1 Department of Biology, University of Massachusetts, Amherst, MA 01003,
USA
2 Department of Neurobiology and Anatomy, University of Utah, Salt Lake City, UT
84112, USA
Author for correspondence (e-mail:
karlstrom{at}bio.umass.edu)
Accepted 3 June 2005
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SUMMARY |
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Key words: Zebrafish, Cyclopamine, Morpholino, Axon guidance, POC, Chiasm, Gfap, Glia, hedgehog, slit, you-too
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Introduction |
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In the vertebrate forebrain, Slit-Robo-mediated repulsion is crucial for
the appropriate dorsoventral position of the optic chiasm
(Rasband et al., 2003). In
mice lacking both Slit1 and Slit2 function, retinal axons cross the midline in
multiple locations around the true chiasm
(Plump et al., 2002
).
Similarly, zebrafish lacking astray (robo2) function show
aberrant retinal axon growth across the midline
(Hutson and Chien, 2002
;
Karlstrom et al., 1996
).
Analyses of these two phenotypes led to the proposal that gradients of Slit
expression descending toward the optic chiasm channel Robo-expressing
axons within the appropriate pathway
(Hutson and Chien, 2002
;
Plump et al., 2002
;
Rasband et al., 2003
;
Richards, 2002b
). Slit1 and
Slit2 have also been shown to channel callosal axons across the cortical
midline in the mouse forebrain (Bagri et
al., 2002
; Shu et al.,
2003d
). It is not known whether the Robo/Slit system also
influences formation of the first forebrain commissures in vertebrates, the
postoptic commissure (POC) and the anterior commissure (AC). However, the fact
that commissures form in astray (robo2) mutants indicates
that Robo2 alone is not necessary for POC and AC midline crossing
(Karlstrom et al., 1996
).
The cellular growth substrate for retinal axons as they cross the midline
consists of neurons, neuroepithelial cells and glia
(Marcus and Easter, 1995;
Mason and Sretavan, 1997
).
CD44/SSEA1-positive neurons border the optic nerve in and across the mammalian
midline, and may provide a barrier that directs RGC axons to cross the midline
in the correct position (Jeffery,
2001
; Marcus et al.,
1995
; Marcus and Mason,
1995
; Mason and Sretavan,
1997
). Other identifiable midline cells include a radial glial
`palisade' (Misson et al.,
1988
) that helps to guide ipsilaterally projecting retinal axons
(Mason and Sretavan, 1997
).
More dorsally, midline glial populations that make up the `glial sling',
`glial wedge', and indusium griseum have all been implicated in midline
crossing in the corpus callosum (Richards,
2002a
; Shu et al.,
2003a
; Shu et al.,
2003b
; Shu et al.,
2003c
; Shu and Richards,
2001
; Silver et al.,
1982
; Silver and Ogawa,
1983
). Little is known about how midline cells influence the
formation of the forebrain commissures.
The zebrafish forebrain provides an easily accessible system for the study
of chiasm and commissure formation. Prior to chiasm formation, the POC forms
in the diencephalon, while the AC forms more anterodorsally in the
telencephalon (Bak and Fraser,
2003; Wilson et al.,
1990
). RGC axons grow across the ventral midline in close
proximity to the POC (Wilson et al.,
1990
). Pioneering growth cones of the POC and optic nerve grow
superficially (Wilson and Easter,
1991
; Wilson et al.,
1990
), and their growth substrate primarily consists of
poorly-characterized neuroepithelial cells and basal lamina
(Burrill and Easter, 1995
;
Wilson and Easter, 1991
;
Wilson et al., 1990
). It is
currently unknown whether these neuroepithelial cells are neural and/or glial
precursor cells, neurons or glia (Burrill
and Easter, 1995
), although some express Glial Fibrillary Acidic
Protein (Gfap) and have a radial glial morphology, and so appear to be
astroglia (Marcus and Easter,
1995
). These Gfap+ cells come in direct contact with POC and optic
axons at the midline at 48 hours post-fertilization
(Marcus and Easter, 1995
).
Several achiasmatic zebrafish mutants have been identified in which
forebrain axons fail to cross the midline and most RGC axons project
ipsilaterally (Karlstrom et al.,
1997; Karlstrom et al.,
1996
). Many of these ipsilateral/midline mutants affect Hedgehog
(Hh)-mediated midline patterning, with detour (dtr) and
you-too (yot) encoding the Hh-responsive transcription
factors Gli1 and Gli2, respectively
(Karlstrom et al., 1999
;
Karlstrom et al., 2003
).
Complex regulation and processing of the vertebrate gli genes results
in either repression or activation of Hh target gene expression
(Koebernick and Pieler, 2002
),
and both dtr(gli1) and yot(gli2) mutations block the ability
of cells to activate Hh signaling
(Karlstrom et al., 2003
). The
cellular and molecular basis for the axon guidance defects in the Hh/midline
mutants is currently unknown. Besides playing an important role in directing
cell differentiation in the forebrain
(Nybakken and Perrimon, 2002
),
Hh proteins directly attract commissural axons to the midline in the mouse
spinal cord (Charron et al.,
2003
; Ogden et al.,
2004
) and may repel RGC axons in the chick forebrain
(Trousse et al., 2001
). It is
thus possible that the midline guidance errors in Hh pathway mutants are due
to a loss of direct Hh-mediated axon guidance. Alternatively, they may be
indirectly caused by cell specification defects in the forebrain
(Karlstrom et al., 2003
;
Sbrogna et al., 2003
).
Although much has been learned about midline guidance in the forebrain, many fundamental questions remain. First, what is the molecular and cellular architecture of the midline growth substrate that leads to proper commissure and then chiasm formation? Second, how do Slits influence the first midline-crossing axons in the forebrain, those of the POC and AC? Finally, given the loss of midline crossing in Hh pathway mutants, does Hh directly or indirectly affect axon guidance in the forebrain? We show that Gfap+ cells express slit1a and form a `bridge' across the midline prior to commissure and chiasm formation, and that Slit molecules help to establish the POC. We show that Hh is not needed directly to guide commissural and retinal axons near the midline, but instead is necessary to establish the proper patterning of midline glia and the patterned expression of slit guidance cues. We propose a model in which slit1a expressed by a glial bridge provides a substrate for axon crossing at the midline, whereas slit2 and slit3 repulsion helps to channel growth cones into commissures.
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Materials and methods |
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Antibodies
Anti-acetylated tubulin (IgG2b monoclonal, Sigma) was used at a dilution of
1:800 to label axons (Wilson et al.,
1990). A polyclonal anti-goldfish-Gfap antibody (generous gift of
Sam Nona, University of Manchester, UK)
(Nona et al., 1989
) was used
at 1:400 to label astroglial cells (Marcus
and Easter, 1995
). F59 (IgG1 monoclonal, Developmental Studies
Hybridoma Bank) is specific for slow muscle myosins in chicken
(Crow and Stockdale, 1986
), and
diluted at 1:10 preferentially labels slow muscle fibers in zebrafish
(Devoto et al., 1996
).
Secondary antibodies from Jackson ImmunoResearch Laboratories were diluted as
follows: FITC goat anti-mouse IgG, 1:200; Cy5 goat anti-mouse IgG, 1:200;
TRITC goat anti-Rabbit, 1:500; FITC goat anti-Rabbit, 1:200.
Immunocytochemistry and in situ hybridization
Embryos were fixed in 4% formaldehyde (Ted Pella) for 2 hours at room
temperature and antibody-labeled, as previously described
(Karlstrom et al., 1999) with
several modifications. Briefly, embryos were dehydrated, incubated in 100%
acetone for 7 minutes at 20°C, rehydrated in PBS+0.2% Triton X-100
(PBS-Tx), digested with 10 µg/ml Proteinase K for 3-5 minutes depending on
age, washed for 3x5 minutes in PBS-Tx, and incubated in blocking
solution (PBS-Tx-2%BSA-5%NGS-1%DMSO) for 1 hour at room temperature. All
primary and secondary antibodies were diluted in blocking solution and applied
for 2 hours at room temperature.
Digoxigenin-labeled anti-sense mRNA probes were synthesized using SP6/T7/T3
Dig RNA Labeling Kits (Roche). The probes used were pax2a
(Krauss et al., 1991),
shh (Krauss et al.,
1993
), ptc1 (Concordet
et al., 1996
), sema3d
(Halloran et al., 1998
),
slit1a (Hutson et al.,
2003
), slit2 and slit3
(Yeo et al., 2001
), robo1,
robo2 and robo3 (Lee et al.,
2001
), and robo4
(Park et al., 2003
). A
standard in situ hybridization protocol
(Jowett, 1997
) was modified to
enable multi-antibody labeling following mRNA probe hybridization. Briefly,
embryos were fixed with 4% formaldehyde (Ted Pella) for 2 hours at room
temperature. Anti-acetylated tubulin and anti-Gfap primary antibodies were
added with anti-DIG antibodies. Following a Fast Red (Roche) color reaction,
embryos were washed for 2x15 minutes with PBS-0.1% Tween20, and then for
3x15 minutes with PBS-Tx, blocked, and incubated in secondary
antibodies. Embryos were never post-fixed.
Embryos were cleared in 75% glycerol and examined using a Zeiss LSM510
laser-scanning confocal microscope at a magnification of 40x or
63x. For lateral views of the forebrain, eyes were removed.
Three-dimensional stacks were acquired using multitracking for double- and
triple-labeled embryos to ensure sequential acquisition and preclude signal
crossover. Stacks ranged from 20 µm to 50 µm in Z-distance with constant
pinhole settings of 105.7 µm (1.0 Airy unit) for all channels and an
(optimal) interval of 0.4 µm. Images are maximum intensity projections
unless otherwise noted. For consistency, throughout this paper we use
`anterior' to mean closer to the telencephalon, and `posterior' for closer to
the diencephalon, despite changes in orientation due to flexure of the nervous
system.
Cyclopamine treatment
At several different time points, wild-type embryos were treated with 100
µM or 200 µM Cyclopamine (Toronto Chemical) in embryo medium + 0.5% EtOH
(Incardona et al., 1998) at
28.5°C. No effect on the assayed phenotypes was seen in control embryos
incubated in embryo medium + 0.5% EtOH. Thirty embryos were treated in 2 ml
volumes in 12-well plates.
Morpholino and mRNA microinjection
Translation-blocking (Nasevicius and
Ekker, 2000) zebrafish slit1a (GACAA CATCC TCCTC TCGCA
GGCAT), slit2 (CATCA CCGCT GTTTC CTCAA GTTCT) and slit3
(TATAT CCTCT GAGGC TGATA GCAGC) morpholinos (MOs, GeneTools) were kept as 10
mg/ml stocks in Danieau's solution (58 mM NaCl, 0.7 mM KCl, 0.4 mM
MgSO4, 0.6 mM Ca(NO3)2, 5 mM Hepes, Phenol
Red) and diluted in Danieau's solution. Injections of 400-500 pl were made
into the yolk of one- to four-cell stage embryos obtained from wild-type
crosses or yotty119 (gli2DR) heterozygous intercrosses.
slit1a MOs were injected at 1 ng, slit2 or slit3 at
2-4 ng, with no phenotypic differences seen between these doses of
slit2 or slit3 MOs. Co-injections of slit2 and
slit3 MOs used 2.5 ng of each. Injected embryos were fixed at 28 hpf
for triple antibody labeling of axons, glial cells and slow muscle fibers.
Overexpression of Slit genes can cause convergent extension defects
(Yeo et al., 2001
), as can
high doses of slit1a, slit2 or slit3 MOs (L.D.H. and
C.-B.C., unpublished). To rule out the possibility that defects in axon
guidance were due to indirect morphogenetic effects, each injected embryo was
stained with F59, and only those that displayed normal body shape, somite
formation and patterning of the superficial slow-muscle layer were used to
score axon and glial defects. Homozygous yot (gli2DR) mutant embryos
were identified by the loss of embryonic slow muscle fibers at 28 hpf
(Stickney et al., 2000
).
Zebrafish shh was transcribed from the T7TSshh plasmid
(Ekker et al., 1995
) and
microinjected as previously described
(Barresi et al., 2000
).
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Results |
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Previous studies reported that Gfap+ cells appear in the midline region
only after forebrain commissures have formed, suggesting that astroglia
probably do not guide axons across the midline
(Marcus and Easter, 1995).
However, our confocal analysis indicates that Gfap+ cells span the midline of
the forebrain at 15 hpf, well before the appearance of any axons in the brain
(Fig. 2)
(Wilson et al., 1990
). Gfap+
cells span the diencephalic midline in the future POC region
(Fig. 2A), and by 18 hpf Gfap+
cells span the midline where both the POC and AC will form
(Fig. 2B). As development
proceeds, Gfap+ cells become increasingly restricted to the locations of the
AC and POC (Fig. 2C, bracket;
Fig. 2D-F). The first POC axons
begin crossing the midline at approximately 23-24 hpf
(Bak and Fraser, 2003
) in
association with Gfap+ cells (Fig.
2C, arrow). RGC axons (Fig.
2E,F, arrowheads) then grow towards the midline close to, but not
in direct contact with, POC axons (Fig.
2E,F, bottom arrow) (Burrill
and Easter, 1995
). A small cluster of Gfap+ cells extends
anteriorly from the glial bridge directly in the position where RGC axons
cross the midline (Fig. 2F,
arrowhead).
Hedgehog signaling does not directly affect axon guidance, but does pattern midline glia
The expression of shh adjacent to the POC and optic chiasm
(Fig. 1D), combined with the
loss of midline axon crossing in Hh mutants (reviewed by
Russell, 2003) and the recent
findings that Shh can directly guide midline crossing axons in mouse
(Charron et al., 2003
) and
chick (Trousse et al., 2001
),
suggest that Shh may directly guide POC and/or RGC axons near the midline of
the forebrain. To test this, we used cyclopamine (CyA) to pharmacologically
block Smoothened-mediated Hedgehog signaling starting just prior to the times
when POC (22 hpf) or RGC (27 hpf) axons grow toward the midline
(Fig. 3A). In embryos treated
with 100 µM CyA from 27 hpf to 36 hpf, RGC axons crossed the midline
normally, forming a wild-type chiasm (Fig.
3E,F). Similarly, the POC and optic chiasm were completely formed
in embryos treated with CyA from 22 hpf to 36 hpf
(Fig. 3D,F). To verify that CyA
was completely blocking Hh signaling at these ages, we examined late-onset
expression of the Hh receptor and the Hh target gene patched1
(ptc1) in the fin bud, which normally begins at 32 hours of
development (Concordet et al.,
1996
; Lewis et al.,
1999
). Treatment with 100 µM CyA from 27 hpf to 36 hpf led to a
complete loss or major reduction of ptc1 expression in the fin bud
(data not shown), whereas 200 µM CyA treatment eliminated all ptc1
expression in the fin bud, indicating a complete block of Hh signaling
(Fig. 3B-F; upper insets)
(Neumann et al., 1999
). Higher
doses of CyA (200 µM) applied between 22-36 hpf also led to a reduction in
the number of RGCs, consistent with a role for Hh in RGC specification
(Neumann and Nuesslein-Volhard,
2000
). However, both POC and RGC axons correctly crossed the
midline in these treated embryos (Fig.
3D,E; lower inset).
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To better understand the relationship between Hh signaling and formation of
the midline glial bridge, we examined yot (gli2DR) mutants that have
a repressed transcriptional response to Hh without a major loss of midline
tissue (Karlstrom et al.,
1999). In yot(gli2) mutants, Gfap+ cells were reduced at
the midline and many cells were aberrantly positioned in the region between
the two commissures (Fig. 4).
This disorganization of Gfap+ cells was apparent before axons are present
(Fig. 4A,D). As seen in the
15-36 hpf CyA-treated embryos, misguided POC axons in yot mutants
were predominantly associated with mis-positioned Gfap+ cells
(Fig. 3C,
Fig. 4F). To assess whether
ectopic expression of Hh would disrupt axon and glial guidance, we injected
Shh-encoding mRNA at the two-cell stage. The POC was reduced and
defasciculated following global Shh overexpression
(Fig. 3G-I, arrows) and Gfap+
cells were similarly expanded along the anteroposterior axis
(Fig. 3G-I, brackets).
Together, these results suggest that Hh signaling is required for the proper
patterning of glial bridges in the forebrain, and that axon errors seen in Hh
pathway mutants may be the indirect effect of disruptions in this axon
substrate.
Hh signaling patterns the forebrain expression of slit1a, slit2, slit3 and sema3d
To better understand how Hh signaling is involved in establishing the
commissural axon growth substrate, we next examined axon guidance molecule
expression in yot (gli2DR) mutants. Given the role for
Robo/Slit-mediated growth cone repulsion in RGC axon guidance
(Hutson and Chien, 2002;
Hutson et al., 2003
), we first
examined the expression of slit1a and slit2/3 during
commissure formation (Fig. 5).
Consistent with the surround repulsion guidance mechanism seen for retinal
axons (Rasband et al., 2003
),
slit2 and slit3 were expressed adjacent to the commissures,
with expression being absent from regions where commissural axons and glial
bridges cross the midline (Fig.
5A,C). In yot(gli2) mutants, slit2 and
slit3 were expanded, with expression filling the commissural regions
normally devoid of slit2/slit3 expression
(Fig. 5B,D). In contrast to
slit2/slit3, slit1a was expressed in the preoptic area and
AC region, with expression overlapping both POC and AC axons
(Fig. 5E). A subset of Gfap+
cells in the glial bridge and the majority of the Gfap+ cells in the AC region
expressed slit1a (Fig.
5G,H, data not shown), although none of these cells expressed
either slit2 or slit3. slit1a expression was reduced in
yot (gli2) mutants in the preoptic area
(Fig. 5F), unlike the expansion
of expression seen for slit2/slit3.
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Slit1a functions distinctly from Slit2 and Slit3 in POC formation
To test whether and how Slit proteins regulate glial bridge and commissure
formation, we next used morpholino antisense oligonucleotide (MO) injections
to reduce Slit function in wild-type and mutant embryos. Inhibition of
slit2, slit3, and slit2/slit3 together in wild-type
embryos resulted in defasciculation of the POC, with many distinct axon
bundles crossing throughout the pre- and post-optic areas
(Fig. 6A-D,K). To semi-quantify
these POC defects, we generated a crossing index of POC formation based on a
scale of 1 to 7 (with 7 being an ideal wild-type commissure and 1 being no
commissure) for individual embryos injected with different MOs
(Fig. 6L). Inhibition of
slit2, slit3, and slit2/slit3 together in wild type
embryos significantly disrupted POC formation
(Fig. 6L, purple bars). Besides
leading to defasciculation, slit2 or slit2/slit3 MO
injections caused some POC axons to wander into inappropriate regions of the
forebrain in most embryos (Table
1). slit3 MO and slit1a MO injections did not
significantly increase axon wandering (Fig.
6A-D,K; Table 1).
In some slit MO-injected embryos the AC was variably reduced, whereas
in others it appeared to be unaffected, despite clear POC defects
(Fig. 6A-E,K).
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Inhibition of Slit2/Slit3, but not Slit1a, rescues POC formation in yot (gli2DR) mutants
Because injection of slit2/slit3 MOs led to axon defects
consistent with the known roles for Slit proteins as axon repellents, we
wondered whether the expansion of slit2/slit3 expression in
yot (gli2DR) mutants could be responsible for the inability of POC
and RGC axons to grow across the midline. To answer this question, we assayed
POC formation in yot (gli2DR) mutants injected with slit MOs
(Fig. 6F-I,K). slit2
and slit3 single MO injections partially rescued POC formation in
yot (gli2DR) mutants (Fig.
6L), whereas injection of both slit2 and slit3
MOs together rescued POC formation to a level that was not significantly
different from that seen in slit2/slit3 MO-injected
wild-type embryos. yot (gli2DR) mutations led to a significant
increase in POC axon wandering, which was further increased after injection of
slit2 and/or slit2/slit3 MOs
(Fig. 6;
Table 1). Together, these
results support the idea that Slit2 and Slit3 act as repellent guidance cues
for POC axons, and that their misexpression in yot (gli2DR) mutants
is a major cause of POC defects seen in this mutant. By contrast,
slit1a MO injection did not rescue POC formation in yot
mutants, and instead caused a further reduction in axon crossing
(Fig. 6J-L). These knockdown
results support the idea that Slit1a functions distinctly from Slit2/Slit3,
with Slit1a being necessary for POC axons to grow across the midline and
Slit2/Slit3 acting as repellent guidance cues.
Inhibition of Slits leads to disorganization and spreading of the glial bridge
Given the slit2/slit3 expression on either side of the
forebrain glial bridges (Fig.
5), and the correlation between Gfap+ cell position and axonal
defects at the midline in yot (gli2DR) mutants (Figs
1,
2,
4), we next wondered whether
Slit molecules might play a role in positioning glial cells in the forebrain.
Indeed, loss of Slit1a, Slit2, Slit3, or both Slit2 and Slit3 function in
wild-type embryos led to an anteroposterior spreading of Gfap+ cells in the
pre- and post optic areas (Fig.
6A-E). This spreading was apparent at 22 hpf, prior to midline
axon growth in the forebrain (Fig.
6A,D, insets).
Glial cells responded more drastically to Slit knockdown when we injected slit MOs into yot (gli2DR) mutants. In this case, injection of slit2, slit3 and slit2/slit3 MOs often led to a complete restoration of the glial bridge across the midline in the POC region (Fig. 6A,F-I). The extent of glial bridge rescue correlated with rescued POC formation. In contrast to Slit2 and Slit3, knocking down Slit1a function did not rescue glial bridge formation in yot (gli2DR) mutants (Fig. 6F,J). Again, in all slit MO-injected yot (gli2DR) embryos, aberrantly positioned axons appeared to be associated with Gfap+ cells.
Overlapping and distinct expression of Robo receptors in commissural neurons and midline glia
To determine which cell populations are potentially capable of responding
directly to Slit cues, we determined whether commissural neurons and midline
glial cells express one or more of the Roundabout (Robo) family of Slit
receptors. Double labeling using different robo in situ probes in
combination with the anti-tyrosine hydroxylase (TH) antibody that labels
commissural neurons of the nucleus of the tract of the POC (ntPOC)
(Holzschuh et al., 2001)
showed that robo1, robo2 and robo3 are expressed in the
majority of ntPOC cells, but that robo4 is not expressed in cell
bodies in the ntPOC region (data not shown).
To determine whether Robo receptors are expressed in cells of the diencephalic glial bridge, we triple labeled 28 hpf embryos with the anti-AT antibodies, anti-Gfap antibodies, and robo1, robo2, robo3 or robo4 in situ probes (Fig. 7). This triple labeling revealed that Gfap+ cells of the glial bridge appear to express robo1 and robo3, but not robo2 (Fig. 7A-L). Interestingly, robo4 expression overlaps with only a subset of Gfap+ cells in the anterior portion of the glial bridge; those Gfap+ cells that come in direct contact with POC axons and RGC axons (Fig. 7M-P). robo4 expression was not seen in Gfap+ cells ventral to the commissure (Fig. 7M). Thus, both glial cells and commissural neurons express different combinations of Robo receptors, and may therefore be capable of responding directly to Slit guidance signals.
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Discussion |
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A distinct function for Slit1a in commissure formation
Several lines of evidence show that Slit1a
(Hutson et al., 2003) may
function distinctly from Slit2/Slit3 during commissure formation in the
zebrafish forebrain. First, slit1a expression is complementary to
slit2/slit3 expression and unlike
slit2/slit3, commissural axons grow in
slit1a-expressing regions (Fig.
5). Second, expression of slit1a is distinctly affected
by the loss of Hh signaling, with slit1a expression being reduced
rather than expanded in yot (gli2DR) mutants
(Fig. 5F). Third, blocking
Slit1a function in wild-type embryos led to a marked reduction in POC crossing
rather than the defasciculation seen in slit2/slit3
morphants (Fig. 6). Fourth,
reducing Slit1a function in yot (gli2DR) mutants failed to rescue
axon crossing in yot (gli2DR) mutants and instead led to an even
further decrease in POC axon crossing (Fig.
6). Interestingly, retinal axons in the optic tract also navigate
across domains of slit1a expression, and knocking down Slit1a
function causes retinal axon guidance errors that are not easily explained by
removal of a repulsive signal (L.D.H. and C.-B.C., unpublished).
|
Slit molecules help to position a glial bridge
Our studies have uncovered a novel role for Slit molecules in the
positioning of astroglia in the vertebrate forebrain
(Fig. 8). We show that Gfap+
cells are positioned in slit1a-expressing regions that do not express
slit2 or slit3 (Fig.
5), and that loss of Slit2/Slit3 (but not Slit1a) function results
in an expansion of the glial bridge in the POC region prior to axon growth
(Fig. 6). In addition, glial
cell defects in yot (gli2DR) mutants were largely rescued following
slit2/slit3 MO knockdown, suggesting that the
mis-positioning of glial cells in yot (gli2DR) mutants is due to the
expanded zones of slit2/slit3 expression in these embryos
(Figs 6,
8). Such a role for Slits in
cell positioning has been shown for a variety of cell types, including glial
cells and mesodermal cells (for reviews, see
de Castro, 2003;
Piper and Little, 2003
). In
the Drosophila CNS, Slit/Robo signaling is required to position glial
cells that later serves to guide longitudinal and commissural axons (reviewed
by Hidalgo, 2003
). A role of
Slits in cell migration has also been shown in mouse, where Slit1 is required
cell autonomously for subventricular neuroblasts to migrate in vitro
(Nguyen-Ba-Charvet et al.,
2004
). The fact that a subset of Robo receptor genes are expressed
in zebrafish forebrain glial cells (Fig.
7, Fig. 8C)
indicates that Robo-mediated Slit repulsion might directly prevent glial cells
from occupying slit2- and slit3-expressing domains, perhaps
by influencing cell migration in a way that is analogous to growth cone
repulsion.
Our confocal analysis of Gfap expression during forebrain development
revealed that two glial bridges span the midline prior to AC, POC and RGC
formation, and that these Gfap-expressing cells are present at the right
position to provide cues for midline axon growth. These astroglial cells send
short fibers perpendicular to the pial surface into the forebrain, and the
developing commissure forms as a ribbon along these fibers (see Movies 1, 2, 3
in the supplementary material) (Marcus and
Easter, 1995). A previous immuno-EM study used the same Anti-Gfap
antibodies to show that retinal and commissural axons directly contact Gfap+
radial glia-like fibers (Marcus and
Easter, 1995
). These anatomical data strongly suggest that the
glial bridge provides the growth substrate for the first commissural axons to
cross the midline (Fig. 8A).
Disruption of the glial bridge correlates with midline axon crossing errors,
and glial cell defects precede axon defects both in yot (gli2DR)
mutants (Figs 3,
4) and in
slit2/slit3 morphants
(Fig. 6D). These functional
data suggest that these glial cells provide a positive or at least permissive
cue for midline axon crossing (Fig.
8A,B). A positive role for glial cells is also supported by the
observation that aberrantly growing axons appear to preferentially grow along
mis-positioned Gfap+ cells rather than following their normal pathway or an
alternative non-glial route. It will now be important to directly test whether
glial cells are both necessary and sufficient for midline guidance of
commissural and retinal axons.
An indirect role for Hh in commissure and glial bridge formation
shh is expressed at the right place and time to influence both
cell fate and axon guidance at the midline in the diencephalon
(Fig. 1E,F, Fig. 8D,E). Secreted Shh was
recently shown to play a direct role in attracting axons to the ventral
midline of the mouse neural tube (Charron
et al., 2003). In addition, in vitro and in vivo experiments in
chick suggest that Shh may inhibit RGC axon growth
(Trousse et al., 2001
).
However, by using CyA to block Smoothened-mediated Hh signaling at different
times, we show that Hh signaling does not appear to directly guide commissural
or retinal axons in the zebrafish forebrain
(Fig. 3). The rescue of POC
formation in yot (gli2DR) mutants by injection of
slit2/slit3 MOs (Fig.
6) also indicates that Hh signaling does not play a major role in
the midline guidance of POC axons. Instead, Hh signaling appears to affect
axon guidance indirectly through its role in patterning of the midline
(Dale et al., 1997
;
Karlstrom et al., 2003
;
Mason and Sretavan, 1997
),
including the formation of the glial bridge and the regulation of axon
guidance-molecule expression (Figs
3,
4,
5).
How can we reconcile our CyA results with the direct inhibitory affect seen
for Shh in chick retinal explants? One possibility is that this particular
guidance function for Shh has not been evolutionarily conserved between fish
and chick. However, this seems unlikely given the high degree of conservation
in other Hh-mediated developmental processes
(Ingham and McMahon, 2001),
and the similar expression of Hh in the chiasm region in chick and fish
(Trousse et al., 2001
)
(Fig. 1E,F). Alternatively,
loss-of-function approaches may be more informative than ectopic expression
studies regarding the direct requirement for Hh in vivo. Thus, although
retinal axons may be able to directly respond to ectopic Hh signals in vitro,
direct Hh-mediated growth-cone guidance may not play a significant role in the
context of other guidance cues present in the chiasm region. The finding that
overexpression of shh in zebrafish not only disrupted AC, POC and RGC
axon crossing but also disrupted glial bridge formation
(Fig. 3G-I) is also consistent
with a primary role for Hh signaling in patterning the axon growth
substrate.
Rescue of POC formation in yot (gli2DR) mutants following slit2/slit3 MO injections, combined with the expansion of slit2/slit3 expression in Hh pathway mutants, suggests that the indirect effect of Hh on glial and axon guidance in the diencephalon is largely mediated by Slit proteins. Hh may regulate Slit gene expression directly, or through its regulation of cell fates. By contrast, loss of Hh or Slit function had little effect on AC formation, indicating that other patterning and guidance molecules are sufficient to pattern the ventral telencephalon and establish the AC. These data are consistent with the known role for Hh in ventral forebrain patterning, and begin to shed light on the downstream molecular targets that result in proper glial position and neural connectivity in this region.
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ACKNOWLEDGMENTS |
---|
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/16/3643/DC1
* Present address: Biology Department, Smith College, Northampton, MA 01063,
USA
Present address: Biology Department, Williams College, Williamstown, MA
01267, USA
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