Department of Medicine and Cell Biology, University of Massachusetts Medical School, 364 Plantation Street, Worcester, MA 01605, USA
* Author for correspondence (e-mail: zheng.bao{at}umassmed.edu)
Accepted 15 December 2003
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SUMMARY |
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Key words: Zic3, Gradient expression, Intra-retinal axon projection, Chick
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Introduction |
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A number of molecules have been implicated to be involved in centrally
directed projection of the retinal axons toward the optic disc. Chondroitin
sulfate proteoglycans (CSPGs), a major component of the ECM, are suggested to
act as inhibitory molecules to prevent the growth of retinal axons toward the
periphery of the rat retina (Brittis et
al., 1992; Snow et al.,
1991
). The CSPGs recognized by a monoclonal antibody CS-56 are
expressed in a gradient in the rat retina, low in the center and high in the
periphery (Brittis et al.,
1992
). However, the distribution of CSPGs in the retina of chick,
quail and cat embryos has been shown to colocalize with the growing optic
axons, suggesting a different role in retinal axon guidance
(McAdams and McLoon, 1995
;
Ring et al., 1995
).
Axonal growth toward the optic fissure also requires expression of cell
adhesion molecules (CAM) of the immunoglobulin superfamily like L1, neural
cell adhesion molecule (NCAM) and neurolin (DM-GRASP). Intravitreal injection
of the antibodies to NCAM disrupted the orderly formation of the fissure and
resulted in massive overshooting of axons which failed to exit at the optic
disc (Thanos and Bonhoeffer,
1984). Injection of Fab fragments to L1 resulted in
defasciculation of axons (Giordano et al.,
1997
), whereas injection of Fab fragments to neurolin caused both
defasciculation and aberrant intraretinal axonal trajectory
(Ott et al., 1998
). In
addition, a number of receptor tyrosine phosphatases have been shown to play a
role in intraretinal axon guidance (Ledig
et al., 1999
).
We report that a gene encoding a zinc-finger transcription factor,
Zic3, is involved in patterning the retina for intraretinal axon
guidance. Zic genes are the vertebrate homologues of the Drosophila
pair-rule gene, odd-paired (opa) and are key regulators of
neural and neural crest development (Aruga
et al., 1998; Brewster et al.,
1998
; Nakata et al.,
1997
). Recently, Zic2 has been shown to pattern binocular
vision that it is both necessary and sufficient to regulate RGC axon repulsion
by cues at the optic chiasm (Herrera et
al., 2003
). Mutations in Zic genes are known to cause
holoprosencephaly and situs abnormalities in human
(Brown et al., 1998
;
Gebbia et al., 1997
).
By in situ hybridization, we have shown that Zic3 is expressed in a periphery-high, center-low gradient in the developing retina, and the gradient expression of Zic3 recedes toward the periphery of the retina correlating with the progression of retinal cell differentiation and axonogenesis. Disruption of gradient expression of Zic3 by retroviral overexpression resulted in mis-targeting of the retinal axons and some axons misrouted to the subretinal space at the photoreceptor side of the retina. Misexpression of Zic3 did not affect neurogenesis or differentiation inside the retina, or grossly alter retinal lamination. By stripe assay, we have further shown that Zic3 may play a role in intra-retinal axon guidance by regulation of the expression of an inhibitory factor(s) to drive the axons to project to the optic disc.
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Materials and methods |
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Immunofluorescent staining on flat-mounts or sections of retina was
essentially as described previously (Jin
et al., 2003). Axons were stained with monoclonal antibody 270.7,
a generous gift from Dr Virginia Lee (University of Pennsylvania Medical
School). Viral infection was confirmed by immunofluorescent staining with
either a monoclonal antibody 3C2 (diluted 1:5) or a polyclonal antibody p27
(SPAFAS. Norwich CT, diluted 1:10,000). Mouse monoclonal antibodies against
Islet-1 (39.4D5), collagen IX (2C2) and visinin were obtained from the
Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, IA), and
used at 1:50, 1:5 and 1:10, respectively. The monoclonal antibody CS-56 was
obtained from Sigma (St Louis, MO) and used at 1:200 dilutions.
Retrovirus and chick embryo injection
A 1.5 kb fragment of cDNA containing the entire coding region of the human
ZIC3 gene (Gebbia et al.,
1997) was cloned into the ClaI site of an avian
replication-competent retroviral vector (RCAS)
(Hughes et al., 1987
;
Morgan and Fekete, 1996
).
RCAS-Zic3 virus was prepared by transfection of a chicken fibroblast line,
DF1, as previously described (Bao et al.,
1999
). All viral stocks were harvested and concentrated to titers
of approximately 5x108 cfu/ml.
Standard specific pathogen-free white Leghorn chick embryos from closed
flocks were provided fertilized by Charles River Laboratories (North Franklin,
Connecticut). Eggs were incubated inside a moisturized 38°C incubator and
staged according to Hamburger and Hamilton
(Hamburger and Hamilton,
1992).
TUNEL assay
Cryosections (20 µm) were prepared from the E7 or E10 retinas injected
with either the control RCAS-GFP or RCAS-Zic3 virus. The DeadEnd Colorimetric
TUNEL system from Promega was used to detect programmed cell death. The assay
was carried out according to the manufacturer's instruction except that the
treatment of proteinase K was changed to 3 µg/ml of concentration for 10
minutes at room temperature to preserve tissue integrity. After TUNEL assay
was completed, the tissue sections were further co-stained with
anti-neurofilament antibody 270.7 and anti-P27 antibodies, to visualize the
axons and the viral antigens, respectively.
Stripe assay
The stripe assay was carried out essentially as described previously
(Walter et al., 1987). For
some experiments, uninjected E8 chick eyecups were dissected out and cut along
at one half radial distance to the center, in order to obtain the central and
peripheral halves of the retina. For other experiments, optic vesicles were
injected with either the RCAS-Zic3 or a control RCAS-GFP virus at HH stage 10
and incubated until E8. Only the central halves of the virus-injected retinas
were collected and homogenized by pressing through 1 ml syringe needles
multiple times in a buffer containing 10 mM Tris-HCl, pH 7.4, 1.5 mM
CaCl2, 1 mM spermidine and protease inhibitor cocktail (Roche).
Nuclei and cytoplasm were removed by centrifugation in step sucrose gradient
for 10 minutes at 50,000 g. The membrane fragments were
collected and washed in PBS and vacuumed onto a polycarbonate filter
(Waterman, pore size 0.1 µm) following the manufacturer's instruction
(Max-Planck-Institute at Tubingen). One type of membrane fragments were
vacuumed on first to form the first stripes and the second type of membrane
fragments mixed with green fluorescent beads (505/515) (Molecular Probes) were
filled in to form the second stripes. After the stripes were formed, the
membranes were coated with 50 µg/ml of laminin (BD Biosciences) for 1-4
hours at room temperature.
E6 chick retinas were dissected and flat-mounted with the ganglion cell side up onto a nitrocellulose filter (Sartoris). Thin stripes of retina were cut and laid onto the polycarbonate filter with the ganglion cell side facing the polycarbonate filter. Retinal explants were cultured in DME/F-12 medium (1:1) containing 10% fetal calf serum, 2% chicken serum and 0.4% methyl cellulose. 40-44 hours later, the explants with the membranes were fixed in 4% paraformaldehyde and stained with an anti-neurofilament antibody (270.7) followed by a Cy3-conjugated donkey anti-mouse antibody (Jackson ImmunoResearch Laboratories).
The fluorescent images of axons and stripes were photographed with a digital camera (SPOT camera). Green images of stripes and red images of anti-neurofilament staining were merged using the SPOT Advanced software. To quantify the results, axons were traced and scanned to obtain digital images. We quantified only those patches that had axons covering eight or more stripes. The number of black pixels of the tracings on the stripes with the fluorescent beads (second stripes) and those on the stripes with no beads (first stripes) was calculated by a public domain NIH Image program. The `preference' of axonal growth was calculated based on the ratio (s/f) of the number of black pixels on the second stripes to that on the first stripes and normalized to the width of the stripes. Control stripe assay experiments were performed to assess the variation of the experimental data by laying down same membrane preps from the central halves of the uninjected E8 retinas in both stripes. Axonal growth preference between two membrane fragments (ratio s/f) was compared with the control stripe assay data (ratio s/f) and statistical significance was analyzed by using the unpaired Student's t-test.
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Results |
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The graded expression pattern of the Zic3 gene was confirmed by in
situ hybridization on the cross sections of the embryonic chicken eye
(Fig. 3). In addition, we found
that Zic3 is highly expressed in the ciliary margin region
(Fig. 3C,E,F), similar as
reported in mouse (Nagai et al.,
1997). Within the retina, Zic3 appears to be expressed in
the undifferentiated progenitor cell population. The graded expression of
Zic3 is consistently periphery-high and center-low in the
undifferentiated cell population at all developmental stages analyzed. At E16,
Zic3 expression is no longer detected inside the retina and is only
retained in the ciliary margin (Fig.
3E,F).
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|
First observed at E6, the number of areas with axonal abnormality increased over time but peaked around E10 (Table 1). In two cases, we observed that a small group of axons turned back 180° to project toward the periphery of the retina (Fig. 4D). In another case, the axons appeared to go around in a circle (Fig. 4C). More commonly (>150 areas), however, the axons had the appearance of being blocked from projecting toward the center of the retina and piled up into a line (Fig. 4E,F). By adjusting the focal plane of the microscope, we found that some axons penetrated the entire thickness of the retina and misrouted to the sub-retinal space (Fig. 5A-F). There, the axons initially followed the same aberrant trajectories as in the ganglion cell side (Fig. 5A,B). Farther away from the site where the axons crossed over, the axons appeared much more disorganized (arrowhead, Fig. 5D). The misrouted axons did not project to the optic disc, indicating that the molecular cues for directing the axons to the center of the retina are not present at the photoreceptor side.
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Misexpression of Zic3 did not grossly affect retinal lamination
To examine whether Zic3 misexpression affects retinal lamination, E10
retinas infected with the RCAS-Zic3 virus were sectioned and stained with
various cell type-specific antibodies. Similar to what is observed in
flat-mount retinas, the axons crossed over to the sub-retinal space at the
photoreceptor side (Fig. 7A-C). However, the optic fiber layer appeared largely normal, although some
protrusions of the axons were observed (arrows,
Fig. 7A,B). By contrast, the
photoreceptor layer often appeared broken, possibly by misrouted axons
(Fig. 7D,G). After examining
all sections from two infected retinas containing 15 areas of abnormal axonal
crossings, we did not observe any gross defect in lamination. However, some
disturbances of retinal layers were observed, at the immediate vicinity of
axon misrouting. Cells in GCL and inner nuclear layer (INL) were seen to
disperse into the deeper layers of the retina toward the photoreceptor side
(Fig. 7H,I). Within the sites
of axon crossing over, the anti-viral GAG antibody staining appeared much
brighter than the adjacent infected areas
(Fig. 7F). Consistent with the
results obtained on flat-mount retina, we also did not observe any significant
change in the number of cells expressing islet 1, Pax6 or visinin in retinal
sections. These results support the idea that Zic3 misexpression appears to
affect intra-retinal axon targeting specifically without grossly altering cell
differentiation and lamination in the retina.
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As shown in Fig. 9B, axons appeared to `prefer' the stripes prepared from the control RCAS-GFP-injected retinas to the stripes prepared from the RCAS-Zic3-injected samples. To quantify the results, the distribution of the axons on the stripes was digitalized and calculated by a NIH Image software and the preference of the axons were accessed by the ratio of axonal growth on the second stripes (RCAS-GFP-injected) to that on the first stripes (RCAS-Zic3-injected) (s/f) normalized to the width of the stripes (see Materials and methods). A total of 15 sample areas (from five independent experiments) were quantified and a mean ratio (s/f) was 5.74.
|
To determine whether an endogenous inhibitory factor is present at higher concentration in the periphery than in the retinal center, we performed stripe assays by laying down the membrane fragments prepared from the peripheral E8 retina as the first stripes and those from the central E8 retina as the second stripes. As shown in Fig. 9C, the axons appeared to grow preferentially on the stripes prepared from the central retina. The results were similarly quantified (mean ratio s/f=5.13, n=15, from five independent experiments). Statistical analysis by the Student's unpaired t-test indicates that the stripes prepared from the central membrane fragments were significantly more preferable to the retinal axons than those from the peripheral membrane fragments (P<0.001). These results suggest that an endogenous negative factor(s) is present at a higher concentration in the peripheral than in the central retina, and Zic3 misexpression may activate the expression of an inhibitory factor to the retinal axons.
Misexpression of Zic3 may activate the expression of a currently unknown guidance factor
We next tested a few candidate molecules that might be downstream of Zic3.
In chicken, Netrin1 is also expressed in the optic fissure and optic
disc. No change in Netrin1 expression was observed in the retinas
infected with the RCAS-Zic3 virus compared with the control-uninjected retinas
(Fig. 10A,B). Similar to
previous reports (McAdams and McLoon,
1995; Ring et al.,
1995
), we also observed that the staining of an anti-chondroitin
sulfate proteoglycans (CSPGs) antibody (CS-56) persists in the center of the
retina in chicken, albeit at a much lower level than in the periphery. This
suggests that CSPGs may colocalize with the growing optic axons and play a
different role in retinal axon guidance. Furthermore, we did not detect any
obvious changes in CS-56 staining, in the areas infected with the RCAS-Zic3
virus compared with that in the uninfected wild-type areas, suggesting that
the expression of CSPGs is not subjected to the regulation by Zic3
(Fig. 10C-F).
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Discussion |
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Because cell differentiation and axonogenesis initiates from the cells at the center of the retina followed by cells at the more peripheral positions in a wave-like fashion, many genes involved in cell growth and differentiation exhibit dynamic expression patterns from center to periphery. However, the expression of these genes usually begins at the central retina, where the retinal cells are actively differentiating at the early stages such as E4.5. The expression of these genes gradually moves toward retinal periphery correlating with the wave of retinal differentiation. By contrast, Zic3 gene is consistently expressed at higher levels at the periphery, even at E4.5. Zic3 did not appear to affect cell differentiation grossly because misexpression did not alter the expression of islet 1, Pax6 and visinin. Furthermore, Zic3 misexpression did not alter the expression of many axon guidance molecules expressed in the retinal cells, including Slit1, Slit2, Robo1, Ephb5 and CSPGs, suggesting that Zic3 misexpression did not change the properties of the retinal cells substantially. The axonal phenotype caused by Zic3 may, therefore, be due to more specific changes in expression of molecules involved in axon guidance.
Members of the Eph family of receptor protein-tyrosine kinases, together
with their ephrin ligands have been shown as graded molecular tags in
establishment of retinotectal map
(Flanagan and Vanderhaeghen,
1998; O'Leary and Wilkinson,
1999
). A number of studies have also shown that the graded
expression of Eph family molecules and ephrins is set up by graded expression
of various transcription factors. Expressed in opposing gradient to
Epha3, two homeobox genes, Soho1 and Gh6 are shown
to specifically repress Epha3 expression in the retina
(Schulte and Cepko, 2000
). At
the D/V axis, a homeobox gene Vax/Vax2 expressed in a gradient in the
ventral retina has been shown to specify the D/V axis of the retina to
influence retinotectal mapping (Mui et
al., 2002
; Schulte and Cepko,
2000
). In mesencephalon, a gradient of engrailed gene expression
defines the rostral/caudal axis of the tectum and set up the gradient
expression of the axon guidance cues, Elf1 and Rags
(Friedman and O'Leary, 1996
;
Itasaki and Nakamura, 1996
;
Logan et al., 1996
;
Retaux et al., 1996
). However,
the role of graded expression of axon guidance cues established by
transcriptional regulation in direction of relatively short-range axonal
projection, has not been fully characterized.
Although the identity of the downstream target gene of Zic3 is currently
unknown, the results of stripe assay suggest that misexpression of Zic3
upregulates the expression of a negative axon guidance cue. This is consistent
with the axonal phenotype we observed by in vivo retroviral misexpression, and
with the fact that Zic3 is normally expressed in a periphery-high,
center-low gradient. A receding periphery-to-center gradient of repellent
under the control of Zic3 may be involved in directing the retinal axon
projection toward the optic disc. Previous studies have shown that the optic
disc is not necessary for centrally directed optic axon growth
(Halfter, 1996;
Harris, 1989
). Removal of the
host optic disc or grafting of an additional optic disc in a host retina in
intraretinal grafting experiment had no obvious effect on the global
navigation of retinal axons. Netrin 1, a known axon guidance molecule that is
expressed in optic disc, has also been shown not directly involved in
long-range guidance of the retinal axons to the optic disc
(Deiner et al., 1997
). These
findings suggest that local environment may play a prominent role in
intraretinal guidance instead of long-range attraction from the optic disc.
Graded expression of attractants or repellents inside the retina is thus
likely to be part of the mechanism that underlies the centrally directed
retinal axon projection. Although our result is consistent with the previous
findings that staining of CS-56 antibody persists in the center of the retina
in chicken (McAdams and McLoon,
1995
; Ring et al.,
1995
), we cannot rule out that a particular constituent of the
CSPGs may still be involved in intraretinal axon targeting or subjected to the
regulation by Zic3. The identities of the core proteins of the CSPGs have
remained undefined.
At the sites of axon crossing-over to the photoreceptor side in the
RCAS-Zic3-infected retinas, we have shown that there were disturbances in cell
distribution and increased cell death. It is possible that the misrouting of
axons is due to physical anomalies generated by increased cell death. However,
it is also possible that the abnormal trajectories of the axons disrupted
normal organization of the retina, which has resulted in increased cell death.
Although we currently do not have definitive evidence to distinguish these
models, several lines of evidence appear to support the latter. First, the
results of stripe assay demonstrate that a negative guidance cue is induced by
misexpression of Zic3. The induction of an inhibitory molecule in vivo can
inhibit the normal centrally directed axon projection and cause misrouting of
the axons. Results of stripe assays comparing the membrane fragments prepared
from the central retina with the peripheral retina further suggest that an
endogenous negative guidance factor is present in higher concentration in the
periphery than in the center. Second, the increase in cell death did not
coincide with viral infection, hence Zic3 misexpression. Despite the fact that
the retinas were infected in over 90% of the total area, the areas with
increased cell death were restricted to the immediate vicinity of the sites
where axons crossed over. If increased cell death were resulted directly from
Zic3 misexpression, a much wider spread of cell death would be expected.
Third, similar phenotypes of axon misrouting to sub-retinal spaces have been
reported for netrin 1 and sonic hedgehog mutant mice
(Dakubo et al., 2003;
Deiner et al., 1997
). Because
both netrin 1 and sonic hedgehog proteins are known to act as axon guidance
cues (Charron et al., 2003
;
Deiner et al., 1997
), the
misrouting of axons to sub-retinal spaces are likely resulted directly from
mis-targeting of the axons. Further study is needed to determine whether there
is a common molecular basis for these similar phenotypes.
It is also intriguing that the phenotype with the axons turning around
constituted only a minor fraction of the total number of the phenotypes. One
possibility is that increased anti-GAG staining intensity was used in most of
the experiments to identify the sites with axonal phenotypes. A small fraction
of the sites with axons turning but no crossing-over might remain undetected.
Second, it is also possible that additional factors are present to keep the
axons in the peripheral-central pathways. It has been shown previously by
intraretinal grafting experiments that the axons are unlikely to grow in the
directions other than the peripheral-central pathways
(Halfter, 1996).
We have previously shown that Slit1 is involved in definition of retinal
axon trajectories by providing intermediate targets to the retinal axons. In
the low Slit1 expression area resulted from overexpression of Irx4,
the axons appeared to turn to avoid the low Slit1 area and were excessively
fasciculated (Jin et al.,
2003). However, the axons still managed to project to the optic
disc, suggesting that Slit1 does not provide overall direction for the retinal
axons to project to the optic disc. In this paper, we have further shown that
Slit1 expression is not under the control of Zic3 regulation,
suggesting that Slit1 and Zic3 are in two separate pathways.
Interestingly, it has recently been reported that Zic2 patterns binocular
vision by specifying the uncrossed retinal projection
(Herrera et al., 2003). It is
common that transcription factors within one gene family play distinct roles
in biological processes by regulating different downstream target genes. In
human, mutations in Zic2 gene have been shown to cause
holoprosencephaly, whereas mutations in Zic3 have been associated
with X-linked situs abnormalities (Brown et
al., 1998
; Gebbia et al.,
1997
). Moreover, Zic2 and Zic3 are expressed in
overlapping but distinct patterns during embryonic development
(Nagai et al., 1997
). In
contrast to Zic2, which is expressed specifically in postmitotic
ganglion cells in ventrotemporal mouse retina but not in chick
(Herrera et al., 2003
), Zic3
is found only in undifferentiated progenitor cells in chick retina. Future
experiments aimed at identification of downstream targets of the Zic family
transcription factors may thus provide further insights in the molecular
mechanisms of the retinal axon guidance.
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ACKNOWLEDGMENTS |
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