1 Departments of Pathology, Anatomy and Cell Biology and Center for Neurobiology
and Behavior, Columbia University, College of Physicians and Surgeons, 630
West 168th Street, New York, NY 10032, USA
2 Cell Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York
Avenue, New York, NY 10021, USA
3 Departments of Visual Science and Molecular Genetics, Institute of
Ophthalmology, University College London, London EC1V 9EL, UK
Author for correspondence (e-mail:
cam4{at}columbia.edu)
Accepted 7 September 2004
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SUMMARY |
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Key words: Foxd1, Foxg1, Brain Factor 1, Brain Factor 2, BF-1, BF-2, Retinal axon divergence, Zic2, EphB1
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Introduction |
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Foxg1 (also named Brain Factor 1, BF-1) and Foxd1 (also named Brain Factor
2, BF-2) are winged helix transcription factors characterized by a DNA-binding
motif originally identified in HNF3 and found in Drosophila forkhead
proteins (Hatini et al., 1994).
Foxg1 and Foxd1 are expressed in adjacent domains in the neural tube at the
time the optic vesicles evaginate. Foxg1 is expressed in the nasal optic cup
and Foxd1 is expressed in a complementary pattern temporally
(Clark et al., 1993
). As
misexpression of Foxg1 and Foxd1 in chick retina results in projection errors
of retinal axons along the anteroposterior axis in the tectum
(Takahashi et al., 2003
;
Yuasa et al., 1996
), this pair
of proteins has been proposed to determine the regional specificity of axon
projection along the nasotemporal retina through the regulation of downstream
targets. Thus, for example, misexpression of Foxg1 in the temporal retina
represses the tyrosine kinase receptor EphA3 and the expression of Foxd1, and
induces the expression of members of the ephrin-A family
(Takahashi et al., 2003
).
In mouse, both Foxg1 and Foxd1 are also expressed in the developing ventral
diencephalon, with the Foxd1 domain including the region in which early
retinal axons establish the optic chiasm, whereas Foxg1 is located more
rostrally. Both genes are therefore well positioned to play a role in the
regionalization of the optic chiasm
(Marcus et al., 1999).
However, despite the recent advances in understanding the role of Foxg1 and
Foxd1 in retinal axial polarity and retino-tectal projections, and in
understanding their relationship to the Eph/ephrin-A family, the extent to
which these transcription factors regulate aspects of retinal specification
and axon navigation in other portions of the retinal axon pathway, such as in
the optic chiasm, remained unexplored. Because the retinofugal projection in
the chick lacks an uncrossed projection at the optic chiasm
(Thanos and Mey, 2001
),
additional studies are needed to determine whether Foxg1 and Foxd1 are
required for patterning the visual projection in animals with binocular vision
such as the mouse. Recent analyses in mice lacking Foxg1 show an increase in
the number of ipsilateral axons, suggesting a role for Foxg1 in the formation
of the optic chiasm (Pratt, 2004).
Mice that lack Foxd1 (Foxd1lacZ/lacZ mice) die at birth due to
kidney malformations but appear to have grossly normal eye development
(Hatini et al., 1996). In this
study we used mice lacking Foxd1 to further study the role of Brain Factor
genes in retinofugal pathway development during the period of chiasm formation
(E14-E18). RGCs that project ipsilaterally are located in the ventrotemporal
(VT) crescent in the mouse retina, whereas axons projecting contralaterally
arise from RGCs throughout the retina. The decision of RGCs from the VT retina
to project ipsilaterally at the optic chiasm is regulated by the zinc-finger
transcription factor Zic2 (Herrera et al.,
2003
), and is subsequently mediated by the pair of tyrosine kinase
factors implicated in axon guidance, EphB1/ephrin-B2, with EphB1 acting as a
receptor in ipsilateral RGC axons, and ephrin-B2 expressed in midline glial
cells at the optic chiasm and functioning as a ligand
(Williams et al., 2003
). Other
guidance factors, such as Slit2 (Erskine et
al., 2000
; Plump et al.,
2002
), chondroitin sulfate proteoglycans
(Chung et al., 2000
) and
ephrin-As (Marcus et al.,
1996a
; Marcus et al.,
2000
) that have been localized in specialized populations of
neurons and glia at the chiasmatic midline, function to pattern the overall
organization of the optic chiasm, rather than in the decussation of RGC axons.
At present, whether any of these, or other, factors are involved specifically
in crossing the midline is unknown.
We report here that the absence of Foxd1 leads to disruption in the shape of the optic chiasm and in the proportion of fibers projecting ipsi-versus contralaterally, and to stalling or misrouting of retinal axons at the optic chiasm. Although eye development and RGC differentiation appear to be normal in the Foxd1 deficient retina, later-expressed genes specifying the ipsilateral RGC axon projection in retina are lost, and regionalization of the ventral diencephalon is altered, both aspects that are important for proper chiasm formation.
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Materials and methods |
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Anterograde and retrograde labeling of RGC axons
Anterograde labeling of the whole retinal projection with DiI, and
retrograde tracing using rhodamine-dextran applied to the optic tract, were
performed as described (Erskine et al.,
2000; Herrera et al.,
2003
; Rachel et al.,
2002
).
To count retrogradely labeled RGCs in the retina ipsilateral to optic tract labeling in Foxd1lacZ/lacZ embryos, a region was delineated in the VT crescent in flattened retinal wholemounts of Foxd1+/+ embryos by two perpendicular lines: one at the central boundary of the ventrotemporal crescent (defined as the region containing all of the dextran-labeled RGCs); and the second perpendicular to this line and to the border of the retina, 300 µm in length. We superimposed these lines onto the ventrotemporal region of Foxd1lacZ/lacZ flattened retinal wholemounts, taking as a reference point the labeled cells closest to the peripheral border of the retina. All of the labeled cells inside the region delimited by these lines were then counted.
Co-culture assays
E14.5 retina and chiasms from embryos from
Foxd1+/+xFoxd1lacZ/lacZ crosses were dissected.
Foxd1+/+ or Foxd1lacZ/lacZ retinal explants were
co-cultured with either Foxd1+/+ or Foxd1lacZ/lacZ
dissociated chiasm cells, as previously described
(Herrera et al., 2003;
Marcus and Mason, 1995
;
Marcus et al., 1996b
;
Wang et al., 1995
;
Williams et al., 2003
).
X-gal histochemistry and immunohistochemistry
Retinal wholemounts were dissected, treated with 3%
H2O2 in PBS to block endogenous peroxidase activity,
washed in PBS, fixed in 2% paraformaldehyde (PFA) and then incubated with 1
mg/ml X-gal diluted in 30 mM K3Fe(CN)6, 30 mM
K4Fe(CN)6 and 2 mM MgCl2 to detect
lacZ expression. The tissue was then processed for
immunohistochemistry with anti-Zic2 antibodies
(Brown et al., 2003) and
anti-rabbit-peroxidase antibodies.
For immunohistochemistry on sections, 4% PFA-fixed cryosections were
incubated with 10% normal goat serum (NGS)-1% Triton X-100 in PBS for 1 hour
at room temperature. Sections were then incubated in anti-Brn3b, anti-Islet1/2
(K4, gift of Dr T. Jessell, Columbia University), anti-SSEA
(Marcus et al., 1995) or
anti-Zic2, for 1 hour at room temperature; washed six times for 30 minutes
each wash in PBS at room temperature; and then incubated with Cy2- or
Cy3-conjugated secondary antibodies (Jackson Immunoresearch) for 1 hour at
room temperature.
In situ hybridization
In situ hybridization, using digoxigenin-labeled riboprobes, was performed
on 20 µm cryosections (Schaeren-Wiemers
and Gerfin-Moser, 1993). Rat cDNA clone IMAGE 1003496 was used as
a template to detect Islet2 mRNA. Ephrin-B2 and EphB1 specific probes were
generated as described by Williams et al.
(Williams et al., 2003
). A
Slit2 probe was generated as described by Erskine et al.
(Erskine et al., 2000
). The
specific probe used to detect Foxg1 mRNA was similar to that
previously described by Hatini et al.
(Hatini et al., 1994
).
Antibody fusion protein binding
The distributions of ephrin-A were visualized by receptor-antibody fusion
protein binding on unfixed frozen sections. Receptor antibody fusion proteins
consisting of the extracellular portions of the EphA receptors fused to the
human IgG1 Fc domain were used. Briefly, 15-µm-thick sections collected on
subbed slides, then blocked for 30 minutes in a solution containing 10% normal
goat serum, 2% BSA and 0.02% Na Azide. Slides were then incubated in 2
µg/ml of receptor fusion protein in 0.5xblocking solution for 1 hour.
The tissue was then fixed in fresh 4% PFA, washed in PBS, heat treated at
70°C for 1 hour to destroy endogenous phosphatase activity, and incubated
for 1 hour in goat anti-human IgG alkaline phosphatase-conjugated secondary
antibody (diluted 1:1000 in 0.5x blocking solution). Sections were color
developed using NBT/BCIP and mounted
(Marcus et al., 1996a;
Marcus et al., 2000
).
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Results |
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During the establishment of the optic chiasm in Foxd1+/+ mice,
pioneer axons arising from dorsocentral retina at E12.5 leave the optic cup
through the optic disc and navigate into the optic stalk to the developing
ventral diencephalon. There, they grow in close relationship to the inverted
V-shaped array of CD44/SSEA neurons, postulated to be important for the
establishment of the correct position of the X-shaped optic chiasm
(Marcus and Mason, 1995;
Mason and Sretavan, 1997
). A
small proportion of these early axons turn to the same side of the brain,
distant from the midline, forming an early uncrossed projection that is
thought to be transient (Guillery et al.,
1995
; Mason and Sretavan,
1997
) (Fig. 1A,
part a).
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By E14.5, many axons have reached the midline area in wild-type embryos, and the typical X-shape of the chiasm is apparent. In addition, the first permanent retinal axons project ipsilaterally from the VT retina (Fig. 1A, part c). In Foxd1lacZ/lacZ embryos, however, the X-shape of the chiasm is more elongated, and the ipsi- and contralateral components seem to be comparable in size (Fig. 1A, part d), in contrast to the predominantly contralateral pattern of the Foxd1+/+ optic chiasm (Fig. 1A, part c).
At E17.5, the altered crossed/uncrossed RGC axon ratio and the difference in the chiasm shape in Foxd1lacZ/lacZ embryos are more evident than at E14.5 (Fig. 1A, parts e,f). At P0, when the development of the optic chiasm is complete in Foxd1+/+ mice (Fig. 1B, part a), four distinctive abnormal optic chiasm phenotypes were observed in the Foxd1lacZ/lacZ embryos. (1) Most RGC axons terminate at the midline of the optic chiasm and end in one or more nodules (Fig. 1B, part b). (2) Some RGC axons traverse the midline and enter the optic tracts, but in an altered crossed/uncrossed ratio, and others terminate in nodules in the optic nerves, rostral to the chiasm and lateral to the midline (Fig. 1B, part c). (3) All RGC axons project ipsi- or contralaterally in an abnormal crossed/uncrossed ratio (Fig. 1B, part d). (4) Most RGC axons project ipsi- or contralaterally, but they split, creating two routes on each side rather than one (Fig. 1B, part e). In some cases, a combination of these phenotypes was observed. The penetrance of each of these phenotypes is shown in Table 1. The major phenotype observed in the Foxd1 deficient chiasm is an increase in the proportion of axons that project ipsilaterally relative to the contralateral component, observed in 85% of the cases. Thus, in the absence of Foxd1, the shape and position of the optic chiasm change, and the behavior of the retinal axons is affected at the midline, with many axons misprojecting or stalling at the midline.
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RGCs that project ipsilaterally arise from the entire retina, rather than only from VT retina
To determine the retinal origin of the increased proportion of RGC axons
that aberrantly project to the ipsilateral optic tract of
Foxd1lacZ/lacZ mice, we retrogradely labeled RGCs in E17.5
Foxd1lacZ/lacZ and Foxd1+/+ embryos with dextran,
applied to one optic tract. In the retina contralateral to the labeled optic
tract, dextran-labeled RGCs were found across the entire retina in
Foxd1+/+ embryos except in the peripheral VT crescent that
presumably contains the RGCs that project ipsilaterally from this eye
(Fig. 2B, part a). In the
Foxd1lacZ/lacZ embryos, labeled cells were likewise located across
the entire retina, but were also found in the most peripheral VT crescent
(Fig. 2B, part b, asterisk).
Moreover, overall there were far fewer labeled cells in the
Foxd1lacZ/lacZ retina compared with the Foxd1+/+ retina,
although this reduction was variable from embryo to embryo
(Table 2).
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Unfortunately, due to technical limitations of forward- and backfilling in the same embryo, is not possible to test whether there is a correspondence between the phenotypes observed by DiI-forward filling and the percentage reduction in the number of retrogradely labeled RGCs. However, we hypothesize that the overall reduction in the number of RGCs observed in mutant compared with in Foxd1+/+ retina reflects the failure of many RGC axons to project past the optic chiasm into the optic tract. Alternatively, the decrease in the RGCs that are backfilled from the optic tract could be a consequence of a reduction in the number of RGCs in the mutant retina. We favor the first possibility, because (1) the optic nerve is robustly labeled upon forward filling from the optic nerve head, and (2) many axons accumulate before and in the chiasm, reflected by axon tangles and knots, which is evidence of a failure of RGC axons to project further than the chiasm (Fig. 1B, parts b and c).
Thus, in the absence of Foxd1, fewer axons reach the optic tracts, probably because many axons are unable to pass through the optic chiasm. Those axons that do traverse the optic chiasm and that project ipsi- or contralaterally arise from the entire retina rather than from their respective topographic locations in the retina. These results indicate that Foxd1 is important for proper routing at the level of the optic chiasm.
Factors mediating the inhibition of uncrossed retinal axons are lost in the Foxd1 deficient retina
To address the question of whether the misrouting of retinal fibers at the
optic chiasm is a consequence of alterations in the retina, in the chiasm, or
in both, we co-cultured Foxd1+/+ and Foxd1lacZ/lacZ
retinal explants with cells dissociated from either Foxd1+/+ or
Foxd1lacZ/lacZ chiasm. Under normal conditions, axons that extend
from VT retinal explants are inhibited by dissociated chiasm cells, whereas
axons from the remainder of the retina grow well on chiasm cells
(Fig. 3A, part a)
(Herrera et al., 2003;
Marcus et al., 1995
;
Marcus and Mason, 1995
;
Marcus et al., 1996b
;
Wang et al., 1995
). Given the
stalling and misrouting seen in contra- and ipsilateral projections in
Foxd1lacZ/lacZ embryos in vivo (forward and backfill experiments),
we expected a general inhibition of all RGC axons, and/or that axons from
contralateral retina would act as ipsilateral axons and vice versa in this
co-culture assay. Our results indicate that in Foxd1lacZ/lacZ
explants, dorsotemporal (DT) axons behaved like Foxd1+/+ RGCs, e.g.
they were not inhibited by Foxd1+/+ or Foxd1lacZ/lacZ
chiasm cells. By contrast, RGCs from Foxd1 deficient VT retina grew well on
chiasm cells from Foxd1+/+ or Foxd1lacZ/lacZ embryos,
rather than being repulsed (Fig.
3A, parts b,d). Interestingly, Foxd1lacZ/lacZ chiasm
cells were able to inhibit Foxd1+/+ VT retinal axons, suggesting
that the chiasm signals that normally repel ipsilateral RGC axons are present
in Foxd1 nulls (Fig. 3A, part
c).
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Gene expression is altered in the Foxd1 deficient retina
Differentiation genes
Retrograde labeling experiments showed that fewer axons traverse the chiasm
in absence of Foxd1. To test the possibility that the total number of RGCs or
that RGC differentiation are affected in Foxd1 deficient embryos, we analyzed
the expression of two markers of differentiated RGCs, Islet1 and Brn3b in the
Foxd1 deficient retina. Islet1 is a LIM-homeodomain transcription factor
expressed in RGCs when they are in S phase of their terminal division
(Brown et al., 2000). Brn3b is
a POU transcription factor involved in the guidance of RGC axons as they exit
from the retina (Liu et al.,
2000
). Brn3b has also been implicated in other axon guidance
events along the retinofugal pathway (Wang
et al., 2002
; Xiang et al.,
1995
). Islet1 and Brn3b expression was unchanged in Foxd1
deficient retina, including in the VT crescent
(Fig. 4A,B, and data not
shown). These results indicate that in the absence of Foxd1, RGC number is
normal and RGC neurons are normally differentiated, in accordance with the
robust DiI staining observed in the optic nerve of Foxd1 deficient mice in
forward filling experiments.
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The tyrosine kinase receptor EphB1 is expressed in the same VT domain as
Zic2 and mediates the response of ipsilateral RGC axons to inhibitory signals
emanating from chiasm cells (Williams et
al., 2003). We have previously postulated that Zic2 might regulate
the tyrosine kinase receptor EphB1
(Williams et al., 2004
). In
Foxd1lacZ/lacZ mice, as with Zic2, EphB1 is not expressed in VT
retina (Fig. 4G,H), suggesting
a role for Foxd1 in the regulation of these two proteins that modulate the
ipsilateral pathway.
Islet2 is a LIM homeodomain transcription factor expressed in a pattern
complementary to Zic2 in the developing retina. Because Islet2 is expressed in
contra- but not in ipsilateral RGCs, it has been suggested that Islet2 could
be involved in the identity and navigation of crossed retinal axons
(Pak et al., 2004). To test
whether changes in Islet2 expression contribute to the misrouting of
contralateral RGCs from contra- to ipsilateral optic tracts in
Foxd1lacZ/lacZ embryos, we performed in situ hybridization with
specific probes to Islet2 in Foxd1lacZ/lacZ and Foxd1+/+
retinal sections. Islet2 expression is normal in the Foxd1 deficient retina.
It is important to note that despite the loss of Zic2 in the VT crescent of
Foxd1 deficient retina, Islet2 maintains its VT-negative pattern
(Fig. 4I,J), demonstrating that
there is a population of cells, now Zic2 negative, that are still present in
the VT crescent of the Foxd1 deficient retina. This result argues that there
is a specific downregulation of Zic2 and EphB1, rather than cell loss at this
region. Moreover, the normal Islet2 expression in the retina suggests that its
expression is not repressed by Zic2 or regulated by Foxd1.
Genes regulating early retinal patterning
Foxd1 was previously reported to be expressed in the temporal half of the
retina at E12.5 (Hatini et al.,
1994). We next investigated whether Foxd1 expression is maintained
at later times; specifically, at E14.5 and E16.5, the period in which Zic2 and
EphB1 are expressed in the VT crescent. To do this, we performed X-gal
staining in Foxd1+/lacZ embryos. Rather than being expressed in the
entire temporal half of the retina, Foxd1 is restricted to the VT quadrant at
E14.5 and E16.5 (Fig. 5A, part
a,e).
|
Because Foxd1 and Foxg1 show adjacent expression domains in the retina
(Hatini et al., 1994), a
pattern suggesting that these two winged helix transcription factors (Foxg1
and Foxd1) might repress each other, we next studied the Foxg1 expression
pattern in Foxd1lacZ/lacZ and Foxd1+/+ embryos. In the
Foxd1 deficient retina, the Foxg1 expression domain expands into the VT
retina, occupying the Foxd1 domain (compare
Fig. 5B, parts a-c with parts
d-f), but does not expand into the dorsotemporal retina, in agreement with
previous data suggesting that Foxd1 represses Foxg1 in retina
(Huh et al., 1999
).
Foxg1 induces ephrin-A2/A5 expression when is misexpressed in the temporal
retina (Takahashi et al.,
2003). Because Foxg1 expression expands into the domain of Foxd1
in the Foxd1 deficient retina, we investigated the pattern of ephrin-A in the
Foxd1 deficient retina. In Foxd1+/+ retina, ephrin-A was found in a
high-nasal low-temporal pattern, whereas in Foxd1lacZ/lacZ retina
ephrin-A was highly expressed in both halves of the retina
(Fig. 5B, parts g,h).
In summary, the absence of Foxd1 in retina leads to the loss of genes known to directly control the uncrossed pathway in the optic chiasm, specifically, Zic2 and EphB1, and to an expansion of Foxg1 and ephrin-A (Fig. 5C).
Expression of regulatory genes and axon guidance factors is altered in the optic chiasm of Foxd1 deficient embryos
Co-cultures of Foxd1+/+ and Foxd1lacZ/lacZ retina and
chiasm cells suggested that RGC axons in the VT retina of
Foxd1lacZ/lacZ embryos display `contralateral axon-like
phenotypes', as they are less repulsed by chiasmatic cells than RGCs in
Foxd1+/+ VT retina. This finding is supported by the loss of two
genes designating the uncrossed projection, Zic2 and EphB1, in the Foxd1
deficient VT retina. However, in vivo, early retinal axons show altered
pathfinding errors as early as E12.5 (Fig.
1A, part b), a finding that cannot be explained by Zic2 or EphB1
downregulation in E14.5 Foxd1 deficient retina, as the first RGCs that form
the optic chiasm derive from dorsocentral retina
(Guillery et al., 1995). One
possible explanation for the early misrouting is that contralateral RGCs are
also de-specified. Unfortunately, to date, only one molecule has been
suggested to be involved in contralateral RGC specification, Islet2
(Pak et al., 2004
), but this
transcription factor shows normal expression in Foxd1 mutant retina. No other
proteins have been directly implicated in the establishment of the
contralateral projection, making it difficult to test for perturbations in the
mechanisms for crossing.
It is also possible that in the absence of Foxd1, the ventral diencephalon is altered and causes axon misrouting. To investigate this possibility, we analyzed the expression pattern of transcription factors and axon guidance molecules implicated in retinal axon divergence or interactions with Foxd1 in the optic chiasm region.
Patterning genes in the ventral diencephalon
Combinatorial domains of regulatory gene expression in the ventral
diencephalon have been implicated in the patterning of retinal axon
projections in the optic chiasm (Marcus et
al., 1999), either by regulating the expression of molecules
involved in axon guidance or by specifying development of the specialized cell
groups that provide cues for guidance
(Marcus et al., 1999
;
Williams et al., 2003
).
The transcription factor Foxg1 is expressed in the pre-optic area, in a
pattern complementary to Foxd1, in a domain where the optic chiasm is formed
(Marcus et al., 1999). The
boundary between these two transcription factors has been proposed to play a
role in the establishment of the optic chiasm. We tested the expression
pattern of Foxg1 in Foxd1lacZ/lacZ embryos and consistently found
that the zone of Foxg1 expression is expanded compared with the pattern in
Foxd1+/+ embryos, invading the supraoptic area normally occupied by
Foxd1. The expansion can be viewed in sagittal
(Fig. 6A, parts a-d) and
frontal sections of Foxd1lacZ/lacZ embryos
(Fig. 6B, parts a,b).
|
|
Slit2, a member of the secreted Slit chemorepellent family that is
necessary for channeling RGC axons during chiasm formation
(Plump et al., 2002), was next
examined in the Foxd1 deficient chiasm. In Foxd1+/+ embryos, Slit2
is strongly expressed immediately dorsal to the optic chiasm and is also
weakly detected ventroposterior to the chiasm, in a patch on either side of
the ventral diencephalon (Fig.
7D, cartoon) (Erskine et al.,
2000
; Plump et al.,
2002
). At E13.5, Slit2 expression appears to be normal in the most
dorsal area of the Foxd1 deficient chiasm. However, its ventroposterior
expression is stronger than in the Foxd1+/+ chiasm
(Fig. 7B, parts a-d). At E15.5,
although less intense, this altered pattern is maintained
(Fig. 7B e-h).
As CD44/SSEA-positive neurons are believed to define the midline and the
posterior border of the future chiasm
(Marcus et al., 1995;
Mason and Sretavan, 1997
;
Sretavan et al., 1994
), we
also investigated the position of this population of early neurons in the
Foxd1 deficient chiasm. CD44/SSEA neurons are located ventral to the optic
stalks and dorsal to the postoptic recess
(Mason and Sretavan, 1997
). At
E13.5 (Fig. 7C, parts a,b) and
at E15.5 (Fig. 7C, parts c,d),
the organization of these early neurons appears to be normal in Foxd1
deficient embryos. However, at E15.5, RGCs axons enter the CD44/SSEA domain
rather than avoid it, as occurs normally in Foxd1+/+ chiasms
(Fig. 7C, parts c,d).
Thus, Foxd1 deficient embryos exhibit aberrant expression of Foxg1, Zic2 and Islet1 transcription factors in the ventral diencephalon, providing evidence for mis-regionalization of the ventral diencephalon. Ephrin-B2, which is repulsive ipsilateral axons, appears to be normal at the Foxd1 deficient chiasm, but Slit2, which mediates inhibition of all RGC axons, is more strongly expressed in the ventral diencephalon in the absence of Foxd1.
To recapitulate, as a consequence of the absence of Foxd1, Zic2 and EphB1 are lost in the VT retina, and Foxg1 ectopically expands into this retinal sector. Accordingly, VT RGCs are less inhibited by chiasm cells in vitro, and the number of ipsilaterally-projecting RGCs from the VT crescent is drastically reduced in the Foxd1 deficient embryos. Moreover, the number of ipsilaterally projecting RGCs from the retinal quadrants outside of the VT crescent increases, and only 10-15% of the normal number of RGCs project past the chiasm into the optic tracts in the Foxd1 deficient embryo, when compared with the wild-type embryo. These data suggest that in Foxd1 deficient embryos, those RGC axons that are able to project into the ipsilateral or contralateral optic tracts do so in a stochastic manner. Our analyses also demonstrate perturbations in the regionalization of the ventral diencephalon, including expansion of Foxg1 and Slit2, and reduction in Zic2 and Islet1. In summary, regionalization and specification of both the retina and ventral diencephalon are disrupted in Foxd1 deficient embryos, and although the relative contribution of alterations of each site is not identified by the present study, these disruptions produce a misallocation of ipsi- to contralateral axons, malformation of the optic chiasm, and reduced retinal projection into the optic tracts.
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Discussion |
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Foxd1 as a pre-patterning gene of ventrotemporal retinal identity
It has been previously reported that Foxd1 is expressed in the temporal
half of the retina at the time that optic vesicles evaginate
(Hatini et al., 1994). At
E14.5, Foxd1 is confined to the VT quadrant rather than the temporal half of
the retina, suggesting a role for Foxd1 in the specification of the
ventrotemporal region of the retina and, in turn, ipsilateral RGC identity.
The expression domain of Foxd1 includes the peripheral VT retina where Zic2 is
expressed. As Zic2 and EphB1, both essential for the specification and
guidance of ipsilateral RGCs, show similar expression in VT RGCs
(Herrera et al., 2003
;
Williams et al., 2003
), we
presumed that the Foxd1 expression domain also includes the EphB1-positive
area. Strikingly, Zic2 and EphB1 are missing in the VT quadrant of Foxd1
deficient mice, indicating that Foxd1 is required for the expression of these
two proteins in this area of the retina. Our data strongly suggests, for the
first time, that Zic2 and EphB1 are linked. Moreover, in accordance with the
loss of Zic2 and EphB1 in this region of the retina, retrograde labeling shows
that there are fewer cells projecting ipsilaterally from the VT retina in
Foxd1 nulls compared with Foxd1+/+ mice. Instead, VT RGCs project
contralaterally, if they do not stall in the chiasm in Foxd1 deficient
mice.
Mix-and-match co-culture assays also support a de-specification of the
ipsilateral phenotype, as in the absence of Foxd1, axons from VT retina are no
longer inhibited by chiasm cells. Therefore, based on expression patterns and
loss-of-function studies, we propose that Foxd1 expression in the retina is
essential for regulation of the molecular cascade directing the ipsilateral
retinal projection, specifically in directing VT RGCs to recognize inhibitory
signals such as ephrin-B2 at the chiasm midline
(Williams et al., 2003). At
present, whether Foxd1 directly regulates Zic2 is not known. Foxd1 might
regulate Zic2 and EphB1 through a non-cell autonomous mechanism or,
alternatively, Foxd1-expressing cells may represent progenitors that will
later express Zic2.
Previous results indicate that Foxg1 regulates ephrin-A2/A5, because
misexpression of Foxg1 in nasal retina induces ephrin-A2/A5 regulation
(Takahashi et al., 2003). In
the case of Zic2 and EphB1, whether the loss of these molecules is a
consequence of the expansion in Foxg1 or the absence of Foxd1 is not known,
but further experiments will address this issue.
Foxd1 function is essential for correct regionalization of the optic chiasm
We observed that at E12.5, pioneer axons of Foxd1 deficient embryos display
an abnormal course once they exit the optic stalk, growing straight to the
optic chiasm midline instead of turning rostrally and ventrally. The loss of
proteins that direct the ipsilateral projection in older VT retina (e.g. Zic2
and EphB1) does not explain the misrouting of the early pioneer axons or the
subsequent increase in RGCs in Foxd1 deficient embryos that aberrantly project
ipsilaterally or are arrested in their growth at the midline. One hypothesis
is that early RGCs and/or contralateral RGCs are mis-specified. Islet2 is
expressed in contra- but not ipsilateral RGCs in both the Foxd1 deficient and
wild-type retina, but it is possible that other molecular mechanisms
underlying RGC axon crossing may be defective.
Another, but not mutually exclusive, hypothesis underlying the chiasm
phenotype in the Foxd1 deficient embryos is that the morphogenesis and
specification of the ventral diencephalon is perturbed. Supporting this view,
the boundary between Foxg1 and Foxd1 is disrupted in the ventral diencephalon
of Foxd1 deficient embryos. This boundary was previously proposed to define
naso-temporal identity (Hatini et al.,
1994) and to mark where the optic chiasm forms
(Marcus et al., 1999
).
Associated with this disruption is the fact that Zic2- and Islet1-expressing
cells are decreased in number and misplaced. Moreover, Slit2 expression
expands at an earlier time than in Foxd1 wild-ype embryos, consistent with the
prevalent stalling and misrouting of RGC axons at the midline. However, the
continued presence of ephrin-B2, important for retinal axon divergence, is in
agreement with the in vitro results indicating that chiasm cells from
Foxd1lacZ/lacZ mice are still able to inhibit Foxd1+/+
VT axons.
The absence of Foxd1 in the ventral diencephalon may affect the formation
of the optic chiasm in two ways. (1) Foxg1 controls the number and type of
cells produced in the cortex (Xuan et al.,
1995; Hanashima et al.,
2002
; Hanashima et al.,
2004
). Thus, it is possible that the Foxg1 expansion in the
ventral diencephalon that results from the loss of Foxd1 affects the number
and/or fate of specific cell types in the midline, thereby indirectly
affecting the organization of the expression pattern of guidance factors they
express, such as Slit2. (2) Foxd1 might directly regulate the expression of
transcription factors and guidance molecules important for RGC growth across
the Foxd1-expressing zone.
Specificity of Foxd1 function in optic chiasm formation
We observed expansion of the Foxg1 expression zone into the Foxd1 domain in
retina, and an induction of ephrin-A in nasal retina. These data are in
agreement with previous reports showing that Foxg1 missexpression in temporal
retina in chick induces ephrin-A5, probably leading to the missprojection of
retinal axons in the tectum (Takahashi et
al., 2003). To study whether the genetic manipulation of Foxd1
affects the retinocollicular projection in mice, and to clarify whether Foxd1
plays a role in the specification of contralateral RGCs, conditional or
tissue-specific mice will be needed. However, in addition to new information
about other genes that act downstream of Foxd1 (i.e. Zic2, EphB1), the total
removal of Foxd1 has revealed that Foxd1 is essential for the correct
formation of the optic chiasm at the midline, but is not required for the
guidance of retinal axons to exit the retina, navigation through the optic
nerves or tracts, or projection to the lateral geniculate nucleus.
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ACKNOWLEDGMENTS |
---|
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Footnotes |
---|
Present address: Department of Pediatrics, Hackensack University Medical
Center, 30 Prospect Avenue, Hackensack, NJ 07601, USA
Present address: Clinical Pharmacology, Merck Research Laboratories,
RY34-A-428, 126 East Lincoln Avenue, Rahway, NJ 07065-0900, USA
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