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The Journal of Neuroscience, July 1, 2000, 20(13):4983-4991
Slit Inhibition of Retinal Axon Growth and Its Role in Retinal
Axon Pathfinding and Innervation Patterns in the Diencephalon
Thomas
Ringstedt1,
Janet E.
Braisted1,
Katja
Brose2,
Thomas
Kidd3,
Corey
Goodman3,
Marc
Tessier-Lavigne2, and
Dennis D. M.
O'Leary1
1 Molecular Neurobiology Laboratory, The Salk
Institute, La Jolla, California 92037, 2 Departments of
Anatomy, and Biochemistry and Biophysics, University of California, San
Francisco, California 94143-0452, and 3 Department of
Molecular and Cell Biology, University of California, Berkeley,
California 94720
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ABSTRACT |
We have analyzed the role of the Slit family of repellent
axon guidance molecules in the patterning of the axonal projections of
retinal ganglion cells (RGCs) within the embryonic rat diencephalon and
whether the slits can account for a repellent activity for retinal
axons released by hypothalamus and epithalamus. At the time RGC axons
extend over the diencephalon, slit1 and
slit2 are expressed in hypothalamus and epithalamus but
not in the lateral part of dorsal thalamus, a retinal target.
slit3 expression is low or undetectable. The Slit
receptors robo2, and to a limited extent
robo1, are expressed in the RGC layer, as are
slit1 and slit2. In collagen gels, axon
outgrowth from rat retinal explants is biased away from
slit2-transfected 293T cells, and the number and length
of axons are decreased on the explant side facing the cells. In
addition, in the presence of Slit2, overall axon outgrowth is
decreased, and bundles of retinal axons are more tightly fasciculated. This action of Slit2 as a growth inhibitor of retinal axons and the
expression patterns of slit1 and slit2
correlate with the fasciculation and innervation patterns of RGC axons
within the diencephalon and implicate the Slits as components of the
axon repellent activity associated with the hypothalamus and
epithalamus. Our findings suggest that in vivo the Slits
control RGC axon pathfinding and targeting within the diencephalon by
regulating their fasciculation, preventing them or their branches from
invading nontarget tissues, and steering them toward their most distal
target, the superior colliculus.
Key words:
axon guidance; axon fasciculation; axon repellents; chemorepellents; robo1; robo2; hypothalamus; retinal ganglion cells
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INTRODUCTION |
Visual information is relayed from
the eye to the brain via the axons of retinal ganglion cells (RGCs).
RGC axons enter the brain at the ventral aspect of the hypothalamus. In
rodents, essentially all RGCs project to their midbrain target, the
superior colliculus (SC) (Linden and Perry, 1983 ). En route to the SC,
RGC axons grow dorsally over the lateral surface of the diencephalon. A
small fraction of RGC axons invade the ventral hypothalamus to form the
retinohypothalamic projection (Johnson et al., 1988; Levine et al.,
1991, 1994). In contrast, approximately one-third of RGC axons later
extend branches into their targets in dorsal thalamus (e.g., the dorsal
lateral geniculate nucleus) (Bhide and Frost, 1991).
The first RGC axons reach the ventral hypothalamus in rats on embryonic
day 14 (E14), grow over the diencephalon, and reach the SC on
E16 (Lund and Bunt, 1976; Bunt et al., 1983; Marcus and
Mason, 1995). Later arriving RGC axons continue to grow over the
diencephalon for several more days (Tuttle et al., 1998). RGC axons
extend over the hypothalamus in a tight fascicle, defasciculate and
spread over the surface of their dorsal thalamic targets, and then
refasciculate into a tight bundle as they approach epithalamus and
turn caudally toward the SC (Fig.
1A) (Tuttle et al., 1998). This
pattern of RGC axon growth over the diencephalon correlates with
in vitro observations that retinal axons can extend over an
inhibitory substrate but in a highly fasciculated manner (Bray et al.,
1980). Defasciculation of RGC axons over dorsal thalamus likely
facilitates their branching and target innervation (Daston et al.,
1996). Consistent with these axonal behaviors, we previously showed
that in vitro the hypothalamus and epithalamus release soluble activities that repel rat and mouse retinal axons, whereas dorsal thalamus releases an activity that promotes their growth (Tuttle
et al., 1998) (Fig. 1B). The molecular identities of
these activities are unknown; however, members of the Slit family of secreted proteins are candidates for the repellent activity.
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Figure 1.
The RGC axon pathway in E17 rat and the
distribution of repellent and attractant activities in diencephalon.
Schematics of sagittal (A) and coronal
(B) views. For clarity, the cortex is outlined as
transparent (Ctx, orange) in
A and is not present in B.
A, The optic tract (opt) from the optic
chiasm (oc) to the SC is represented in
purple. RGC axons are tightly fasciculated as they
course over the surface of the hypothalamus (Hy) but
begin to defasciculate when they reach ventral thalamus, spread out
dramatically over the lateral surface of dorsal thalamus
(dTh), and later form and extend branches into their
dorsal thalamic target nuclei (and to a lesser extent ventral
thalamus). As RGC axons extend beyond the dorsal thalamus and approach
epithalamus (Epi), they refasciculate on the surface of
diencephalon and turn caudally to grow toward the SC. (Adapted
from Tuttle et al., 1998). B, Summary of collagen gel
coculture studies of Tuttle et al. (1998) that showed that the
hypothalamus and epithalamus release soluble repellent activities
(represented by  signs), and the lateral part of the dorsal
thalamus (i.e., the target nuclei for RGC axons) releases a
growth-promoting activity (represented by + signs) for retinal axons.
These findings were obtained by coculturing multiple E15 rat retinal
explants (purple) at a distance from different
parts of whole living vibratome coronal sections of E15, E17, or E19
rat diencephalon (dorsal is up, and lateral is to the
left).
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In Drosophila, the transmembrane receptor Roundabout (Robo)
is expressed on axons, plays a role in preventing ipsilaterally projecting axons from crossing the midline and commissural axons that
have crossed the midline from recrossing (Seeger et al., 1993; Kidd et
al., 1998; Zallen et al., 1998). Mutant analysis suggests that Robo is
a receptor for a midline repellent activity, subsequently shown to be
Slit. In mammals, three slit genes, slit1, slit2, and slit3, and two Slit receptors,
robo1 and robo2, have been identified (Itoh et
al., 1998; Nakayama et al., 1998; Brose et al., 1999; Li et al., 1999).
Slit2 has been shown to be a chemorepellent for olfactory bulb,
hippocampal (Nguyen Ba-Charvet et al., 1999), and spinal motor axons
(Brose et al., 1999). The objective of the present study was to
evaluate the role of the Robos and Slits in the patterning of RGC
projections within the embryonic rat diencephalon and whether the slits
can account for the hypothalamic and epithalamic repellent activities.
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MATERIALS AND METHODS |
Animals
Sprague Dawley rats from E13 to E17 were obtained from
timed-pregnant females (Harlan Sprague Dawley, Indianapolis,
IN). Embryos were staged according to Butler and Juurlink
(1987). The day of insemination was designated as E0.
In situ hybridization. Timed-pregnant rats (E13-E17) were
anesthetized with sodium pentobarbital and cesarean-sectioned to expose
the embryos. Embryos were decapitated, and the heads were fixed by
immersion in 4% paraformaldehyde (Pfa). After cryoprotection (30%
sucrose), the heads were coronally sectioned at 20 µm on a cryostat.
The sections were fixed for 20 min in 4% Pfa, rinsed twice in PBS, and
then delipidated in 2× standard SSC at 65° C for 30 min.
After two rinses in water and PBS, the slides were acetylated for 10 min in 0.25% acetic anhydride in 0.1 M
ethanolamine and then rinsed in PBS and alcohol-dehydrated. After
drying, the sections were incubated overnight in a humidified chamber
with 90 ml of hybridization buffer per slide (hybridization buffer is
50% formamide, 10% dextran sulfate, 1× Denhardt's solution, 0.3 M NaCl, 10 mM Tris, pH 7.5, 10 mM sodium phosphate, pH 6.8, and 5 mM EDTA). Antisense RNA probes were added to a
concentration of between 20,000 and 50,000 cpm/µl hybridization
buffer. After hybridization, the sections were washed 30 min at 55°C
in 5× SSC with 20 mM  -mercaptoethanol
(-me), 30 min in 1× SSC (with 20 mM -me)
at 65°C, 45 min at 65°C in 1× SSC, 50% formamide, and 20 mM -me, and 2 hr at 37°C in 0.5× SSC, 50%
formamide, and 20 mM -me. They were then
treated with RNase A (20 µg/ml) for 30 min at 37°C, washed three
times for 15 min in 1× SSC at 60°C and 5 min in 0.2× SSC at
room temperature. Finally, sections were dehydrated, dried, and dipped
in Kodak (Eastman Kodak, Rochester, NY) NT-B2 emulsion. After 5-8 d,
the slides were developed, stained with DAPI (Sigma, St. Louis, MO),
and mounted in Permount. Probes were labeled with
[35S]UTP by in vitro
transcription with T7 RNA polymerase from fragments of Slit1,
Slit2, Slit3, Robo1, or Robo2 subcloned into PBS S/K (Stratagene,
La Jolla, CA).
Preparation of explants
Timed-pregnant rats (E15) were anesthetized with sodium
pentobarbital and cesarean-sectioned to expose the embryos. Embryos were decapitated, the eyes were removed, and retinas were dissected from surrounding tissues and cut into eight radially symmetric pieces
without regard to the retinal axes. Explants were then cocultured
overnight in DMEM-F12 with 2 mM glutamine, 0.1%
penicillin-streptomycin, 20 mM HEPES, 0.6%
D-glucose, and 10% rat serum (growth medium), supplemented
with 10 µM cytosine arabinoside (AraC). Retinal axon growth is greater and more radially symmetric, and non-neuronal cell
migration from the explants is reduced in cultures exposed briefly to
AraC. At the concentrations and exposure times used here, AraC does not
appear to have detrimental effects on neurons (Martin et al., 1990).
The following day, explants were rinsed in DMEM-F12 and plated in
growth medium without AraC as described below.
Preparation of aggregates of
slit2-transfected cells
293T cells were transfected with slit2-myc cDNA using
SuperFect Transfection Reagent (Qiagen, Hilden, Germany). The
expression vector was constructed by inserting a sequence encoding
human Slit2 into the expression vector pSecTagB. pSecTagB
without insert was used for mock transfections. Several days before
transfection, confluent 60 mm plates were split 1:10. Subconfluent
cells were then transfected as follows: 4 µg of DNA was diluted into
DMEM to a final volume of 150 µl, and then 20 µl of SuperFect
Transfection Reagent was added to the DNA solution. The samples were
incubated for 5-10 min before adding 1 ml of DMEM with 0.1%
penicillin-streptomycin and 10% FBS (cell growth medium). Cell growth
medium was removed from the cells and replaced with the transfection
solution. Two to 3 hr later, the transfection solution was removed, the
cells were rinsed three times with PBS, and then 5 ml of cell growth medium was added to the dishes. After 24 hr, cells were harvested, centrifuged to a pellet, and resuspended in 100-300 µl of cell growth medium (see below). Agar blocks containing cells were then prepared as follows: 2% low melting point agar in DMEM-F12 was added
to a 35 mm dish and allowed to gel. A 1 cm square was cut out and
removed, and the dish was placed on a warming block. Thirty-five microliters of cells and 35 µl of molten 2% agar in DMEM-F12
was added to this cavity, mixed, and allowed to gel. Small cubes were then cut from the agar and placed in cell growth medium and cultured overnight. Cell blocks were then rinsed in L15-glucose and cut into
smaller pieces (~300-400 µm cubes) before plating.
Preparation of collagen gel cocultures
Collagen was prepared from adult rat tails. Cocultures were set
up as follows: 900 µl of collagen solution was mixed with 100 µl of
10× MEM and 13-20 µl of a 1 M solution of sodium
bicarbonate and placed on ice. Twenty-five microliters of collagen was
pipetted onto the bottom of four-well dishes (Nunc, Naperville, IL) and allowed to gel. Retinal explants were then placed onto this base with a
Pasteur pipette, excess L15-glucose was removed, and 75 µl of
collagen was added on top. With forceps, retinal explants and
slit2-myc- or vector (mock)- transfected 293T cell
aggregates were positioned ~150-300 µm apart. The collagen was
then allowed to gel before adding 500 µl of DMEM-F12 containing
0.1% penicillin-streptomycin, 0.6% D-glucose,
2 mM glutamine, and 5% rat serum. Explants were cultured in a humidified 37°C, CO2
incubator for 1-1.5 d.
Immunocytochemistry
Collagen gel cocultures were fixed for at least 1 d in 4%
paraformaldehyde plus 1% glutaraldehyde at 4°C, rinsed numerous times in 0.1 M phosphate buffer, and then blocked for 2-4
hr in 10% lamb serum in PBS with 0.1% Triton X-100 (blocking buffer). Cocultures were then incubated 3-4 d at 4°C in mouse monoclonal anti- tubulin antibody (Amersham Pharmacia Biotech, Arlington Heights, IL), diluted 1:500 in blocking buffer, rinsed numerous times
in PBS, and incubated in Cy3-conjugated anti-mouse secondary antibody
(Amersham Pharmacia Biotech), diluted 1:1000 in blocking buffer for
2 d. Cultures were then rinsed in PBS and stored at 4°C.
Analysis of axon outgrowth in collagen gel cocultures
After fixation, retina 293T cell cocultures were labeled with an
anti- tubulin monoclonal antibody (see above) and photographed using
a 4× or 6.3× lens on a Nikon (Tokyo, Japan) Microphot fluorescence microscope. The micrographs were then scanned into Adobe Photoshop (Adobe Systems, San Jose, CA). Quantitation was done on the scanned images. All data were analyzed using Student's t test.
Number and length of axon bundles. On each image, a line was
drawn through the center of the retinal explant, dividing it in two
sectors, one facing toward and one away from the 293T cells (see Fig.
6). Axon bundles were counted in both sectors, and their lengths were measured.
Directional effects. To determine whether retinal axons turn
in response to Slit2, the axons angle of deviation away from the 293T
cells was measured in the half of the explant facing toward the 293T
cells (see Fig. 7A) as follows. A base line, perpendicular to the one dividing the explant in two sectors and to the surface of
the 293T cells, was drawn. Another line, the expected direction of
growth (Gx), was drawn through the center of the explant and the base of the axon bundle. A third line, representing the actual direction of growth (Ga) was then drawn through the base and
the tip of the axon bundle. The absolute value of the angle between the
baseline and Ga (AGa) was then subtracted from
the absolute value of the angle between the baseline and Gx
(AGx). The difference is negative when the axon bundle
extends away from the 293T cells and positive when it grows toward them.
Fasciculation. Scanned images were transferred into Scion
(Frederick, MD) Image (beta 3b). The optical density was measured as
average pixel values (0-256) in a circular area centered at a position
that corresponded to 75% of the length of the bundle from the
explant. The diameter of the circle was adjusted to fit the width of
the fascicle. All fascicles in the focal plane were measured,
regardless of whether they projected toward or away from the explant.
The background value of the image was measured as the average of
several areas in the collagen gel at similar distances from the explant
but devoid of axon bundles. The bundle values minus the mean background
value were averaged for each coculture, and the average per coculture
was calculated.
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RESULTS |
Expression of Slits in diencephalon
To determine whether the Slits may be involved in the controlling
the pathfinding and innervation patterns of RGC axons within the
diencephalon, we examined the expression patterns of the mammalian slit
homologs slit1, slit2, and slit3,
relative to the developing RGC axonal pathway. In situ
hybridizations were performed on coronal sections of E15 and E17 rat
brains, ages when RGC axons grow over the diencephalon and a repellent
activity for retinal axons is released by the hypothalamus and
epithalamus (Tuttle et al., 1998).
At E15, slit1 is strongly expressed in the ventromedial part
of ventral thalamus and throughout much of the hypothalamus, with
particularly high levels in the dorsal two-thirds (Fig.
2A). Slit2
is highly expressed in medial ventral thalamus, as well as in discrete
areas within the dorsal and ventral regions of hypothalamus (Fig.
2C). In addition, slit1 is expressed weakly (Fig.
2A) and slit2 is expressed strongly (Fig.
2C) in epithalamus and just lateral to the dorsal thalamic
ventricular zone. Neither slit1 (Fig. 2A)
nor slit2 (Fig. 2B) are expressed in the
lateral aspect of dorsal thalamus, a target of RGC axons.
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Figure 2.
Expression of the slits in
embryonic rat diencephalon. Coronal sections through the diencephalon
of E15 (A, C, E) and E17
(B, D, F) rat
brains showing slit1 (A,
B), slit2 (C,
D), and slit3 (E,
F) expression detected with
S35-labeled riboprobes. Dorsal is up.
Sections were stained with bisbenzimide. Each photo is a single
exposure using both dark-field and UV fluorescence illumination.
A, At E15, slit1 is expressed in the
ventromedial part of ventral thalamus, as well as throughout the dorsal
two-thirds of hypothalamus. slit1 expression is also
detected in medial dorsal thalamus and epithalamus. B,
At E17, slit1 continues to be expressed throughout most
of hypothalamus, as well as in medial ventral and dorsal thalamus, and
epithalamus. C, At E15, slit2 is highly
expressed in medial dorsal thalamus and at lower levels in epithalamus,
medial ventral thalamus, and in distinct parts of dorsal and ventral
hypothalamus. D, At E17, slit2 is most
highly expressed in the dorsal thalamic, epithalamic, and ventral
hypothalamic ventricular zones, as well as throughout ventral
hypothalamus. In contrast, slit3 is expressed at low or
nondetectable levels in diencephalon at both E15
(E) and E17 (F).
Arrowheads mark the optic tract. Scale bars, 200 µm.
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At E17, both slit1 (Fig. 2B) and
slit2 (Fig. 2D) continue to be highly
expressed within the hypothalamus, with slit2 expression highest in the ventral half. At this age, a small wedge of
slit1 expression is present in the lateral aspect of dorsal
thalamus (Fig. 2B). In addition, slit1 is
expressed strongly in epithalamus and in a thin band at the midline of
dorsal thalamus; slit2 expression is high in the epithalamic
and dorsal thalamic ventricular zones. In contrast, at both E15 (Fig.
2E) and E17 (Fig. 2F),
slit3 expression in the diencephalon is very low (e.g.,
ventral hypothalamus and dorsal thalamus, except its lateral aspect) or
nondetectable relative to slit1 and slit2.
These expression patterns of the Slits correlate with the fasciculation
and innervation patterns of RGC axons within the diencephalon (for
description, see Fig. 1). In summary, at the levels of the diencephalon
over which the optic tract courses, we find that the combined
expression of the Slits is high in the hypothalamus (high
fasciculation, restricted sparse innervation), declines to lower levels
in the lateral part of ventral thalamus (tract begins to
defasciculate, restricted modest innervation), declines further to
nondetectable levels throughout most of the lateral part of dorsal
thalamus (tract is defasciculated, heavy innervation), and then
increases to high levels in the epithalamus (high fasciculation and
tract turns caudally, no innervation). These findings are consistent
with a role for Slit1 and/or Slit2 as diencephalic repellents for RGC axons.
Expression of Slit receptors in retina
If slit1 and slit2 act as repellents for RGC
axons, we would expect that robo1 and/or robo2,
receptors for the Slits (Brose et al., 1999; Yuan et al., 1999), would
be expressed by RGCs. To test this possibility, in situ
hybridizations were performed on sections through E13, E15, and E17 rat
retinas, ages that cover the majority of the period of RGC axon growth.
At all ages examined, robo1 is expressed most strongly in
the retinal marginal zone (Fig.
3A-C). In addition, beginning
at E15, scattered cells in the RGC layer in central retina are well labeled (Fig. 3B,G). By E17,
robo1 expression is present at low levels throughout the
retina, with punctate dense labeling in the forming RGC layer,
indicative of scattered highly expressing cells (Fig. 3C).
robo2 is expressed in central retina at E13, with especially
high levels on the vitreal side, the location of the forming RGC layer
(Fig. 3E,D). Expression of
robo2 spreads peripherally such that at E17 robo2
is expressed throughout the retina, again with highest expression in
the forming RGC layer (Fig.
3F,H).
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Figure 3.
Expression of the Slit receptors,
robo1 and robo2, in embryonic rat retina.
Coronal sections of E13 (A, D), E15
(B, B', E,
E'), and E17 (C, F)
rat retinas showing robo1 (A-C)
and robo2 (D-F) expression
detected with S35-labeled riboprobes. Dorsal is
up. The sections are counterstained with bisbenzimide.
Each photo is a single exposure using both dark-field and UV
fluorescence illumination. B' and E' are
higher power views of the regions marked with an arrow
in B and E, respectively. At 13 (A), E15 (B, B'),
and E17 (C), robo1 is expressed in
the retinal marginal zone (arrowheads). Scattered cells
that appear to be highly expressing slit1 are detected
in the ganglion cell layer in central retina at E15
(arrows in B, B') and at
E17 (arrow in C). At E13
(D) and E15 (E,
E'), robo2 is expressed in central
retina, with highest levels in the ganglion cell layer
(arrows). It is also highly expressed in the epithelium
at E13 (D, arrowhead). At E17,
robo2 is expressed throughout the retina, with highest
levels in the ganglion cell layer (arrow in
F). OpN, Optic nerve. Scale bars,
200 µm.
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These data demonstrate that, at the time RGC axons are extending over
the diencephalon, robo2, and to a lesser extent
robo1, receptors for Slit1 and Slit2, are expressed in the
RGC layer. Given the high, uniform expression of robo2 in
the RGC layer, we conclude that most if not all RGCs express it.
However, because only a small proportion of cells in the RGC layer
highly express robo1, it is difficult to be certain that
they are RGCs because a sizeable proportion of cells in the adult RGC
layer are displaced amacrine cells (Jeon et al., 1998). In summary, our
findings suggest that Slit1 and/or Slit2, acting predominantly through
Robo2, may act as diencephalic repellents for RGC axons in
vivo.
Expression of Slits in retina
We also examined the expression of slit1,
slit2, and slit3 within the retina at E13, E15,
and E17. At all ages examined, both slit1 (Fig.
4A-C) and
slit2 (Fig. 4D-F) are most highly
expressed in the RGC layer. Expression is first detected at E13 in
central retina but spreads peripherally over time, apparently following the central to peripheral gradient of the generation of RGCs (Morest, 1970). In addition, a domain of high slit2 expression
surrounds RGC axons coursing through the optic nerve (Fig.
4E,F). In contrast, slit3 (Fig. 4G-I) is not expressed to any
significant degree in the retina at any of the ages examined. At E17,
slit1 exhibits a graded, high ventral to low dorsal
expression pattern. These data suggest that in vivo, Slit1
and Slit2, acting predominantly through Robo2, and to a lesser extent
through Robo1, may be involved in intraretinal development. In
addition, if both Robo and Slit proteins are present on RGC axons, they
might modulate interactions between RGC axons.
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Figure 4.
Expression of Slits in embryonic rat
retina. Coronal sections of E13 (A, D,
G), E15 (B, E,
H), and E17 (C, F,
I) rat retinas showing slit1
(A-C), slit2
(D-F), and slit3
(G-I) expression detected with
S35-labeled riboprobes. Dorsal is up.
The sections are counterstained with bisbenzimide. Each photo is a
single exposure using both dark-field and UV fluorescence illumination.
At E13 (A, D) and E15 (B,
E), slit1 (A,
B) and slit2 (D,
E) expression is highest in the ganglion cell layer in
central retina (arrows), but by E17, both
slit1 (C) and slit2
(F) are expressed throughout the RGC layer
(arrows). Note that slit1 expression is
more restricted to the ganglion cell layer than slit2
expression. Arrowheads in E and
F indicate expression of slit2 at the
optic disk and bounding the optic nerve (OpN).
slit2 expression at E17 shows a high ventral to low
dorsal graded pattern (C). slit3
expression is not detected in retina at E13 (G),
E15 (H), or E17
(I). Scale bars, 200 µm.
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Slit2 biases and inhibits retinal axon growth in collagen gels
To determine the effects of Slit2 on RGC axon growth, we
cocultured in three-dimensional collagen gels explants of retina at a
distance from aggregates of 293T cells transiently transfected with
slit2-myc cDNA or with vector cDNA as a control. Similar analyses were not done for Slit1 because we have not been able to
produce sufficient levels of Slit1 protein by transfection. Explants of
retina were prepared from E15 rat embryos, an age when RGC axons are
extending over the diencephalon in vivo and are responsive
to the hypothalamic and epithalamic repellent activities in
vitro (Tuttle et al., 1998).
Axon outgrowth from retinal explants is robust when cocultured with
mock-transfected cells (Fig.
5A-D), whereas when
cocultured with slit2-transfected cells (Fig.
5E-H), outgrowth is substantially decreased.
Overall, axon growth appears to be biased away from the cells, and
often bundles of axons originate on the explant side facing the
slit2-transfected cells (Fig. 5G). We
quantified several features of axon outgrowth in the cocultures,
including the bias in outgrowth, the length of axon bundles, and the
total number of axon bundles, using the scheme presented in Figure
6A. When retina is
cocultured with mock-transfected cells (n = 18), 35%
more axon bundles extend from the side facing toward the cells compared
with the side away from them (Fig. 6B). In contrast, when retina is cocultured with slit2-transfected cells
(n = 20), 30% fewer axon bundles emanate from the side
facing toward the cells (Fig. 6B). Comparison between
the two types of cocultures reveals that the ratio of toward to away is
decreased by ~50% in the Slit2 cocultures. Thus, Slit2 substantially
alters the distribution of axon outgrowth from retinal explants,
resulting in a biased outgrowth away from the source of Slit2. In
addition, Slit2 results in a decrease in the length of retinal axon
bundles. In cocultures with mock-transfected cells, axon bundles are
the same length on the explant sides toward and away from the cells, whereas in the Slit2 cocultures, they are 35% shorter on the side toward the slit2-transfected cells compared with the away
side (Fig. 6C). This decrease in the length of axon bundles
suggests that Slit2 slows the rate of retinal axon extension.
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Figure 5.
Axon outgrowth from retinal explants is biased
away from, and inhibited by, slit2-transfected cells.
Explants from E15 rat retinas were cocultured for 1-1.5 d in collagen
gels at a distance from aggregates of 293T cells transfected with human
slit2-myc (Slit2) or, as a control, with
the parental plasmid (Mock). Cocultures were
immunostained with an anti--tubulin antibody (see Materials and
Methods) and photographed under fluorescence illumination. The 293T
cells are to the bottom. Shown are a representative
series of explants to show the range and bias of axon outgrowth.
A-D, When cocultured with mock-transfected cells,
retinal axon outgrowth is robust and exhibits a modest bias toward the
cells. All 18 explants had outgrowth in the mock cocultures.
E-H, In contrast, when cocultured with
slit2-transfected cells, retinal axon outgrowth is
substantially decreased and is biased away from the cells. Five of 20 explants in the Slit2 cocultures had no outgrowth (data not shown). The
arrow in G marks axon bundles that arise
from the side of the explant facing the
slit2-transfected cells but extend away for them. Scale
bar, 200 µm.
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Figure 6.
Quantification of effects of Slit2 on biasing and
inhibiting retinal axon outgrowth in vitro.
A, Quantitation scheme. Cocultures were labeled with an
anti--tubulin antibody, and the number and length of axon bundles
were quantified in the hemiretina sector facing toward or away from the
293T cells. Quantitation was done blind to transfected cell type on 18 cocultures with mock-transfected 293T cells (575 axon bundles) and 20 cocultures with slit2-transfected 293T cells (92 axon
bundles). B, Axon outgrowth is biased away from
slit2-transfected cells. The ratio of the number of axon
bundles extending from the explant side facing the 293T cells compared
with the side facing away from the cells is decreased by ~50% in the
retina Slit2 compared with the retina mock cocultures
(p < 0.04; Student's t
test). C, Axon length is decreased by Slit2. The ratio
of the length of axon bundles on the side of the explant facing toward
compared with the side facing away from the cells is decreased by
~35% in the retina Slit2 compared with the retina mock cocultures
(p < 0.05; Student's t
test). D, Slit2 decreases overall axon outgrowth. The
total number of axon bundles extending from retinal explants is
decreased by 84% in the retina Slit2 compared with the retina mock
cocultures (p < 4 × 10 9; Student's t test).
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|
The overall axon outgrowth from retinal explants is also diminished in
the presence of slit2-transfected cells. Counts of the total
number of axon bundles emanating from retinal explants in the
cocultures shows that the number is decreased by 84% in the cocultures
with slit2-transfected cells compared with mock-transfected cells (Fig. 6D). Thus, Slit2 is a potent inhibitor of
retinal axon growth in vitro. The growth inhibition of Slit2
appears to be concentration-dependent because, on the explant side
facing the slit2-transfected cells in which the amount of
Slit2 protein should be greater, fewer axon fascicles emerge (Fig.
6B) and they are shorter (Fig. 6C).
Although retinal axon outgrowth is fasciculated in both sets of
cocultures, the axon bundles generally appear to be denser, that is
more tightly fasciculated, in the presence of Slit2 (Fig. 5). To assess
the degree of fasciculation, we digitally measured the optical density
of the axon bundles immunostained using an anti--tubulin antibody,
with the rationale being that more tightly fasciculated axon bundles
should exhibit a greater optical density (see Materials and Methods).
Retinal explants cocultured with mock-transfected cells had an average
pixel value of 100 ± 1.5 (on a scale of 0 to 256), whereas those
cocultured with slit2-transfected cells had an average pixel
value of 114.7 ± 5.9. This difference was highly significant
(p < 0.005; Student's t test). We
therefore conclude that retinal axons are more tightly fasciculated in
the presence of Slit2.
The findings described above demonstrate that Slit2 inhibits retinal
axon growth and alters the distribution of outgrowth from retinal
explants, resulting in a biased growth away from the source of Slit2.
To determine whether Slit2 also has a directional affect on retinal
axon growth, especially whether it repels retinal axons, we measured
the angle of deviation of axons away from slit2-transfected cells (Fig. 7A). We found no
significant difference in axon turning when retinal explants are
cocultured with slit2-transfected compared with
mock-transfected cells (Fig. 7B). Thus, although Slit2
biases and inhibits retinal axon growth in vitro, we are
lacking formal criteria that Slit2 is also a chemorepellent for retinal
axons.
View larger version (23K):
[in this window]
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|
Figure 7.
Quantitation of the effect of Slit2 on the
directional growth of retinal axons in vitro.
A, Quantitation scheme. Quantitation was done blind to
transfected cell type on 13 randomly selected cocultures of each type
(those with no outgrowth were not used); 575 axon bundles were analyzed
in the cocultures with mock-transfected cells, and 32 axon bundles were
analyzed in the slit2-transfected cell cocultures. The
axons angle of deviation away from the 293T cells was measured in the
"toward" hemiretina sector as follows: a line (Gx),
representing the expected direction of growth, was drawn through the
center of the explant and the base of the axon bundle. A second line
(Ga), representing the actual direction of growth, was
then drawn through the base and the tip of the axon bundle. The
absolute value of the angle between the base line (thick dashed
line) and Ga (AGa) was then
subtracted from the absolute value of the angle between the baseline
and Gx (AGx). The difference is negative
when the bundle extends away from the 293T cells and positive when it
extends toward them. B, Retinal axons are not repelled
by Slit2 in collagen gel cocultures. No significant difference is found
in the angle of deviation of axon bundles away from the 293T cells
between the retina mock and the retina Slit2 cocultures
(p = 0.22; Student's t
test).
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DISCUSSION |
Our previous work showed that the hypothalamus and epithalamus
release a soluble repellent activity for rat and mouse retinal axons
and that the distribution of this activity correlates with the
fasciculation and innervation patterns of RGC axons within the
embryonic diencephalon (Tuttle et al., 1998). Here, we presented evidence that the axonal chemorepellents Slit1 and Slit2 comprise the
diencephalic repellent activity. At the time RGC axons extend over the
diencephalon, slit1 and slit2 are expressed in
the hypothalamus and epithalamus but not in RGC target nuclei in dorsal
thalamus, and most if not all RGCs express the Slit receptor
robo2, and a limited number might express robo1.
In addition, our in vitro assays show that Slit2 is a potent
inhibitor of retinal axon growth. These findings, together with those
of Erskine et al. (2000) and Niclou et al. (2000), suggest that the
slits have crucial roles in RGC axon pathfinding from the eye to the midbrain.
Within the retina, we find that the Robo receptors and slit1
and slit2 are coexpressed in the RGC layer, with
slit1 exhibiting a high ventral to low dorsal graded
expression. Other axon guidance ligands and receptors are also
coexpressed in the retina, often in a graded manner in the RGC layer,
including EphA and EphB receptors and their ephrin-A and ephrin-B
ligands (Cheng et al., 1995; Kenny et al., 1995; Marcus et al.,
1996; Braisted et al., 1997; Holash et al., 1997; Monschau et al.,
1997). The graded expression of the ephrin-A ligands appears to
regulate the pool of functional EphA receptors on RGCs via receptor
phosphorylation and influence RGC axon guidance by enhancing the
gradient of functional receptors (Connor et al., 1998; Hornberger et
al., 1999). Although the role of the coincident expression of
the Slits and Robos in RGCs is undefined, it may serve to modulate
axon-axon interactions within the RGC axon pathway and thereby
influence the fasciculation of RGC axons and their responses to Slits
expressed along the pathway (see below).
It seems likely that robo2 is the primary mediator of RGC
axon response to the Slits, because it is expressed at much higher levels in the RGC layer than robo1. However, the relative
contributions of Slit1 and Slit2 in influencing RGC axon growth within
the diencephalon is less clear. We have shown that Slit2 inhibits
retinal axon growth and is expressed in the hypothalamus and
epithalamus at the time RGC axons extend over the diencephalon.
slit1 is also highly expressed in the hypothalamus and
epithalamus in a pattern that partially overlaps with that of
slit2, but thus far we have not tested its function.
However, Slit1 is a repellent for olfactory axons (Yuan et al., 1999)
and migrating olfactory progenitor cells (Wu et al., 1999). Therefore,
it is likely that both Slit1 and Slit2 are components of the
hypothalamic and epithalamic repellent activities.
The expression patterns of slit1 and slit2, and
our findings that RGC axon growth is both inhibited and more tightly
fasciculated in the presence of slit2, relate well to the fasciculation
and innervation patterns of RGC axons within the diencephalon. RGC axons are tightly bundled and invade sparsely if at all parts of the
diencephalon that express the slits, i.e., the hypothalamus and
epithalamus, and are defasciculated and innervate parts that do not
express the slits, i.e., their target nuclei in the lateral part of
dorsal thalamus. The fasciculation pattern of the RGC axons in relation
to domains of ;slit expression is consistent with the demonstration
that retinal axons are able to extend over an inhibitory substrate
in vitro but in a highly fasciculated manner (Bray et al.,
1980). Thus, one possible role for the Slits expressed in the
hypothalamus and epithalamus is to promote the fasciculation of RGC
axons. The defasciculation of RGC axons over dorsal thalamus may be
allowed by its lack of Slit expression and could be enhanced by a
Slit-mediated repulsion between RGC axons because RGCs appear to
express Robos and Slits. The potential action of the Slits on RGC axon
fasciculation may be similar to the influences of the ephrin-A axonal
repellents on the fasciculation of cortical axons (Winslow et al.,
1995) and retinal axons (McLaughlin and O'Leary, 1999).
Because the hypothalamus expresses slit1 and
slit2, it may seem counterintuitive that RGC axons enter the
brain at its ventral aspect and extend dorsally over its surface. It is
likely though that the Slit-mediated inhibition is balanced by
activities that promote and direct RGC axon growth, for example the
chemoattractant netrin-1 (De la Torre et al., 1997; Deiner et al.,
1997). This idea is supported by analyses of RGC axon pathfinding in
mice deficient for the homeodomain protein Vax1 (Bertuzzi et al.,
1999). vax1 is expressed in discrete populations of
cells of the optic stalk, nerve and chiasm, and ventral hypothalamus.
In vax1 mutants, RGC axons fail to form a chiasm and very
few penetrate the brain. Instead, at their entry point in ventral
hypothalamus, RGC axons form a "Probst-like" bundle that is capped
by an aberrant dense cluster of cells, which are normally
vax1+. Thus, a domain that is
normally growth permissive for RGC axons, is inhibitory in the mutants:
a change that correlates with a dramatic decrease in
netrin-1 expression but maintained expression of
slit1 (Bertuzzi et al., 1999). Similarly,
netrin-1 expressed at the optic disk and in the optic nerve
may neutralize any inhibitory effects on RGC axon growth exerted by
Slit2 expressed at the optic disk and bounding the nerve (Fig.
3E,F), which may help
explain why the optic nerve is reduced or absent in netrin-1
mutant mice (Deiner et al., 1997). In normal development, the
expression of slit2 surrounding the optic nerve may serve to
channel RGC axons from the eye to the brain.
RGCs innervate their targets via branches that form along RGC axons at
specific locations within the optic tract (Bhide and Frost, 1991; Simon
and O'Leary, 1992a,b). Our findings suggest that the Slits may prevent
RGC axons from forming and extending branches into nontarget tissues.
This could be achieved in two ways. First, the Slits could directly
inhibit branch formation and extension, similar to apparent role of
ephrin-A in inhibiting retinal axon branching (Roskies and
O'Leary, 1994; O'Leary et al., 1999). This action is the
opposite of that of the N-terminal fragment of Slit2, which promotes
the elongation and branching of dorsal root ganglia axons in
vitro (Wang et al., 1999). A second way would be a result of
Slit-mediated modulation of axon fasciculation; the defasciculation of
RGC axons over dorsal thalamus should facilitate their branching and
innervation of their target nuclei, whereas their branching over the
hypothalamus should be diminished by their tight fasciculation. This
suggestion is supported by the finding that the branching of
corticospinal axons is delayed and diminished by the selective removal
of polysialic acid from their surfaces, which results in an increase in
their fasciculation (Daston et al., 1996).
As RGC axons approach the domain of slit1 and
slit2 expression in the epithalamus, they not only
refasciculate but also turn and grow caudally toward the SC, suggesting
that the Slits may act in vivo as repellents for RGC axons.
Although our in vitro data indicate that Slit2 inhibits
retinal axon growth, our analysis of axon turning failed to provide
evidence that Slit2 is a chemorepellent for retinal axons. Although we
previously referred to the unidentified hypothalamic and epithalamic
activities as repellents for retinal axons (Tuttle et al., 1998), we
did not perform a quantitative analysis of axon turning as we have done
for Slit2. However, we did find that retinal axon growth is biased away
from the hypothalamus and epithalamus in vitro, similar to
our finding here that retinal axon growth is biased away from
slit2-transfected cells. Although our data do not reveal a
directional, i.e., repellent, effect of Slit2 on retinal axons, the
Slits might nonetheless act in vivo as repellents and
promote RGC axon turning. We suspect that axon turning in the collagen
cocultures is impeded by the fasciculation of rat retinal axons, which
is enhanced in the presence of Slit2. This situation has similarities
to the effect of chemoattractant netrin-1 on spinal commissural axons:
although netrin-1 can cause the turning of spinal axons under some
conditions in vitro (Kennedy et al., 1994; Hong et al.,
1999), in collagen gels it induces the outgrowth of axon bundles but
does not affect their directionality (Kennedy et al., 1994).
We conclude that Slit1 and Slit2 play a role in the pathfinding of RGC
axons and the patterning of their projections within the diencephalon
by regulating the fasciculation of RGC axons within the optic tract,
preventing them or their branches from invading nontarget tissues and
steering them toward their distal target, the SC. It will be of
interest to determine the requirement of the Slits for the development
of RGC projections and to identify other molecules that cooperate with
the Slits to define the path of RGC axons through the diencephalon and
promote their innervation of their dorsal thalamic targets.
| |
FOOTNOTES |
Received Feb. 29, 2000; revised April 17, 2000; accepted April 18, 2000.
This work was supported by National Institutes of Health Grant EY07025
(D.D.M.O'L.). C.G. and M.T.L. are Investigators of the Howard Hughes
Medical Institute. T.R. is supported by the Swedish Brain Foundation
and the Swedish Medical Research Council.
Drs. Ringstedt and Braisted contributed equally to this work
Correspondence should be addressed to Dennis D. M. O'Leary,
Molecular Neurobiology Laboratory, The Salk Institute, 10010 North Torrey Pines Road, La Jolla, CA 92037. E-mail: doleary{at}salk.edu.
| |
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TOP
ABSTRACT
INTRODUCTION
