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First published online 8 February 2006
doi: 10.1242/dev.02272
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Neuroscience Training Program, and Departments of Zoology and Anatomy, University of Wisconsin, Madison, WI 53706, USA.
* Author for correspondence (e-mail: mchalloran{at}wisc.edu)
Accepted 4 January 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Semaphorin, Axon guidance, Retinal ganglion cell, Optic chiasm, Zebrafish
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Initial studies of RGC axon guidance at the chiasm identified the midline
of the ventral diencephalon as a source of extrinsic guidance cues. Live
imaging of mouse RGC growth cones showed behavioral differences between
ipsilaterally and contralaterally projecting growth cones as they contacted
midline tissue (Godement et al.,
1994
; Sretavan and Reichardt,
1993). In vitro, explants of midline tissue inhibited outgrowth
from retinal cultures that normally gave rise to uncrossed axons, but did not
affect explants that normally gave rise to crossed axons
(Wang et al., 1995),
suggesting that the midline tissue is selectively inhibitory to ipsilaterally
projecting RGC axons. The chiasm region contains a population of cells that
express the surface molecule CD44, which can affect the relative size of the
crossed and uncrossed projections in mouse
(Lin and Chan, 2003).
Disruption of chondroitin sulfate proteoglycans, another family of
cell-surface molecules expressed at the chiasm, reduced the uncrossed
projection in mouse (Chung et al.,
2000). EphrinB2 is also expressed at the midline and acts as a
repulsive cue for axons that project ipsilaterally
(Nakagawa et al., 2000).
Together, these studies show that the midline tissue is a source of cues that
help to determine the laterality of RGC projections. Interestingly, RGC axons
in a zebrafish mutant lacking the Slit receptor Robo2 make ipsilateral errors
in addition to other guidance errors
(Hutson and Chien, 2002).
However, this study and others in mice suggest that the main role of Slit/Robo
signaling is to channel axons to the correct location of the optic chiasm
rather than to provide laterality information
(Erskine et al., 2000;
Plump et al., 2002;
Ringstedt et al., 2000).
Recent work indicates that the laterality decision is also regulated by
intrinsic factors in RGCs. In mouse, the zinc-finger transcription factor Zic2
is expressed exclusively by ipsilaterally projecting RGCs, and is both
necessary and sufficient for RGC axon repulsion by midline chiasm explants
(Herrera et al., 2003).
Zic2-positive RGCs also express the receptor tyrosine kinase EphB1, which
appears to mediate repulsion from ephrinB2 at the midline to cause these axons
to project ipsilaterally (Williams et al.,
2003). Interestingly, Xenopus tadpoles lack retinal EphB1
and midline ephrinB2 expression, and have an entirely crossed retinal
projection. During Xenopus metamorphosis, upregulation of ephrinB at
the midline and EphB in a restricted part of the retina coincides with the
development of an ipsilateral component of the visual pathway
(Nakagawa et al., 2000).
Together, these studies suggest that RGC axons project contralaterally unless
otherwise specified to respond to repulsive cues at the midline. However, it
is not yet clear what drives the large majority of RGC axons to cross the
midline. Two winged helix transcription factors, Foxd1 and Foxg1, are
expressed in complementary patterns in both retina and ventral diencephalon,
and appear to be involved in development of the contralateral projection as
mice deficient in either displayed increased ipsilateral projections
(Herrera et al., 2004;
Pratt et al., 2004). Another
recent study showed that expression of the LIM-homeodomain transcription
factor islet 2 is restricted to contralaterally projecting RGCs in mouse
(Pak et al., 2004). However,
no guidance cues at the midline have yet been shown to promote the crossing of
RGC axons.
Here, we provide evidence that a secreted semaphorin, Sema3d, expressed at the chiasm guides the midline crossing of RGC axons in zebrafish. Zebrafish are an excellent system in which to study the development of the contralateral RGC projection, as all RGC axons cross the midline. Disruption of normal Sema3d through either global overexpression or knockdown induced aberrant ipsilateral projections without affecting other pathfinding decisions. Sema3d overexpression slowed or prevented RGC axon growth through the midline region, whereas Sema3d knockdown reduced the ability of RGC axons to leave the midline by increasing growth cone retraction in the optic tract. We propose that midline Sema3d is important for RGC axon extension from the chiasm midline into the contralateral optic tract.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Embryo age is defined as hours post-fertilization
(hpf) or days post-fertilization (dpf). For live imaging experiments, embryos
were kept in 0.003% 1-phenyl-2-thiourea after 22 hpf to block pigment
formation. For heat induction, embryos were transferred to a 39°C water
bath for one hour.
In situ hybridization and immunohistochemistry
A digoxigenin-UTP labeled riboprobe for sema3d was synthesized by
in vitro transcription and hydrolyzed to an average length of 200-500 bases by
limited alkaline hydrolysis (Cox et al.,
1984). Whole-mount in situ hybridization was performed as
described previously (Halloran et al.,
1999). For double labeling, anti-acetylated tubulin (Sigma, St
Louis, MO) at a dilution of 1:1000 was added to an overnight incubation in
AP-tagged anti-digoxigenin antibody (Roche, Indianapolis, IN). Following the
AP reaction, antibody labeling was completed with the Vectastain Mouse IgG ABC
immunoperoxidase labeling kit (Vector Laboratories, Burlingame, CA).
For whole-mount immunohistochemistry, embryos were fixed in 4% paraformaldehyde for one hour at room temperature, treated with 0.1% collagenase (Sigma, St Louis, MO) in PBS, blocked in 5% sheep serum and 2 mg/ml BSA in PBS, and incubated in Zn-5 (Zebrafish International Resource Center, Eugene, OR) at a dilution of 1:500. Antibody labeling was completed with the Vectastain Mouse IgG ABC immunoperoxidase labeling kit (Vector Laboratories, Burlingame, CA).
Morpholino antisense
Morpholino oligonucleotides previously designed against the sema3d
translation start site (Liu et al.,
2004) were synthesized by Gene Tools (Corvallis, OR). The same
sequence with four mismatched bases was used as a control. Morpholinos were
diluted in Danieau buffer and 1% Phenol Red
(Nasevicius and Ekker, 2000).
RGC axon effects were seen using morpholino concentrations of 100-250 µM;
higher concentrations caused high mortality and non-specific effects. For all
experiments, approximately 1 nl of a 100-250 µM solution was injected into
embryos at the one- to four-cell stage.
DiI injections and imaging
For axon labeling, embryos were mounted in 1% low-melting point agarose. A
0.5% solution of DiI
(1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine
perchlorate, Molecular Probes, Eugene, OR) in dimethylformamide was pressure
injected into the retina. For whole eye fills, embryos were fixed in 4%
paraformaldehyde and dye was injected into multiple parts of the RGC layer and
allowed to transport overnight. Confocal series were collected on a Zeiss
Axiovert 100M microscope with the BioRad 1024 Lasersharp Confocal software
package, and processed with Metamorph software (Universal Imaging Corporation,
West Chester, PA).
For live imaging, small amounts of DiI were injected into ventronasal, nasal or dorsonasal retina and allowed to transport for one hour at room temperature. Timelapse movies were captured on a Nikon E600FN upright microscope with a Photometrix CoolSnapHQ camera at 28-30°C. For Sema3d overexpression experiments, a single z-plane was collected every minute for up to 14 hours. For Sema3d knockdown experiments, a z-stack of three to seven planes covering the extent of the growth cone was captured every two minutes for up to 23 hours.
Quantification of guidance errors
For quantification of the extent of ipsilateral tectal innervation, the
tectal neuropil was outlined from a simultaneously imaged channel containing
only autofluorescence. This tectal outline was overlaid onto reconstructions
of confocal stacks of dorsally imaged RGC axon projections. A threshold was
set for each image to match the distribution of visible projections, and the
extent of ipsilateral innervation was determined as the percentage of tectal
area over threshold. All analyses were performed blind to embryo treatment and
groups were compared using two-tailed Student's t-tests.
For the identification of guidance errors in Sema3d morpholino-injected embryos, we analyzed confocal stacks of ventrally imaged DiI-labeled axons in the optic chiasm. Because Sema3d morpholino-injected embryos had reduced or absent tectal innervation when compared with control embryos, analysis was restricted to guidance errors visible in ventral views of the chiasm region. All analyses were performed blind to embryo treatment and groups were compared using binomial z-tests.
For analyses of live axon behaviors and growth rates, the distance between the eyes was divided into three regions: the `midline region' was defined as the middle 20% of the intereye distance, spanning the chiasm midline; `before midline' was defined as the region between the injected eye and the midline region; and `after midline' was defined as the region between the midline region and the uninjected eye. Growth cone advance, retraction or pausing was recorded for each timelapse frame then analyzed by region. Overall axon growth rates were calculated for each region by measuring total distance traveled and total time spent in each region. Outgrowth was only measured in the first half of the `after midline' region due to the dorsal turn of the optic projection medial to the contralateral eye. Groups were compared using two-tailed Student's t-tests.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
sema3d, a secreted semaphorin, is expressed in the ventral
diencephalon from 16 hpf (Halloran et al.,
1999) until at least 5 dpf, at which point all RGC axons have
reached the tectum (Burrill and Easter,
1994; Karlstrom et al.,
1996; Stuermer,
1988). Throughout the period when RGC growth cones cross the
midline (approximately 36-48 hpf), sema3d is expressed by cells both
anterior and posterior to the region of the forming chiasm
(Fig. 1B) and by midline cells
in the ventral diencephalon just dorsal to where the RGC axons cross
(Fig. 1C). This expression
pattern is maintained throughout visual pathway development, although by 5 dpf
the sema3d-expressing cells dorsal to the axons have shifted slightly
posterior because of tissue growth and shape changes
(Fig. 1D). Thus,
sema3d is expressed at the ventral midline throughout the development
of the optic chiasm.
Neuropilins (Npns) are receptor components for class 3 semaphorins. In
zebrafish, Npn1a and Npn2b are involved in at least some Sema3d-mediated axon
guidance events (Wolman et al.,
2004). We have previously shown that npn1a, npn1b and
npn2a are expressed by RGCs throughout RGC axon extension
(Liu et al., 2004), indicating
that RGCs should be capable of responding to Sema3d encountered at the midline
of the forming optic chiasm.
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). Tg(hsp70:sema3dmyc) embryos were
heat shocked at 36, 42 and 48 hpf to induce Sema3d overexpression throughout
RGC axon midline crossing, and were fixed at 5 dpf, after the tectum is
innervated. Non-heat-shocked Tg(hsp70:sema3dmyc) embryos
and heat-shocked wild-type embryos were used to control for the presence of
the transgene and the heat-shock treatment. RGC projections were visualized
with unilateral DiI whole-eye fills. Global Sema3d expression induced aberrant RGC projections to the ipsilateral tectum. At 5 dpf, dorsal imaging of tecta revealed ipsilateral projections in 97% (n=32) of Sema3d-overexpressing embryos but only 32% (n=25) of wild-type controls (P<0.000001; data not shown). Because the number of misguided axons varied between embryos, we also analyzed the extent of ipsilateral projections by calculating the percentage of ipsilateral tectal area covered by axons. Representative wild-type (Fig. 2A) and Sema3d-overexpressing (Fig. 2B,C) embryos are shown to illustrate the range of ipsilateral innervation observed. At 5 dpf, axons covered a significantly larger area of the ipsilateral tectum following Sema3d overexpression, an average of 11% of the tectal area, compared with 1-4% in control groups (Fig. 2E).
Ventral views of the optic chiasma of Sema3d-overexpressing embryos showed ipsilateral axons in the region of the optic tract (Fig. 2D). Analysis of individual confocal optical sections allowed us to trace these axons to the midline region, indicating that the axons projected to the midline before turning ipsilaterally. This observation suggests that ubiquitous Sema3d expression causes a specific axon guidance error at the chiasm midline to direct RGC axons into the ipsilateral optic tract.
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We found that global Sema3d expression selectively decreased RGC axon growth rate at the chiasm midline. Through the midline region, growth cones in Sema3d-overexpressing embryos advanced at only 7.7±1.9 µm/hour, which was significantly slower than control growth rates of 13.2±1.5 µm/hour (Fig. 3A, see also Movies 1 and 2 in the supplementary material). By contrast, outgrowth rates were not significantly changed by Sema3d overexpression either before or after the midline (Fig. 3A). Representative growth cones from control and Sema3d-overexpressing embryos are shown in Fig. 3. In just over three hours, this control growth cone extended from the midline well into the optic tract (Fig. 3C), whereas the growth cone in a Sema3d-overexpressing embryo remained near the midline (Fig. 3D).
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Ubiquitous Sema3d expression increases RGC growth cone complexity and axonal dynamics in the midline region
Our live analysis also revealed dramatic increases in RGC growth cone
morphological and behavioral complexity at the midline in response to Sema3d
overexpression. Consistent with previous studies
(Bovolenta and Mason, 1987;
Chan et al., 1998;
Hutson and Chien, 2002;
Mason and Erskine, 2000;
Mason and Wang, 1997), control
growth cones projecting toward the midline displayed a simple, streamlined
shape with short filopodia extending from the leading edge of the growth cone
(Fig. 4A, see Movie 1 in the
supplementary material). Upon reaching the midline region, control growth
cones became more complex with multiple filopodia extended toward the
contralateral side (Fig. 4B).
Before the midline, growth cones in Sema3d-overexpressing embryos also had
simple morphologies, and were indistinguishable from control growth cones
(Fig. 4C). Upon reaching the
midline region, these growth cones exhibited a larger increase in
morphological complexity than control growth cones, extending longer filopodia
in multiple directions, including back towards the ipsilateral side
(Fig. 4D, see Movie 2 in the
supplementary material). Growth cones in three Sema3d-overexpressing embryos
exhibited an extremely complex morphology, with long and active filopodia
projecting in all directions (Fig.
4E,F, see also Movie 3 in the supplementary material). These
growth cones remained stalled near the midline for 6-11 hours, spending more
than 90% of their time paused and with overall growth rates of 0 µm/hour.
Some of the filopodia of these growth cones also had unusually long lifetimes,
with individual processes persisting for up to five hours.
The axon shafts proximal to the growth cones also responded to Sema3d overexpression with dynamic morphological changes, shifting to form convoluted trajectories (Fig. 4G). These axon movements occurred in 33% (n=15) of Sema3d-overexpressing embryos, but in only 15% (n=20) of control embryos. Axon movements in Sema3d-overexpressing embryos were also larger and continued for longer periods of time than did those in controls, and suggest that exposure to ubiquitous Sema3d may alter adhesion of the axons or their substrate. Axons also extended interstitial filopodium-like processes that occurred along 86% (n=14) of axons in Sema3d-overexpressing embryos, but along only 25% (n=20) of axons in control embryos. These processes emerged along all regions of the axon but were most numerous and persistent at the midline (Fig. 4H).
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;
Chan et al., 1998;
Mason and Wang, 1997),
suggesting that exposure to ubiquitous Sema3d may cause RGC axons to pause at
the midline and search for guidance information.
Sema3d knockdown induces ipsilateral RGC axon guidance errors and reduces midline crossing
In addition to Sema3d overexpression, we examined the effect of
loss-of-function on RGC projections, as a more specific test of Sema3d
function. We used a morpholino antisense knockdown approach with a morpholino
oligonucleotide that has been previously shown to block Sema3d translation
(Liu et al., 2004;
Wolman et al., 2004). We
injected morpholinos into newly fertilized embryos (see Materials and methods
for doses), and fixed embryos at 2.5, 3.5 or 5 dpf.
RGC axons in Sema3d morphant (morpholino-injected) embryos showed three primary axon phenotypes. First, as in Sema3d-overexpressing embryos, a subset of axons turned ipsilaterally instead of crossing the chiasm midline and extended in the region of the ipsilateral optic tract (arrows, Fig. 5D-F,I). In ventral images of the chiasm region, ipsilateral guidance errors were visible in significantly more Sema3d knockdown embryos than control embryos at all three ages examined (Fig. 5J). With all ages combined, ipsilateral projections in the chiasm region occurred in 56% of Sema3d morphants, but in only 10% of embryos injected with a four-base mismatch control morpholino. The ipsilateral phenotype was also stronger in Sema3d morphants, which frequently had several turning axons (Fig. 5F,I), whereas control morpholino-injected embryos typically had only one ipsilateral axon.
Second, some RGC axons turned and extended anteriorly from the correct pathway (arrowheads, Fig. 5D,F). This type of guidance error was not seen following Sema3d overexpression. At 2.5 dpf, anterior projections occurred in 25% of Sema3d morpholino-injected embryos but were not seen in control embryos (Fig. 5J). Interestingly, at later ages, anterior projections occurred at similar low frequencies of 7-23% in both control and Sema3d morphant embryos.
Third, Sema3d knockdown reduced the midline crossing of RGC axons. RGC axon bundles crossed the chiasm midline in all control embryos at 2.5 dpf, but reached the midline in only 82% of Sema3d morphant embryos at the same age (Fig. 5J); this suggests that RGC axon growth may be delayed in the absence of Sema3d. Within axon bundles that did reach the midline by 2.5 dpf, growth cones were visible at the midline in 52% of Sema3d morphants (Fig. 5G), but in only 10% of controls, indicating that Sema3d knockdown may increase growth cone pausing or stalling at the midline, as was seen in the timelapse movies of Sema3d-overexpressing embryos. Similar results were found at 3.5 dpf (Fig. 5J). In some cases, large numbers of axons extended to the midline but failed to extend beyond it (Fig. 5H), further suggesting that Sema3d knockdown induces pausing or stalling at the midline. Sema3d morphants had noticeably smaller RGC axon bundles at all ages, and a small number of DiI eye fills did not label any axons, indicating that Sema3d knockdown may occasionally reduce or severely delay RGC axon outgrowth.
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Timelapse imaging reveals increased midline pausing and aberrant projections following Sema3d knockdown
We used live timelapse imaging to examine how Sema3d knockdown affects RGC
growth cone behavior to generate guidance errors. Growth cones or axons were
imaged in 23 Sema3d morphant embryos for up to 23 hours with z-stacks
collected every 2 minutes.
In the region before the midline, RGC growth cones in Sema3d morphant embryos maintained a simple morphology (data not shown), similar to growth cones in both control and Sema3d-overexpressing embryos. Interestingly, 42% (n=12) of growth cones imaged before the midline paused briefly (30-90 minutes) just before reaching the midline. From the midline onward, growth cones in Sema3d morpholino-injected embryos (n=16) displayed three behaviors not seen in controls (Fig. 6A). First, 44% of growth cones crossed the midline then paused in the proximal optic tract throughout the remainder of the imaging session (2-8 hours). Similar to the stalling behavior caused by Sema3d overexpression, these long pauses were characterized by shape changes, increased morphological complexity, and dynamic filopodial extensions and retractions. However, unlike growth cone stalling in Sema3d-overexpressing embryos, these long pauses were separated by brief periods of forward growth. Second, 25% of growth cones in Sema3d morphants repeatedly advanced and retracted between the midline and the proximal optic tract (Fig. 6B, see Movie 4 in the supplementary material). Periods of extension and retraction were separated by pauses, and these growth cones made no net forward progress despite being extremely active. Third, another 13% of growth cones engaged in repeated extensions and retractions after crossing the midline but still made slow progress along the optic tract. This increased incidence of growth cone retractions distinguished Sema3d knockdown from Sema3d overexpression, which, as discussed earlier, did not change the frequency of growth cone retractions. The final 19% of growth cones grew across the midline and extended into the dorsal optic tract with no unusual pausing or retractions.
Sema3d knockdown also increased the incidence of interstitial projections along RGC axon shafts similar to those caused by Sema3d overexpression. Projections occurred in 74% (n=23) of Sema3d morphant embryos, compared with 25% of control axons (described in overexpression experiments). These projections were very active and emerged from all regions of the axon to project anteriorly or posteriorly from the pathway (Fig. 6C). Anterior projections arising near the midline were the most complex, dynamic and persistent, and frequently included processes directed back toward the midline or ipsilateral side (Fig. 6C, see Movie 5 in the supplementary material). Other anterior projections extended in the region of the ipsilateral optic tract; these processes arose both from axons whose growth cones had already extended into the contralateral optic tract and from axons whose growth cones were still approaching the midline (Fig. 6D). In two embryos, anterior interstitial projections developed into new growth cones that appeared to extend in the ipsilateral optic tract (Fig. 6E, see Movie 6 in the supplementary material). Ipsilateral growth cones were imaged in a total of 26% of Sema3d morphant embryos. Interestingly, some of the ipsilateral projections, including the two de novo ipsilateral growth cones, appeared to emerge from axons that had growth cones simultaneously extending in the contralateral optic tract (Fig. 6E, Movie 6 in the supplementary material). Although RGC axons have never been described to branch in the optic chiasm, these movies suggest that individual axons might extend growth cones to both sides of the brain following Sema3d knockdown. It remains to be seen whether a single axon can maintain bilateral projections or if one projection will retract in favor of the other. Alternatively, from our analysis, we cannot rule out the possibility that the simultaneously imaged ipsilateral and contralateral growth cones arose from separate but very tightly apposed axons.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our data support a model in which RGC axons encounter Sema3d at the midline of the ventral diencephalon and are repelled down a gradient away from the midline and into the contralateral optic tract. This proposed model has three independent requirements: (1) RGC axons respond to Sema3d as a repulsive cue; (2) RGC axons become responsive to Sema3d upon reaching the midline; and (3) RGC axons derive directional information from a gradient of Sema3d expression.
First, changes in RGC axon outgrowth and behavior following Sema3d
manipulation strongly suggest that RGC axons are repelled by Sema3d. Sema3d
knockdown impaired the ability of growth cones to leave the midline, which was
revealed through live analysis to be caused by either intermittent advance or
repeated extension and retraction. These growth cone behaviors could indicate
that Sema3d knockdown caused the loss of either a repulsive signal propelling
axons away from the midline or a permissive signal allowing axon advance
through the midline region. However, RGC axons in Sema3d morphants also
extended aberrant processes into the anterior region of normal Sema3d
expression, suggesting that Sema3d normally inhibits growth into these areas.
A repulsive role of Sema3d is also consistent with previous work in our
laboratory showing that RGC growth cones were inhibited or repelled by
localized ectopic patches of Sema3d near the optic tract
(Liu et al., 2004). Effects
observed after ubiquitous Sema3d overexpression, including slowed growth and
increased growth cone pausing in the midline region, are not consistent with a
simple permissive role of Sema3d but could be indicative of increased
inhibition. Finally, increased interstitial projections following Sema3d
overexpression may reflect a repellent environment; this is supported by in
vitro studies showing that RGC axons undergo extensive back-branching after
growth cone collapse in response to both uniform application and point sources
of known repellent cues (Campbell et al.,
2001; Davenport et al.,
1999). Together, our findings are most consistent with a role for
Sema3d as a repulsive cue for RGC axons.
Second, our findings suggest that RGC axons become sensitive to Sema3d-mediated repulsion only after reaching the chiasm midline. Normally, RGC growth cones first encounter Sema3d at the midline, implying that the Sema3d expression pattern could determine the onset of responsiveness. However, even in Sema3d-overexpressing embryos, in which RGC axons grow through Sema3d-expressing tissues before the midline, RGC growth cones showed no significant behavioral or guidance responses to the ubiquitous Sema3d until they reached the midline region. Both Sema3d overexpression and knockdown caused dramatic increases in growth cone exploratory behavior at the midline, suggesting the disruption of guidance information normally used by the growth cones in this region. Interestingly, although all phenotypes indicative of repulsion occurred at the midline, our proposed model does not exclude the possibility that Sema3d may also play some role in promoting RGC axon growth to the midline. Some Sema3d morphant embryos showed delayed or reduced RGC axon outgrowth or brief growth cone pausing before the midline. If Sema3d does help to attract axons to the midline, our findings indicate that RGC axons must then switch their responsiveness to Sema3d from attraction to repulsion upon crossing the midline.
Midline axon guidance is inherently asymmetric, requiring axons to approach
then leave the same tissue, and studies of other midline systems show
precedence for changes in growth cone responsiveness at the midline.
Commissural axons in Drosophila ventral nerve cord and vertebrate
spinal cord and hindbrain are initially attracted to midline Netrin, but lose
this responsiveness after reaching the midline and become repelled by midline
Slit to extend to the contralateral side
(Harris et al., 1996;
Kennedy et al., 1994;
Kidd et al., 1999;
Kidd et al., 1998a;
Kidd et al., 1998b;
Mitchell et al., 1996;
Shirasaki et al., 1998;
Shirasaki and Murakami, 2001;
Zou et al., 2000). RGC axons
are also capable of regulating their guidance responses; in Xenopus,
RGC axons acquire sensitivity to Sema3a as a repellent after crossing the
chiasm midline (Campbell et al.,
2001). Alternatively, cells can change the nature of their
response to a single cue. For example, migrating mesoderm cells in
Drosophila switch their response to Slit from repulsion to attraction
at different points in their pathway
(Kramer et al., 2001).
Similarly, Xenopus RGC axons are attracted to Netrin as they exit the
eye, lose responsiveness to Netrin as they cross the midline, and become
repelled by Netrin as they approach the optic tectum
(Shewan et al., 2002). Both
sensitivity and modality of growth cone responses to particular cues can be
modulated by a number of factors, including expression levels of specific
receptor components (Hong et al.,
1999; Julien et al., 2005;
Keleman et al., 2002;
Wolman et al., 2004),
activation of other surface molecules
(Hopker et al., 1999;
Kantor et al., 2004;
Sabatier et al., 2004;
Stein and Tessier-Lavigne,
2001; Stoeckli et al.,
1997), and levels of intracellular signaling molecules such as
cyclic nucleotides (Polleux et al.,
2000; Song et al.,
1998; Song et al.,
1997), indicating that both intrinsic and extrinsic factors are
probably involved in determining how RGC growth cones respond to midline
Sema3d.
Third, our results support the hypothesis that RGC growth cones rely on a
gradient of Sema3d to provide the directional information needed to guide
projections contralaterally. Global Sema3d overexpression and knockdown both
alter the normal midline pattern of Sema3d expression. Likewise, the two
Sema3d manipulations resulted in many similar midline axon phenotypes,
including ipsilateral projections, growth cone pausing and increased
morphological complexity. These phenotypes indicate that Sema3d-mediated
guidance information was lost at the midline following both overexpression and
knockdown, demonstrating that the presence or absence of Sema3d is not
sufficient to convey guidance information but that the correct pattern of
expression is also needed. Although the majority of RGC axons were eventually
able to extend away from the midline despite changes in midline cues, the
formation of ipsilateral projections suggests that the directionality of this
guidance decision was impaired. RGC axons are known to use gradients of
guidance cues, including Sema3d and others, for retinotopic mapping on the
surface of the optic tectum or superior colliculus
(McLaughlin and O'Leary,
2005). The importance of gradients for proper RGC guidance in the
tectum further suggests that cue gradients may also be important for guidance
at the midline.
Together, our findings show that midline Sema3d plays an important role in
the development of the contralateral visual pathway. Our model does not
exclude the possibility that Sema3d may act in concert with other guidance
factors. For example, some guidance effects of Sema3a and Sema3b are mediated
by receptor complexes formed by neuropilins and immunoglobulin superfamily
cell adhesion molecules (Sema3a by Npn1 with L1, and Sema3b by Npn2 with
NrCAM) (Castellani et al.,
2000; Castellani et al.,
2002; Falk et al.,
2005). Additionally, many RGC axons still cross the midline after
our manipulations of Sema3d, indicating that Sema3d is probably not the only
factor directing RGC laterality in the zebrafish. Several zebrafish mutants
with ipsilateral guidance errors were identified in a large-scale mutagenesis
screen (Karlstrom et al.,
1996). Many of these mutations affect patterning and development
of the diencephalon midline, suggesting that they may influence guidance cues
indirectly. Interestingly, in a Gli2 mutant, yot, midline
sema3d expression is reduced or absent in the ventral diencephalon,
and both RGC axons and those of another ventral commissure, the postoptic
commissure, fail to cross the midline
(Barresi et al., 2005;
Karlstrom et al., 1996).
Further analysis of these mutants may identify other cues important for
laterality of RGC projections during formation of the optic chiasm.
Live imaging of RGC axons and growth cones in combination with manipulations of a putative guidance factor, Sema3d, demonstrates how live cell behaviors can elucidate the nature and mechanisms of guidance cue function in vivo. Here, disruption of the normal Sema3d expression pattern in the ventral diencephalon through global overexpression or knockdown caused phenotypes that appear very similar in fixed embryos, but which can be distinguished through differences in growth cone behaviors and dynamics. These experiments have allowed us to identify Sema3d as an important extrinsic cue for guiding the midline crossing of RGC axons to develop the correct laterality of the zebrafish visual system.
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ACKNOWLEDGMENTS |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/133/6/1035/DC1
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-tubulin immunohistochemistry (brown) at 36 hpf (B) and 38 hpf (C) show
sema3d expression at the midline of the ventral diencephalon,
anterior, posterior and dorsal to where RGC axons cross the midline.
(D) sema3d expression is maintained in the diencephalon
through 5 dpf. Arrowheads show the location of RGC axons, and asterisks
indicate the same region of sema3d in B and C. AC, anterior
commissure; ON, optic nerve; POC, postoptic commissure. Scale bars: 100 µm
in A; 50 µm in B,D; 25 µm in C.
