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The Journal of Neuroscience, July 1, 2000, 20(13):4975-4982
Retinal Ganglion Cell Axon Guidance in the Mouse Optic Chiasm:
Expression and Function of Robos and Slits
Lynda
Erskine1,
Scott
E.
Williams1,
Katja
Brose2,
Thomas
Kidd3,
Rivka A.
Rachel1,
Corey S.
Goodman3,
Marc
Tessier-Lavigne2, and
Carol A.
Mason1
1 Departments of Pathology and Anatomy and Cell
Biology, Center for Neurobiology and Behavior, Columbia University,
College of Physicians and Surgeons, New York, New York 10032, 2 Howard Hughes Medical Institute, Departments of Anatomy,
and Biochemistry and Biophysics, University of California, San
Francisco, California, 94143-0452, and 3 Howard Hughes
Medical Institute, Department of Molecular and Cell Biology, University
of California, Berkeley, California 94720
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ABSTRACT |
The ventral midline of the nervous system is an important choice
point at which growing axons decide whether to cross and project
contralaterally or remain on the same side of the brain. In
Drosophila, the decision to cross or avoid the CNS
midline is controlled, at least in part, by the Roundabout (Robo)
receptor on the axons and its ligand, Slit, an inhibitory extracellular matrix molecule secreted by the midline glia. Vertebrate homologs of
these molecules have been cloned and have also been implicated in
regulating axon guidance. Using in situ hybridization,
we have determined the expression patterns of robo1,2
and slit1,2,3 in the mouse retina and in the region of
the developing optic chiasm, a ventral midline structure in which
retinal ganglion cell (RGC) axons diverge to either side of the brain.
The receptors and ligands are expressed at the appropriate time and
place, in both the retina and the ventral diencephalon, to be able to
influence RGC axon guidance. In vitro,
slit2 is inhibitory to RGC axons, with outgrowth of both
ipsilaterally and contralaterally projecting axons being strongly
affected. Overall, these results indicate that Robos and Slits alone do
not directly control RGC axon divergence at the optic chiasm and may
additionally function as a general inhibitory guidance system involved
in determining the relative position of the optic chiasm at the ventral
midline of the developing hypothalamus.
Key words:
axon guidance; diencephalon; hypothalamus; optic chiasm; Robo; retinal ganglion cell; Slit
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INTRODUCTION |
In the mouse visual system, retinal
ganglion cell (RGC) axons from the two optic nerves grow toward one
another and either cross the ventral midline of the diencephalon
(developing hypothalamus) or turn away from it, forming an x-shaped
fiber pathway, the optic chiasm. Contralaterally projecting RGCs are
distributed throughout the retina, whereas ipsilaterally projecting
cells are found exclusively in the ventrotemporal crescent (for review,
see Guillery et al., 1995 ). In addition to being essential for the
establishment of normal binocular vision, this system provides an
excellent model for studying axon guidance at the ventral midline of
the CNS.
Using dye tracing and video microscopy, the trajectory and dynamic
behavior of RGC axons as they navigate through the mouse optic chiasm
has been described and, in the midline region in which the axons
diverge, specialized populations of radial glia and neurons (CD44/SSEA
neurons) have been identified (Sretavan and Reichardt, 1993; Godement
et al., 1994; Sretavan et al., 1994; Marcus and Mason, 1995; Marcus et
al., 1995; Mason and Wang, 1997) (for review, see Mason and Sretavan,
1997). Interactions between these cells and the RGC axons have been
implicated in both directing axon divergence and determining the
position of the chiasm on the developing hypothalamus (Wizenmann et
al., 1993; Sretavan et al., 1994, 1995; Wang et al., 1995, 1996).
However, the molecular nature of the underlying guidance signals
remains primarily unknown.
In Drosophila Roundabout robo mutants, axons that
normally grow ipsilaterally project across the midline, and all axons
aberrantly cross and recross multiple times (Seeger et al., 1993).
Roundabout (Robo) defines a novel family of highly conserved
guidance receptors (Kidd et al., 1998a; Zallen et al., 1998). Genetic
and biochemical evidence has demonstrated that Slit, an inhibitory
extracellular matrix molecule secreted by the midline glia, is a ligand
for Robo (Battye et al., 1999; Brose et al., 1999; Kidd et al., 1999; Li et al., 1999). To date, three robo and three
slit mammalian homologs have been identified and shown to be
expressed within the developing nervous system. In vitro,
Slits both inhibit CNS axon outgrowth and neuronal migration and
promote sensory axon growth and branching (Holmes et al., 1998; Itoh et
al., 1998; Kidd et al., 1998a; Ba-Charvet et al., 1999; Brose et al.,
1999; Hu, 1999; Li et al., 1999; Wang et al., 1999; Wu et al., 1999; S. Yuan et al., 1999; W. Yuan et al., 1999; Zhu et al., 1999).
To determine whether these molecules play a role in chiasm formation,
we examined the expression and potential function of Robos and Slits in
the developing mouse visual system. Both the receptors and ligands are
expressed in dynamic patterns in the retina and ventral diencephalon.
In vitro, Slit2 is inhibitory to RGC axon outgrowth.
However, no differential effect on ipsilaterally and contralaterally
projecting axons was found. One function of Robos and Slits may be to
prevent RGC axons from growing into particular regions of the brain,
thereby determining the position on the diencephalon at which the optic
chiasm develops.
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MATERIALS AND METHODS |
Embryos. All experiments were performed using
C57BL/6J mice maintained in a timed-pregnancy breeding colony. Noon of
the day on which a plug was found was considered embryonic day 0.5 (E0.5). Pregnant mothers were anesthetized with a mixture of
ketamine and xylazine, and the embryos were removed by cesarean
section. Embryonic age was confirmed by comparing external appearance
and crown-rump length with the criteria given by Theiler (1972). For DiI labeling, immunohistochemistry, or in situ
hybridization, embryos were fixed overnight in 4% paraformaldehyde in PBS.
DiI labeling of RGC axons. To label fully the optic nerve
and tract, a small crystal of DiI (D282; Molecular Probes, Eugene OR)
was placed over the optic disk of one eye, and the embryos were stored
at 37°C for 4 d in PBS containing 0.1% sodium azide. Labeled
heads were sectioned horizontally or coronally at 100 µm on a
vibratome, and the DiI was photoconverted to a permanent brown reaction
product as described previously (Marcus et al., 1995).
Immunohistochemistry. Fixed heads of embryos at E12.5-E17.5
were sectioned horizontally or coronally at 100 µm on a vibratome (Fig. 1) and immunostained as
described previously (Marcus et al., 1995). Monoclonal antibody
RC2 labels specialized midline radial glia at the optic chiasm (Marcus
et al., 1995; Marcus and Mason, 1995) (Fig. 1B), Mab
480-1.1, against stage-specific embryonic antigen 1 (SSEA-1), labels
the early born CD44/SSEA-1 neurons posterior to the optic chiasm
(Marcus et al., 1995; Marcus and Mason, 1995) (Fig.
1B), and K4 (guinea pig polyclonal against Islet1/2)
marks the RGC layer in embryonic retinas (Thor et al., 1991; R. Rachel, L. Erskine, and C. A. Mason, unpublished observations). Antibodies were a gift of Dr. T. M. Jessell (Columbia University, New York, NY) and were used at a final concentration of 1:3 (RC2 and
480-1.1) or 1:10 000 (K4).
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Figure 1.
Anatomy of the region of the developing
optic chiasm during the major period of RGC divergence.
A, Head of an E14.5 mouse embryo indicating the planes
of section used in this study. B, Schematic diagrams of
horizontal and coronal sections through the optic chiasm of an E14.5
mouse embryo. The path of crossed and uncrossed RGC axons is shown in
relation to the specialized cell types (radial glia, dark gray
hatching; CD44/SSEA neurons, light gray) present
in this region. All axons grow into and contact the radial glia before
either crossing the midline, at the tip of the SSEA-1 positive
region, or turning back into the ipsilateral optic tract (Marcus et
al., 1995). D, Dorsal; V, ventral;
A, anterior; P, posterior;
N, nasal; T, temporal.
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In situ hybridization. In situ hybridization,
using digoxigenin-labeled riboprobes, was performed on 100 µm
vibratome sections according to the method of Laufer et al. (1997). Rat
cDNAs encoding robo1,2 and slit1,2,3 (Kidd et
al., 1998a; Brose et al., 1999) were used as templates for riboprobe synthesis.
Photography. Sections were mounted in 90% glycerol in PBS
and photographed using Kodak 64T or Ektachrome 400 color slide film on a Zeiss Axioplan microscope. Slides were scanned into a
Macintosh computer, and the figures were prepared using Adobe Photoshop.
Collagen gel cultures. Retinal explants from E14.5 mouse
embryos were cultured as described previously (Wang et al., 1996), except that a 50:50 mix of bovine dermis and rat tail collagen (Collaborative Research, Bedford, MA) was used. Explants were cocultured, 100-300 µm apart, with aggregates of untransfected COS
cells or COS cells transfected, using Lipofectamine Plus (Life Technologies, Grand Island NY), with the vector plasmid or with human Slit2 (hSlit2) fused at its C terminus to a myc-tag (Brose et al., 1999). Heparin (50 ng/ml; Sigma, St. Louis, MO) was
added to the medium because this has been reported to augment the
release of Slit from the plasma membrane (Brose et al., 1999). Heparin alone had no effect on RGC axon outgrowth (data not shown). After 24 hr, the cultures were fixed with 4% paraformaldehyde in PBS, and the
neuronal processes were visualized using an anti- -tubulin monoclonal
antibody (Sigma), followed by a Cy3-conjugated goat anti-mouse
antibody. Labeled cultures were mounted on slides in Gelmount and
photographed using T-max 400 film on a Zeiss Axioplan microscope.
The extent of axon outgrowth was quantified using the public domain NIH
Image analysis system to measure the area covered by the RGC axons.
This measurement takes into account both the number and length of the
axons, parameters that could not be accurately measured individually in
these three-dimensional cultures. Because outgrowth was not radial,
care was taken to ensure the correct orientation of the explants (see
Fig. 6), and only the outgrowth originating from the half of the
explant facing the cells was quantified.
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RESULTS |
The expression of robos and slits in the
developing mouse retina and ventral diencephalon was determined at four
ages, each of which represents a significant time point during the
development of the optic chiasm: E12.5, shortly after the onset of RGC
genesis and the age at which the first axons begin to grow into the
diencephalon; E14.5 and E15.5, the principle period of RGC genesis and
axon divergence; and E17.5, after ipsilaterally projecting RGCs have ceased to be born. Generation of contralaterally projecting RGCs continues until birth (Dräger, 1985; Colello and Guillery, 1990; Marcus and Mason, 1995; Marcus et al., 1995). Identical expression patterns were found at E14.5 and E15.5; thus, data from only one of
these ages (E14.5) is presented. At all ages examined, no
slit3 expression was detected in either the retina or the
region of the developing optic chiasm (data not shown). This is in
contrast to the findings of W. Yuan et al. (1999), who reported
expression of slit3 in the developing lens and pigmented
epithelium of the mouse retina.
Expression patterns of robos and
slits in the mouse retina
At E12.5, robo2 and slit1 are expressed in
the dorsocentral region of the retina, the position in which the first
RGCs to be born are located. Robo1 and slit2 are
not expressed at this age (Fig.
2A-E). By E14.5, in
addition to robo2 and slit1, robo1 and
slit2 also are expressed throughout the RGC layer (Fig.
2F-J). The mRNA for robo1 is
restricted to a scattered subpopulation of cells, whereas
robo2 and the slits appear to be expressed by most cells in the RGC layer (Fig. 2, compare P,
Q). The robo1-positive cells are distributed
throughout the retina (Fig. 2G) and therefore are unlikely
to represent cells with a particular projection phenotype at the optic
chiasm (ipsilaterally projecting RGCs are found exclusively in the
ventrotemporal crescent of the retina). At this age, slit1 mRNA is expressed in a high ventral, low dorsal gradient (Fig. 2I). No nasal-temporal gradients were detected (data
not shown). At E17.5, robo2 expression is maintained within
the RGC layer, whereas slit1 is, at best, only very weakly
expressed (Fig.
2K,M,N). Slit2 is clearly restricted to the inner nuclear layer (Fig.
2O,R), as is robo1 at this age (Fig.
2L).
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Figure 2.
Expression patterns of robos and
slits in the developing mouse retina. Coronal sections
of E12.5 (A-E), E14.5
(F-J), and E17.5
(K-O) retinas stained with an antibody
against the LIM homeodomain proteins, Islet 1/2 (an early RGC
marker; A, F, K) or
by in situ hybridization using digoxigenin-labeled
riboprobes for robo1 (B,
G, L), robo2
(C, H, M)
slit1 (D, I,
N), or slit2 (E,
J, O). Boxed regions in
G, H, and O are shown at
higher power in P, Q, and
R respectively. Dotted line in
R marks the central lumen of the retina. Scale bar:
A-O, 500 µm; P, R, 80 µm. Dorsal, Top; ventral, bottom.
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Expression patterns of robos and
slits in the region of the developing optic chiasm
At E12.5, the first RGC axons enter the brain in which they
establish the correct position and shape of the optic chiasm. These
early axons grow into the diencephalon and then course ventrally before
extending toward the midline along the border of the CD44/SSEA neurons
(Marcus and Mason, 1995) (Fig.
3A-D). Later in development, RGC axons make distinct pathway choices at the midline, thereby projecting to targets on both sides of the brain (Figs.
4A-D, 5A).
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Figure 3.
Expression of robos and
slits in the ventral diencephalon of E12.5 mouse
embryos. A-D, Serial horizontal sections showing both
DiI-labeled RGC axons (brown) and SSEA-1
(black). In the most dorsal section
(A), no RGC axons are present.
Asterisk marks the third ventricle. More ventrally
(B), a few axons can be seen at the junction of
the optic stalk and the brain (arrow). In
C and D, the region of the future optic
chiasm, RGC axons have entered the diencephalon and appear to be
growing along the border of the SSEA-1-positive cells but have not yet
crossed the midline. E-T, Comparable serial sections
with those in A-D after in
situ hybridization to show patterns of robo1
(E-H), robo2
(I-L), slit1
(M-P), and slit2
(Q-T) expression. Orientation is the same as in
Figure 1B (anterior, top;
posterior, bottom). Scale bar, 500 µm.
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Figure 4.
Expression of robos and
slits in the ventral diencephalon of E14.5 mouse
embryos. A-D, Serial horizontal sections
double labeled with DiI to show the RGC axons (brown)
and SSEA-1 (black), which marks the CD44/SSEA neurons
posterior to the optic chiasm. In the more dorsal sections
(A, B), RGC axons are present in the
optic nerve (A) and at the junction of the optic
nerve and the brain (B). The axons then grow more
ventrally before diverging to form the x-shaped optic chiasm
(C, D). The site at which the axons
diverge is marked by a thin raphe of the CD44/SSEA neurons.
E-T, Comparable serial sections with those in
A-D after in situ
hybridization to show patterns of robo1
(E-H), robo2
(I-L), slit1
(M-P), or slit2
(Q-T) expression. U-W, Coronal
sections labeled with photoconverted DiI to show the RGC axons
(U), the monoclonal antibody RC2 (labels radial
glia; V), or after in situ
hybridization for slit2
(W). Asterisks in
V and W marks the RGC axons.
Arrows point to strong staining of RC2 and
slit2 in the radial glial cell bodies. Orientation is
the same as in Figure 1B. Scale bar, 500 µm.
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Figure 5.
Expression of robos and
slits in the ventral diencephalon of E17.5 mouse
embryos. A, Horizontal section, at the level of the
optic chiasm, double labeled to show both the RGC axons
(brown) and SSEA-1 (black).
B-E, Comparable sections after in situ
hybridization to show the patterns of expression of
robo1 (B), robo2
(C), slit1
(D), or slit2
(E). Anterior, Up; posterior,
down. Scale bar, 250 µm.
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At E12.5, robo1 is expressed in a domain posterior and
medial to the CD44/SSEA neurons (Fig. 3E-H), which
express robo2 (Fig. 3I-L). Slit1 is
expressed around the junction of the optic nerve and the brain (Fig.
3M,N), with strongest
expression dorsal to the site at which the optic stalk joins the
diencephalon (Fig. 3B, arrow). Slit1
also is weakly expressed in a subset of the CD44/SSEA neurons (Fig.
3O,P). In the more dorsal region of the developing optic chiasm, the domain of slit1 expression lies
some distance posterior to the axons (Fig. 3C,O).
However, more ventrally, slit1 is expressed in a region
directly adjacent to the path taken by the RGC axons (Fig.
3D,P). Slit2 is strongly expressed
at the ventral midline of the diencephalon in the region in which the RGC axons enter the brain and turn to grow ventrally (Fig.
3Q-T). The domain of slit2 expression
includes the position of the glial knot, a glial structure at the
midline of the diencephalon that has been proposed to delimit the
pathway of the ingrowing RGC axons (Silver, 1984).
Later in development, robo1 is more weakly expressed
throughout the diencephalon, both posterior to the CD44/SSEA neurons and around the third ventricle (Figs. 4E-H,
5B). Robo2 continues to be expressed in the
CD44/SSEA neurons (Fig. 4I-L), a pattern that is
maintained until at least E17.5 (Fig. 5C). At E14.5,
slit1 is still expressed around the junction of the optic
nerves and the brain (Fig. 4, compare B,
N) and, in a similar pattern to robo2, in
the CD44/SSEA neurons (Fig. 4N-P). By E17.5,
slit1 can no longer be detected at the junction of the brain
and optic nerve and is only weakly expressed by the CD44/SSEA neurons
(Fig. 5D). Slit2 expression is maintained at the
ventral midline of the diencephalon, in a region directly dorsal to the
site of axon divergence (Fig. 4Q-T). At all ages
examined, no slit2 mRNA was seen at the more ventral level
at which the axons cross the midline (Fig. 4S,T, 5E).
The cell bodies of the midline glia resident in the ventral
diencephalon lie dorsal to the site at which the retinal axons diverge.
Only their radial processes are found among the RGC axons (Marcus et
al., 1995, their Fig. 1E) (Fig.
1B). In coronal sections, the mRNA for
slit2 appears to be expressed by a subset of these specialized midline radial glial cells, suggesting that Slit2 protein
may be present on their radial processes and consequently at the site
at which the axons diverge (Fig. 4U-W).
Unfortunately, no good antibodies are currently available to confirm
this expression pattern. Overall, these results indicate that Robos and
Slits are expressed at the appropriate time and place to be able to regulate RGC axon guidance.
In vitro, Slit2 inhibits RGC axon outgrowth
To test whether Slit2 can regulate RGC axon outgrowth, explants,
taken from each of the four retinal quadrants, were cocultured in
collagen gels with aggregates of cells expressing hSlit2. Explants were
taken from the most peripheral part of E14.5 retinas because, at this
age, this region of the ventrotemporal retina contains a high
percentage of RGCs whose axons will project ipsilaterally at the optic
chiasm; the remainder of the retina is composed predominately of
contralaterally projecting RGCs (Dräger, 1985) (Fig.
6A). Thus, a direct
comparison could be made of the effect of hSlit2 on ipsilaterally and
contralaterally projecting RGC axons.
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Figure 6.
Effect of hSlit2 on RGC axon outgrowth in
vitro. A, Schematic diagram of a flat-mounted
E14.5 mouse retina. RGCs that project contralaterally are found
throughout the retina, whereas ipsilaterally projecting cells are
restricted to the ventrotemporal crescent (black
region). To enable a comparison of the behavior of crossed and
uncrossed RGC axons, explants were prepared only from the most
peripheral part of each retinal quadrant. Consequently, growth from
these explants was not radial but originated only from the cut edge
(indicated by the dotted lines). D, Dorsal; V, ventral; N,
nasal; T, temporal. B-E, Explants from
dorsotemporal retina cultured alone (B), with
clusters of mock-transfected cells (C), or with
clusters of cells transfected with hSlit2 (D,
E). Explant in E is oriented such that
growth is directed away from the Slit-expressing cells. Scale bar, 500 µm. F, Extent of RGC axon outgrowth from explants
cocultured with mock-transfected (open bars) or
hSlit2-expressing COS cells (filled bars).
Numbers above bars indicates number of
explants. *p < 0.001 compared with growth in the
presence of the mock-transfected cells (Student's unpaired
t test). Data were pooled from five independent
experiments. G, Extent of RGC axon outgrowth in the
presence of hSlit2 expressed as a percentage of the outgrowth seen in
cultures containing mock-transfected cells.
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When cultured alone, outgrowth from these explants was not radial but
grew out predominately from the cut edge, with little or no outgrowth
emanating from the peripheral side (Fig. 6B). The
amount of outgrowth from the cut half of the explant was quantified by
measuring the area of the dish covered by the RGC axons (Fig. 6F; see Materials and Methods). This measurement
takes into account both the number and length of the axons growing into
the collagen.
Coculturing explants with aggregates of mock-transfected cells had no
effect on neurite outgrowth (Fig. 6C). However, in cultures containing hSlit2, RGC outgrowth was markedly decreased (Fig. 6D). In particular, fewer axons appeared to grow from
the explants into the collagen. We refer to this as inhibition rather
than repulsion because, particularly in control cultures, we could not
measure the angles turned by individual axons (Fig.
6B,C) and thus definitively show
that axons were being repelled by the Slit-expressing cells. Compared
with outgrowth in the presence of mock-transfected cells, outgrowth
from all four retinal quadrants was significantly decreased (Fig.
6F). This was unlikely to be attributable to
hSlit2 having a general toxic effect on the explants. When explants
were cultured such that growth was directed away from the
Slit-expressing cells, Slit2 had no effect on axon outgrowth (Fig.
6E; data not shown).
The extent of outgrowth from each retinal quadrant, cultured in the
absence of hSlit2, was not identical (Fig. 6F,
open bars). The extent of inhibition induced by hSlit2
therefore was normalized by dividing the amount of outgrowth seen in
the presence of hSlit2 by that seen in cultures containing
mock-transfected cells (Fig. 6G). hSlit2 induced a 47%
decrease in the amount of outgrowth from ventrotemporal retina
(ipsilaterally projecting RGC axons) and a 51, 40, and 42% decrease in
the outgrowth from dorsotemporal, ventronasal, and dorsonasal retina,
respectively (all sources of contralalerally projecting RGC axons).
These results indicate that hSlit2 is inhibitory to all RGCs and, at
least in vitro, is not a more potent inhibitor of
ipsilaterally than contralaterally projecting axons.
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DISCUSSION |
The decision to cross or turn away from the midline is an
important intermediate step in the projection of many axons to their targets. One place at which this occurs is the midline of the mammalian
diencephalon in which RGC axons from each eye diverge at the optic
chiasm. Here we report that Robo receptors and their Slit
ligands, molecules important for midline guidance in
Drosophila (Kidd et al., 1999), are expressed at the
appropriate time and place in the mouse retina and diencephalon to be
able to influence RGC axon guidance at the optic chiasm. In
vitro, Slit2 was found to be a potent inhibitor of RGC axon
outgrowth. However, no differential effect on ipsilaterally and
contralaterally projecting RGCs was evident. Together with the findings
of Niclou et al. (2000) and Ringstedt et al. (2000), these results
implicate Robos and Slits as important regulators of RGC axon guidance
and suggest that, during optic chiasm development, they may play
additional roles to controlling guidance across the midline.
Robos and slits are expressed in the
retina and ventral diencephalon at the time when the optic chiasm is
developing
In the retina, robo2 is expressed by RGCs before any
axons have reached the ventral midline of the diencephalon (Figs.
2C, 3C) and continues to be strongly expressed
during later stages of development (Fig.
2H,M). In contrast,
robo1 is not detected until after a number of axons have
started to cross the midline and then only in a subset of cells (Figs.
2B,G, 4C,D).
This suggests that Robo2 is likely to be the principle player of these
two receptors in terms of RGC axon guidance.
In the diencephalon, the Slits are expressed at three sites, all of
which have been identified as regions in which RGC axons make guidance
decisions and coincide with zones defined by domains of transcription
factor expression (Marcus and Mason, 1995; Marcus et al., 1999). First,
slit1 is expressed around the junction of the optic nerve
and the brain, with the strongest expression dorsally (Figs.
3B,M,
4B,M,N). Netrin,
which can promote the growth of RGC axons (Wang et al., 1996), also is
expressed at this site (Deiner and Sretavan, 1999). In Vax1 mutants,
netrin expression is lost from this region, whereas
slit1 expression is maintained, and this is associated with
a failure of RGC axons to grow into the diencephalon (Bertuzzi et al.,
1999). This indicates that this coexpression of netrin and Slit1 is
important for RGC axon guidance and suggests a mechanism by which axons
may be directed toward the midline of the diencephalon in which, unlike
other regions of the CNS, no chemoattractant activity has been detected (Wang et al., 1996). First, netrin may act to attract the RGC axons,
which, upon reaching the brain, lose their ability to respond to this
signal (Shirasaki et al., 1998). Slit1 repulsion would then dominate
and direct the RGC axons away from the optic nerve, toward the ventral midline.
Slit1 also is expressed in the CD44/SSEA neurons (Fig.
4N-P). These neurons define an inhibitory zone
posterior to the chiasm into which RGC axons never extend (Sretavan et
al., 1994; Marcus and Mason, 1995; Marcus et al., 1995). The
first RGC axons appear to grow along the border of these cells thereby
establishing the correct position and shape of the forming optic chiasm
(Marcus and Mason, 1995). Several molecules inhibitory to RGC outgrowth have been shown to be expressed by these neurons and may act to prevent
RGC axons from growing back into this area (Sretavan et al., 1994;
Marcus et al., 2000). Whether Slit1 also is involved in preventing
axons from growing into this area remains to be determined. Indeed, in
some regions, slit1 is expressed some distance posterior to
the front of the growing axons (Fig. 4C,O) (but
see Fig. 4D,P). This raises the
possibility that the Slit1 on the CD44/SSEA neurons may regulate the
guidance of only a subset of the RGC axons or is involved in other
aspects of hypothalamic development.
Finally, slit2 is expressed on a subset of the specialized
radial glia present at the ventral midline of the diencephalon (Figs.
3Q, 4Q,W). All RGC axons grow
into and contact the radial processes of these cells (Marcus et al.,
1995), making Slit2 a good candidate for a factor controlling RGC axon
divergence at the midline.
Slit2 and RGC axon guidance
Slit2 is expressed at the correct time and place to be
able to regulate axon crossing at the developing optic chiasm. However, when presented alone in vitro, Slit2 does not have a
differential effect on ipsilaterally and contralaterally projecting RGC
axons; both are strongly inhibited (Fig. 6). One possibility is that Slit2 alone is not enough to direct divergence but that additional factors are required. Other axon guidance molecules, such as
Nr-CAM (M. Lustig, C. A. Mason, M. Grumet, and T. Sakurai,
unpublished observations) and Eph/ephrin receptors and ligands
(Bertuzzi et al., 1999; Marcus et al., 2000) are expressed on the glial
cells present at the ventral midline of the mouse diencephalon. In the future, it will be important to determine whether these molecules can
synergize with Slit2 and thereby control RGC axon divergence at the midline.
Another possibility is that the lack of a differential response of
crossed and uncrossed RGC axons to Slit2 reflects an inappropriate regulation of Robo expression in our culture system. In
Drosphila a third molecule, commisureless (Comm), is
required for midline guidance (Seeger et al., 1993) Comm is expressed
by the midline glia and, as axons approach the midline, downregulates
Robo on their growth cones. Depending on their original level of Robo expression, axons can now cross or are still repelled from the midline
by Slit (Tear et al., 1996; Kidd et al., 1998b). In the absence of
comm, all axons are repelled by the midline (Seeger et al.,
1993). Thus, appropriate regulation of Robo expression, perhaps by a
Comm-like molecule, may be required to induce a differential response of ipsilaterally and contralaterally projecting RGC axons to Slit2.
Alternatively Slit2 could be involved in regulating other aspects of
optic chiasm development. RGC axons grow into the diencephalon and
then, at the border of the slit2 domain, a region
corresponding to the glial knot (Silver, 1984), turn ventrally before
approaching the midline (Figs.
3B,R,
4B,R). By preventing axons from
growing directly across the midline, Slit2 could determine the exact
position along the dorsoventral axis at which the optic chiasm develops and may from part of the molecular basis for the proposed barrier function of the glial knot.
Robos and slits colocalize both in
the retina and the ventral diencephalon
In addition to their expression in the ventral diencephalon, both
slit1 and slit2 are expressed within the retina
(W. Yuan et al., 1999) (Fig. 2). Colocalization of ephrin ligands with their Eph receptors also occurs within the RGC layer of the chick and
mouse retina and may act to modulate RGC axon guidance (Marcus et al.,
1996; Dütting et al., 1999; Hornberger et al., 1999). Overexpression of ephrinA ligands within the chick retina leads to the
establishment of an ectopic ipsilateral pathway at the optic chiasm and
the misrouting of axons within the tectum. In a similar manner, Slit in
the retina could function to modulate RGC axon guidance at the optic
chiasm. Alternatively, Slit expression in the retina may reflect a role
for the Robo-Slit guidance system in intraretinal development or
function to prevent RGC axons from extending into deeper layers of the
retina. Additional studies will be required to distinguish between
these possibilities.
Robo2 and slit1 also colocalize on the CD44/SSEA neurons.
These neurons are present in the diencephalon before the first RGC axons grow into the brain and extend their axons dorsally within the
diencephalon (Sretavan et al., 1994). Thus, the simplest explanation for the expression of robo2 by these neurons is that it is
required by them to make their own guidance decisions. Slit1 could
interact with the Robo2 coexpressed by these cells to modulate their
guidance and/or be involved in regulating the development of other axon tracts within the hypothalamus (see above).
Conclusions
We have shown that robos and slits are
expressed in the developing mouse visual system in a manner consistent
with their being important regulators of RGC axon guidance and that
in vitro Slit2 is inhibitory to RGC axon outgrowth. Several
different sites at which Slits may influence RGC axon guidance at the
optic chiasm were identified: around the junction of the optic nerve
and the brain, the CD44/SSEA neurons, and the midline radial glia.
These results suggest that Robos and Slits may be involved in
regulating the growth of RGC axons from the optic nerve into the brain
and in determining the position at which the optic chiasm develops on
the ventral midline of the hypothalamus. Additional work will be
required to determine whether they are involved in regulating axon
divergence at the midline.
| |
FOOTNOTES |
Received Dec. 28, 1999; revised March 8, 2000; accepted March 17, 2000.
This work was supported by National Institutes of Health Grants EY
12736 and PO NS 30532 (C.A.M.), and American Paralysis Association
Grant GB1-9801-2 (C.S.G.). K.B. is the recipient of National Science
Foundation Predoctoral and University of California, San Francisco,
Chancellor's Fellowships, and L.E. is a recipient of EMBO and
Human Frontier Science Program Long-Term Fellowships. T.K. is a
Postdoctoral Associate, and C.S.G. and M.T.L. are investigators with
the Howard Hughes Medical Institute. We thank Dr. Mary Morrison for
critical comments during the course of this work, Drs. Neil Vargesson
and Ed Laufer for advice and guidance on in situ
hybridization, Dr. Samantha Butler for advice on the collagen gel
assay, and Gul Dolan and Richard Blazeski for technical assistance.
Correspondence should be addressed to Lynda Erskine, Department of
Pathology, 14-509 Physicians and Surgeons Building, Columbia University, College of Physicians and Surgeons, 630 W. 168th Street, New York, NY 10027. E-mail: le63{at}columbia.edu.
| |
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TOP
ABSTRACT
INTRODUCTION
