cultures were prepared based on a
method described previously (Wang et al.,
1996). Collagen (10-20 µl) was spread evenly onto circular
glass coverslips and allowed to set at 37°C. Equal-sized retinal explants
from peripheral DN or VT retina (regions shown in
Fig. 1C) were placed on top of
the collagen in 20 µl of serum-free culture medium containing 0.5%
methylcellulose to aid adhesion. Explants were incubated at 37°C for 2
hours, allowing them to adhere to the collagen gel. Tissue that would provide
optic chiasm cells was cut from the ventral surface of the brain and included
a region extending 
200 µm anterior, posterior and lateral to the
decussation so as to include the Foxg1-expressing region. Chiasm
tissue was dissociated using papain (Worthington Biochemical Corporation,
#LK003160) and cells were resuspended in a 1:1 mixture of rat-tail and bovine
collagen before being added to the retinal explants at 50,000
cells/mm2. After 2 hours, fresh serum-free culture medium without
methylcellulose was added to the cultures, which were then incubated at
37°C for 48 hours. Co-cultures were fixed in 4% PFA in PBS after 48 hours
followed by neurofilament and Brn3a immunohistochemistry. The co-cultures were
blocked in 10% goat serum, 0.2% Triton-X-100 in PBS (PBSTx-100) for 90 minutes
at room temperature prior to overnight incubation at 4°C with primary
antibodies: rabbit neurofilament (1/200) (Biomol International) and mouse
Brn3a (1/300) (Chemicon International). Following washes in 0.1% PBSTx-100 at
room temperature, the cultures were incubated overnight at 4°C with
secondary antibodies: goat anti-mouse Alexa Fluor 546 (1/500) (Molecular
Probes) and goat-anti-rabbit Alexa Fluor 488 (1/500) (Molecular Probes).
Following 0.1% PBSTx-100 washes, cultures were incubated in TO-PRO-3 (1/2,000)
for 1 hour and mounted in a 9:1 solution of glycerol: PBS. The densities of
chiasm cells that were viable, judged by high-power examination of nuclei
after culture, were counted in three sampling boxes adjacent to the explant
border: mean densities were 28,000-38,000 cells/mm2 and did not
vary significantly between any of the different co-culture combinations by
ANOVA (P=0.653) (see Fig. S1 in the supplementary material). Neurite
outgrowth was quantified for all retinal explants by obtaining measures of its
amount and length (see Fig. S2 in the supplementary material). The amount of
outgrowth was estimated by surrounding each explant by a line at 23, 45, 68 or
91 µm from its edge [e.g. yellow polygon in Fig. S2B (see supplementary
material) 45 µm from edge] and calculating the percentage of its
circumference that was covered by neurites crossing it (see Fig. S2C,D in the
supplementary material). This estimate is referred to as `percentage axon
coverage'. A measure of length was obtained by calculating the mean of the
lengths of the five longest neurites for each culture. One-way ANOVA was used
to assess the significance of differences between groups and, where
differences were significant (P<0.05), Tukey tests were used to
assess differences between pairs of groups (n values stated in
Results are numbers of explants).
Ephb1 and Foxd1 in situ hybridization
Digoxigenin-labelled antisense riboprobes were from mouse Ephb1
and Foxd1 cDNAs. Ephb1 in situ hybridization was on 10 µm
paraffin sections (Christoffels et al.,
2000). Foxd1 in situ hybridization was on 100 µm vibrotome
sections (Erskine et al.,
2000).
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Pratt et al.,
2004; Xuan et al.,
1995)]. Dorsal retina showed widespread lacZ staining
(Fig. 1E). Moving ventrally,
lacZ staining retreated progressively nasally
(Fig. 1F-J), until it was
absent from the entire retina in extremely ventral sections
(Fig. 1K). A diagram of
Foxg1 activation deduced from these data in the E14.5 retina is shown
in Fig. 1B (results were the
same at E13.5 and E15.5; not shown). The border between
Foxg1-positive and Foxg1-negative regions runs at an angle
to the dorsoventral axis, such that Foxg1 is expressed in DN regions,
declining towards the VT retina, where Foxg1 expression is entirely
absent.
Zic2, Ephb1 and Foxd1 expression are altered in the Foxg1-/- nasal retina
The Foxg1-/- retina produces an increased ipsilateral
projection, much of which arises ectopically from nasal retina. Previous work
has shown that at E14.5-16.5, when VTC axons are navigating ipsilaterally, the
zinc-finger transcription factor Zic2 and the axon guidance receptor EphB1
determine the navigation of ipsilaterally projecting RGC axons from the VTC
(Herrera et al., 2003;
Williams et al., 2003). Here,
we have considered the possibility that in the absence of Foxg1 these
ipsilateral determinants are upregulated in nasal retina.
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).
In E14.5 Foxg1+/+ retina
(Fig. 2A-H), Zic2-expressing
cells were predominantly in VT retina, with strongest expression in a cluster
in the RGC layer adjacent to the strongly Zic2-expressing ciliary marginal
zone (CMZ) (Fig. 2C,D,G), as
described before (Herrera et al.,
2003). In other regions of Foxg1+/+ retina,
weak staining for Zic2 was seen in small numbers of cells in the RGC layer,
although most Zic2-positive cells were outside the RGC layer and were Brn3a
negative (Fig. 2E,F,H).
Quantification of all cells staining positive for Zic2 in the RGC layer
(irrespective of their level of staining) in six evenly spaced horizontal
sections (Fig. 3A), is shown in
Fig. 3B,C (light blue bars show
Foxg1+/+ data): in Foxg1+/+ embryos,
there were more Zic2-expressing cells in VT retina than in other quadrants, as
described previously (Herrera et al.,
2003).
In E14.5 Foxg1-/- retina (Fig. 2I-P), Zic2 is still expressed in the RGC layer of VT retina (Fig. 2L,P) and quantification in this region revealed no significant difference in proportions of Zic2-positive cells compared with those in Foxg1+/+ embryos at E14.5 and E16.5 (right-hand bars in Fig. 3B,C). However, large increases in the proportion of Zic2-expressing cells were visible in the RGC layer of DN (Fig. 2J,N) and ventronasal (VN) (Fig. 2K,O) retina. Quantification in Foxg1+/+ and Foxg1-/- DN and VN retina confirmed that there were significantly greater proportions of Zic2-expressing cells in Foxg1-/- nasal retina (Fig. 3B,C). Quantification of numbers of Brn3a-expressing RGCs at E14.5 and E16.5 revealed no significant differences between Foxg1+/+ and Foxg1-/- retinae, ruling out the possibility that changes in numbers of RGCs account for increased Zic2 expression (see Fig. S3 in the supplementary material).
To investigate whether ectopic expression of Zic2 was associated with
ectopic Ephb1 expression in Foxg1-/- nasal
retina, the distribution of Ephb1 mRNA was revealed using in situ
hybridization (Fig. 4). In
Foxg1+/+ embryos, strong staining for Ephb1 was
observed in RGCs in the VTC (Fig.
4B,C), in agreement with previous reports
(Williams et al., 2003). In
Foxg1-/- mutants, Ephb1-expressing RGCs were
observed in VT retina, as in Foxg1+/+ embryos
(Fig. 4F,H). High levels of
Ephb1 expression were also observed in clusters of nasal RGCs
(Fig. 4D-G, arrows in D,F, box
in E) the distributions of which matched those of Zic2-expressing RGCs in
mutant nasal retina.
Previous studies have shown that loss of the transcription factor Foxd1,
the expression of which is complementary to that of Foxg1 in normal retina,
results in loss of Zic2 and EphB1 from the VTC, suggesting that Foxd1 might be
an upstream activator of Zic2 and Ephb1
(Herrera et al., 2004). We
used in situ hybridization to test whether Foxd1 expression expands
into DN retina. Sections from Foxg1+/+ embryos confirmed
the expected expression of Foxd1 in temporal retina
(Fig. 4I-K). In
Foxg1-/- mutants, Foxd1 was expressed more widely
in both temporal and nasal retina (Fig.
4L-N). In ventral sections, staining for Foxd1 expression
was strongest temporally (Fig.
4N), but in more dorsal sections there was very strong ectopic
nasal expression (Fig.
4L,M).
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;
Pratt et al., 2004;
Xuan et al., 1995) and do not
show abnormal proportions of ipsilateral projections
(Pratt et al., 2004) (N.M.T.,
T.P. and D.J.P., unpublished). For this reason, and to avoid the need to
genotype embryos prior to culture (whereas E14.5 Foxg1-/-
embryos are easily recognized morphologically, genotyping is required to
distinguish Foxg1+/- from Foxg1+/+
embryos), both genotypes were used to provide Foxg1-expressing tissue
that is referred to here as Foxg1+/±. The DN
retinal explants were dissected from the centre of the region that normally
expresses Foxg1, whereas the VT explants were dissected from
Foxg1-negative retina, as shown in
Fig. 1C. Explants were
dissected from equivalent regions in Foxg1+/± and
Foxg1-/- retinae.
Retinal axon outgrowth in the absence of chiasm cells
To investigate whether intrinsic growth differences exist between
Foxg1+/± and Foxg1-/- RGC
axons, retinal explants were cultured alone without chiasm cells.
Fig. 5A-D shows typical
confocal images of Foxg1+/± and
Foxg1-/- explants from DN and VT retina. Fluorescence
immunohistochemistry reveals expression of the axon marker neurofilament
(green) and the POU-homeodomain transcription factor Brn3a (red), expressed by
postmitotic RGCs (Pan et al.,
2005). Brn3a-expressing RGCs were present and appeared healthy in
all retinal explants.
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), demonstrating that axons from the VT retina, many of which
normally project ipsilaterally at the chiasm, are capable of prolific growth
in the absence of chiasm cells. This was also true for DN
Foxg1-/- retina versus VT Foxg1-/-
retina. Importantly, we found no significant differences in the amounts of
outgrowth or the lengths of the longest neurites between
Foxg1+/± and Foxg1-/- DN or
Foxg1+/± and Foxg1-/- VT
explants (Fig. 5E,F). In view
of these results, differences in the growth of axons from
Foxg1+/± and Foxg1-/- DN
explants or from Foxg1+/± and
Foxg1-/- VT explants in the presence of
Foxg1+/± or Foxg1-/- chiasm
cells described in the following sections can be attributed to differences in
the interaction of these explants with chiasm cells.
DN retinal axon outgrowth on chiasm cells
Co-cultures were prepared in the following combinations: (1) DN
Foxg1+/± retina with
Foxg1+/± chiasm cells; (2) DN
Foxg1-/- retina with Foxg1+/±
chiasm cells; (3) DN Foxg1+/± retina with
Foxg1-/- chiasm cells; (4) DN Foxg1-/-
retina with Foxg1-/- chiasm cells (compositions of
co-cultures will be notated as Foxg1+/± retina
Foxg1+/± chiasm,
Foxg1-/- retina Foxg1+/±
chiasm, etc.).
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Foxg1+/± chiasm co-culture. Prolific neurite growth
was seen from all sides of the explant, reaching far into the surrounding
chiasm cells. By contrast, DN Foxg1-/- retina
Foxg1-/- chiasm co-cultures consistently displayed limited
neurite growth into the surrounding chiasm cells: neurites were short and
highly fasciculated, and a large proportion wrapped around the explant
(Fig. 6D). DN
Foxg1-/- retina Foxg1-/- chiasm
co-cultures showed a significantly lower percentage of axon coverage and
shorter neurite lengths than did DN Foxg1+/± retina
Foxg1+/± chiasm co-cultures
(Fig. 6E,F, compare 1st and 4th
bars). This in vitro result reflects the increased avoidance of the
Foxg1-/- chiasm by nasal Foxg1-/-
retinal axons in Foxg1-/- embryos in vivo
(Pratt et al., 2004).
Culturing Foxg1+/± retina with
Foxg1-/- chiasm cells, or Foxg1-/-
retina with Foxg1+/± chiasm cells, enabled us to
investigate the effect on retinal axon growth of removing Foxg1 from
chiasm cells or from the retina. Fig.
6B shows an example of a typical DN
Foxg1+/± retina Foxg1-/-
chiasm co-culture. In comparison with the DN
Foxg1+/± retina
Foxg1+/± chiasm co-culture shown in
Fig. 6A, fewer and shorter
neurites were observed. This is reflected in a significant reduction in
percentage axon coverage and mean neurite length
(Fig. 6E,F, compare 1st and 3rd
bars). In addition, DN Foxg1-/- retina
Foxg1-/- chiasm co-cultures had significantly lower mean
percentage axon coverage compared with DN Foxg1-/- retina
Foxg1+/± chiasm co-cultures
(Fig. 6E, compare 2nd and 4th
bars). These results indicate that loss of Foxg1 from chiasm cells reduces
their ability to support retinal axonal growth.
Our data indicate that DN axons grow better in the presence of chiasm cells than in their absence (mean percentage axon coverage and lengths of longest neurites were roughly double in the presence of chiasm cells) (Fig. 5E,F and Fig. 6E,F, compare 1st bars). Loss of Foxg1 from chiasm cells reduces the growth of Foxg1-expressing DN axons to levels below those seen from Foxg1-expressing DN axons grown in the absence of chiasm cells (compare 1st bars in Fig. 5E,F with 3rd bars in Fig. 6E,F). This suggests that chiasm cells might normally play an active Foxg1-dependent role in supporting the growth of DN axons, and that the loss of Foxg1 from the chiasm converts this positive role to an inhibitory one.
The effect of removing Foxg1 from DN retina can be seen in
Fig. 6C, which shows a typical
DN Foxg1-/- retina
Foxg1+/± chiasm co-culture. Fewer neurites were
seen compared with DN Foxg1+/± retina
Foxg1+/± chiasm co-cultures. Quantification showed
a significant reduction in percentage axon coverage
(Fig. 6E, compare 1st and 2nd
bars) although there was no significant reduction in the lengths of the
longest neurites (Fig. 6F,
compare 1st and 2nd bars). Amounts of outgrowth from
Foxg1-/- DN retina cultured with
Foxg1+/± chiasm cells remained similar to those
from Foxg1-/- DN retinal explants cultured without chiasm
cells (compare 2nd bars in Fig.
5E,F with 2nd bars in Fig.
6E,F).
Data presented above are for measurements at 45 µm from the explant. Data on percentage axon coverage at other distances, from 23 µm to 91 µm, are shown in Fig. S4 in the supplementary material. Predictably, average percentage axon coverage fell as the distance from the retinal explant increased, but the differences between the different types of co-culture remained the same at each point of measurement.
In summary, our data provide functional evidence that Foxg1 is required in its normal region of expression in the DN retina to support the growth of DN RGC axons across chiasm cells: whereas the addition of Foxg1+/± chiasm cells enhanced outgrowth from Foxg1+/± DN retina in culture, addition of Foxg1+/± chiasm did not have this effect on Foxg1-/- DN retina. This is likely to be explained by changes in expression of Zic2 and Ephb1 in Foxg1-/- DN retina (see above). In addition, removing Foxg1 from the chiasm reduced the growth of DN Foxg1+/± and Foxg1-/- retinal axons to levels below those of DN Foxg1+/± and Foxg1-/- retinal axons grown without chiasm cells, suggesting that the failure of many DN axons to penetrate the chiasm and instead to enter the ipsilateral optic tract in Foxg1-/- mutant mice might be explained by a chiasmatic defect.
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Foxg1+/± chiasm; (2) VT
Foxg1-/- retina Foxg1+/±
chiasm; (3) VT Foxg1+/± retina
Foxg1-/- chiasm; (4) VT Foxg1-/-
retina Foxg1-/- chiasm. Previous work has shown
that, in the presence of chiasm cells, wild-type VT retinal axons are shorter
and fewer in number compared with axons from wild-type DN retina, reflecting
the fact that many are repelled from the chiasm into the ipsilateral optic
tract in vivo (Herrera et al.,
2003; Herrera et al.,
2004; Marcus et al.,
1995; Marcus and Mason,
1995; Marcus et al.,
1996; Wang et al.,
1995). We observed the same result: values for percentage axon
coverage and neurite length from VT retinae grown with chiasm cells were much
lower (66-93% less) than from DN retinae grown with chiasm cells and from VT
retinae grown without chiasm cells (compare values in Figs
5,
6,
7). Analysis of variance
(ANOVA) indicated that, unlike data from the DN retinal co-cultures, there was
no statistically significant effect of varying the genotype of the retina or
the chiasm across the four co-culture combinations shown in
Fig. 7A-F. Our data provide no
evidence for a defect in the ability of Foxg1-/- VT RGC
axons to grow on chiasm cells. |
DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) but,
despite this, mutant retina retains a normal volume and a normal number of
RGCs (Pratt et al., 2004). We
investigated the molecular changes in the retina underlying the increased
ipsilateral routing of RGC axons in Foxg1-/- embryos and
found a significant increase in the number of nasal RGCs expressing the
ipsilateral determinant Zic2. RGCs expressing Ephb1 were also found
in nasal retina and are most probably responsible for the increased
ipsilateral routing of Foxg1-/- nasal retinal axons owing
to repulsion by ephrin B2 at the chiasm
(Williams et al., 2003).
Together, these findings suggest that: (1) Foxg1-expressing nasal
retina is competent to express a genetic program, including the ipsilateral
determinants Zic2 and EphB1, which are normally expressed only in
Foxg1-negative VTC cells as their axons navigate the chiasm; and (2) Foxg1
normally represses this ipsilateral program within nasal RGCs to prevent the
ipsilateral routing of their axons.
Patterning the retina
Previous descriptions have considered that the normal expression of
Foxg1 is limited mainly to nasal retina before and at the age when
retinal axons are navigating the chiasm
(Pratt et al., 2004;
Hatini et al., 1994;
Huh et al., 1999). Data
presented here indicate a slight modification: the boundary of expression runs
at an angle to the dorsoventral axis of the retina so that the expression
domain of Foxg1 is centred in DN retina. This means that the
Foxg1-positive domain is complementary to the domain of expression of
the transcription factor Foxd1, which is centred in VT retina
(Herrera et al., 2004).
Previous studies have shown: (1) that loss of Foxd1 results in loss of Zic2
and EphB1 from the VTC, suggesting that Foxd1 is an upstream activator of
Zic2 and Ephb1 (Herrera
et al., 2004); and (2) that loss of Foxd1 results in an expansion
of Foxg1 into VT retina, suggesting that Foxd1 represses Foxg1 in the
retina (Herrera et al., 2004).
In light of our present findings indicating that Foxg1 represses Zic2 and
EphB1, the loss of Zic2 and EphB1 expression in Foxd1-/-
mutants might be explained by the expansion of Foxg1 into VT retina.
Could loss of Foxg1 cause an upregulation of Zic2 and EphB1 in DN retina via an ectopic DN expression of Foxd1? Our results suggest that this is possible, as Foxd1 expression expands into the DN retina of Foxg1-/- mutants. Based on current evidence, there are several possibilities to explain the actions of Foxg1 and Foxd1 in the normal retina: (1) Foxd1 might be a direct upstream activator of Zic2 and Ephb1, and Foxg1 might prevent Zic2 and Ephb1 expression in DN retina indirectly by preventing expression of Foxd1; (2) Foxg1 might be a direct upstream repressor of Zic2 and Ephb1, and Foxd1 might allow expression of Zic2 and Ephb1 in VT retina indirectly by preventing expression of Foxg1; (3) both Foxd1 and Foxg1 might be direct regulators (positive and negative, respectively) of Zic2 and Ephb1 expression.
In normal retina, the expression domains of Zic2 and EphB1 are restricted
peripherally in VT retina to cells in the VTC that are in the process of
forming permanent ipsilateral projections. Later, Zic2 is rapidly
downregulated once these projections have formed, while EphB1 expression
becomes more widespread in the retina, where it may regulate other processes
(Herrera et al., 2003;
Williams et al., 2003). We
observed characteristically restricted expression of Zic2 and Ephb1
not only in VT retina but also in DN retina of Foxg1-/-
mutants, where ectopic expression of Zic2 and Ephb1 was mainly in its
peripheral region. This suggests that the sequence of events that generate
ipsilateral projections from VT retina might also be followed in DN retina of
Foxg1-/- mutants.
Foxg1 represses ipsilateral axon guidance from nasal retina
Our results indicate that Foxg1 is required by DN retina for its
axons to grow to their normal extent on chiasm cells in vitro and hence for
them to penetrate the chiasm to grow contralaterally in normal numbers in
vivo. This might occur because Foxg1 normally represses expression of an
ipsilateral determinant in DN retina or activates expression of a
contralateral determinant in DN retina. Our evidence supports the former by
revealing a significant increase in Zic2- and Ephb1-expressing cells
in Foxg1-/- nasal retina. In the VTC, expression of Zic2
by RGCs is sufficient for their axons to project ipsilaterally
(Herrera et al., 2003). Our
findings suggest that abnormal expression of Zic2 by RGCs in DN retina can
also redirect their axons ipsilaterally. This is in excellent agreement with
recently reported findings by Garcia-Frigola et al.
(Garcia-Frigola et al., 2008),
who showed that misexpression of Zic2 in RGCs outside the VTC during a
specific time-window around E13.5 is sufficient to direct the axons of those
RGCs ipsilaterally. In E14.5 and E16.5 Foxg1-/- embryos,
we found seven- to eightfold increases in numbers of Zic2-expressing cells in
DN retina, very close to the eightfold increases in ipsilateral projections
reported previously in E15.5 Foxg1-/- embryos
(Pratt et al., 2004). In the
normal VTC, Zic2 is thought to act via positive transcriptional regulation of
the EphB1 axon guidance receptor (Williams
et al., 2003; Herrera et al.,
2003; Lee at al.,
2008; Garcia-Frigola et al.,
2008) and our results suggest that this same mechanism occurs in
the DN retina in the absence of Foxg1. Our results indicate that DN cells are
competent to express an ipsilateral program characteristic of the VTC in the
absence of Foxg1 protein.
A likely model is that Foxg1 in DN retina normally represses Zic2;
thus, reducing expression of its downstream target Ephb1 and
preventing repulsive EphB1-ephrin B2 interactions between retinal axons and
chiasm cells, although a parallel direct effect of Foxg1 on Ephb1
expression can not be excluded. Other hypotheses are possible but less
attractive. Previous findings have identified a link between the contralateral
projection and the transcription factor islet 2
(Pak et al., 2004) and
cell-adhesion molecule Nr-CAM (Williams et
al., 2006). Both islet 2 and Nr-CAM are expressed in
contralaterally projecting RGCs and mice lacking these genes display an
increased ipsilateral projection. However, these abnormal projections arise
from RGCs confined to the VTC and, in the case of Nr-CAM mutants, are
generated later than the abnormal ipsilateral projections in
Foxg1-/- embryos (Pak
et al., 2004; Williams et al.,
2006). It seems probable, therefore, that any loss of islet 2 or
Nr-CAM in Foxg1-/- retinae would not explain in a simple
way the respecification of normally contralaterally projecting nasal RGCs to
an ipsilateral fate, as observed in Foxg1-/- embryos
(Pratt et al., 2004).
Foxg1 controls axon guidance at the chiasm
The extra ipsilateral projections in Foxg1-/- embryos
arise from temporal as well as nasal RGCs
(Pratt et al., 2004). The
greatest increases in numbers of Zic2-expressing cells were confined to nasal
retina, where Foxg1 is normally expressed, with only a small but significant
increase in dorsotemporal (DT) retina at E16.5, which is too late to explain
the increased ipsilateral projection from the temporal retina of
Foxg1-/- embryos. This increased projection from temporal
retina is probably caused by defects at the optic chiasm. This might be due to
changes in the biochemistry of chiasm cells or might arise as a secondary
consequence of the altered routing of a large proportion of axons from the
nasal retina.
Our studies provide direct evidence that, in addition to its action in the
retina, Foxg1 also functions as a contralateral determinant by regulating the
environment at the chiasm. Our culture work indicated that
Foxg1-/- chiasm cells are less supportive than
Foxg1+/+ chiasm cells of retinal axons growing across
them. Current hypotheses on the mechanisms guiding the laterality of RGC axons
at the chiasm focus on the importance of inhibitory interactions that repel
some axons into the ipsilateral tract
(Nakagawa et al., 2000;
Williams et al., 2003). On the
other hand, our findings indicated that DN axons grow better in the presence
of chiasm cells than in their absence, suggesting that chiasm cells might play
an active role supporting the growth of DN axons across them. Interestingly,
previous studies using a similar culture approach found no such evidence for a
growth-promoting effect of chiasm cells on DN axons; in some cases, the
presence of chiasm cells inhibited the growth of DN axons
(Wang et al., 1995;
Williams et al., 2003;
Williams et al., 2006). There
are several differences between the methods we used and those of others. A
potentially crucial difference is that, unlike previous workers, we placed
chiasm cells in a collagen gel rather than on a laminin substrate. Previous
work has shown that laminin can convert growth cone attraction to growth cone
repulsion (Hopker et al.,
1999); it is possible that our results are explained by the
release of a growth-promoting molecule whose actions are reversed or negated
depending on the substrate used.
Loss of Foxg1 from chiasm cells removes their ability to support the growth
of Foxg1-expressing DN axons. One possible explanation for this is that Foxg1
at the chiasm normally upregulates the expression of growth-promoting
molecules. Alternatively, or in addition, Foxg1 at the chiasm might prevent
the expression of growth-inhibiting molecules, and/or modifiers of those
inhibitory molecules, that might otherwise counteract the growth promoting
activity of chiasm cells. Evidence that the second possible mechanism
contributes to the net action of Foxg1 comes from our finding that there is
less outgrowth from retinal explants grown on chiasm cells lacking Foxg1 than
from chiasm cells cultured with no chiasm cells. Given the potential
complexity of the effects of Foxg1 at the chiasm and the fact that previous
work has not shown changes in the expression of obvious candidate molecules,
including ephrin B2, CD44, SSEA-1 (Pratt
et al., 2004) and Zic2 (N.M.T., T.P. and D.J.P., unpublished), a
systematic unbiased approach to identifying molecular changes at the chiasm in
the absence of Foxg1 is now indicated.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/24/4081/DC1
|
ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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