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First published online 2 October 2008
doi: 10.1242/dev.023663
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Departamento de Neurobiología Molecular Celular y del Desarrollo, Instituto Cajal, CSIC and CIBER de Enfermedades Raras (CIBERER), Avda. Dr Arce 37, Madrid 28002, Spain.
* Author for correspondence (e-mail: bovolenta{at}cajal.csic.es)
Accepted 9 September 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Growth cone, Morphogen, Chiasm, Boc, Transcriptional regulation
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Sanchez-Camacho et al.,
2005), in many cases exerting more than one function on the same
cell type. A prime example of these multiple activities of Hh is in the
generation, differentiation and axon growth of vertebrate retinal ganglion
cells (RGCs), which are responsible for transmitting visual information from
the retina to the brain (Amato et al.,
2004).
In non-mammalian vertebrates, FGF signalling triggers the onset of
neurogenesis that generates the first neurons, the RGCs
(Martinez-Morales et al.,
2005). Newborn RGCs begin to express and release Shh, a member of
the Hh family, which further propagates the differentiation wave, establishing
a stable source of Shh within the eye
(Esteve and Bovolenta, 2006).
Once generated, RGCs extend an axon that exits the eye through the optic disc
and navigates towards the ventral midline of the diencephalon, where axons
from the two eyes meet to form the optic chiasm. In animals with panoramic
vision, such as birds and fish, the entire axonal population from one eye
projects to the contralateral side of the brain
(Erskine and Herrera, 2007;
Mason and Sretavan, 1997).
Although Shh is strongly expressed along the ventral midline of the entire
CNS, its expression is downregulated at the preoptic region just when retinal
axons are approaching, whereas it is maintained at the chiasm borders
(Trousse et al., 2001).
Retroviral-mediated ectopic expression of Shh in the embryonic chick preoptic
area impairs growth of the retinal fibres
(Trousse et al., 2001).
Similarly, inappropriate expression of Shh at the optic chiasm, as observed in
the Pax2a (Pax2.1; noi) zebrafish mutant,
correlates with abnormal ipsilateral turning of RGC axons
(Macdonald et al., 1995),
indicating that local downregulation of Shh might be crucial for RGC midline
crossing. Consistently, RGC growth cones respond with a reversible and
re-inducible collapse upon repeated Shh applications, leading to the
hypothesis that Shh diffusing from the chiasm borders might serve to restrict
anteroposteriorly the growth of visual axons
(Trousse et al., 2001).
Furthermore, lowering or increasing Shh activity in the chick retina causes
disorganisation of the fibre layer, indicating that adequate intraocular
levels of the morphogen are also directly or indirectly required to direct RGC
axon growth towards the optic disc (Kolpak
et al., 2005).
Thus, RGCs are cells that secrete and perceive Shh, and their axons extend
from the retina to the midline between two different Shh sources: the RGCs
themselves and the preoptic area at the midline. Shh, however, is transported
along the RGC axons (Traiffort et al.,
2001), which presumably secrete the morphogen along their path, as
detectable levels of Shh protein have been found in optic nerve extracts
(Wallace and Raff, 1999).
Consistent with an axonal release, pharmacological and genetic interference
with RGC-derived Shh disrupts the development of a specialised group of glial
cells at the optic disc (Dakubo et al.,
2003; Morcillo et al.,
2006), as well as the proliferation and migration of
oligodendrocyte precursors (Merchan et
al., 2007) and the differentiation of astrocytes in the optic
nerve (Wallace and Raff,
1999). It is therefore logical to assume that RGC growth cones
must be exposed to Shh even before reaching the midline.
Whether RGC-derived Shh contributes to axonal outgrowth remains unexplored.
Likewise, whether mammalian RGC axons respond to Shh has not been analysed.
This is a relevant question because there seem to be some species-specific
differences between mice, fish and chick in Shh-mediated retinal
differentiation (Esteve and Bovolenta,
2006), although the absence of contralateral projection in
Pax2-null mice - in which, as in fish mutants, Shh is abnormally
expressed at the optic chiasm (Torres et
al., 1996) - favours a conserved role for Shh at the chiasm
midline. Furthermore, in mammals, both the specification and the midline
behaviour of a subset of RGCs differ from those of fish and birds in that a
proportion of axons originating in the temporal region of the retina do not
cross the midline but instead project ipsilaterally, assuring binocular vision
(Erskine and Herrera, 2007;
Mason and Sretavan, 1997).
Establishment of this ipsilateral projection requires the expression of Zic2,
a zinc-finger transcription factor that is specifically expressed in
ipsilateral but not contralateral projecting RGCs (I-RGCs and C-RGCs,
respectively) (Herrera et al.,
2003; Pak et al.,
2004), and the activity of the EphB and ephrin B signalling
proteins, which are localised to the I-RGC and chiasm, respectively
(Williams et al., 2003).
Whether Zic2-positive RGCs also express and sense Shh is unclear.
Here we have analysed whether Shh provides guidance information to the mouse RGC axons in their growth from the eye to the chiasm, and whether there are differences between the I- and C-RGC axonal populations, uncovering in the process differential non-autonomous (from the chiasm area) and cell-autonomous (from the RGC) functions of Shh.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Animals
were handled according to Spanish (RD 223/88) and European (86/609/ECC)
regulations.
In situ hybridisation and immunohistochemical procedures
Mouse embryos were fixed in 4% paraformaldehyde (PFA) in PBS (PFA/PBS)
overnight at 4°C and sectioned by vibratome (40 µm) or cryostat (20
µm) in the coronal plane. Sections were hybridised with the following
digoxigenin-labelled probes using standard protocols: Shh, Ptc1, Ptc2,
Hip1, Boc, Gli1, Gli2 and Gli3. Immunostaining of tissue
sections and explant cultures was performed by standard protocols using the
following antibodies: anti-Shh (5E1; ascitic liquid 1:200; DSHB), anti-Zic2
(1:10,000), anti-Isl2 (1:5000), anti-Gfap antiserum (Dako; 1:2000),
anti-tubulin βIII (Tuj1; Tubb3) (Promega; 1:1000), anti-SSEA-1 (Fut4)
(MC-480; 1:500), anti-Pax2 (Zymed Laboratories; 1:2000), anti-GFP (Molecular
Probes; 1:1000) and Alexa Fluor 488- or 594-conjugated fluorescent secondary
antibodies (Molecular Probes; 1:1000).
Retinal explants and collapse assays
Ventrotemporal (VT, predominantly containing I-RGCs) or dorsonasal (DN,
containing C-RGCs) explants from E14.5 retinas were embedded in collagen gel
matrix in the presence or absence of soluble or bead-immobilised Shh (1
µg/ml) as described (Trousse et al.,
2001). In some cases, cyclopamine (10 µM, Toronto Research
Chemicals, Toronto, Canada) was added to the medium. After 48 hours, explants
were fixed in 4% PFA and stained with anti-Tuj1 antibody or Alexa Fluor
594-conjugated phalloidin (Molecular Probes; 1:40). Neurite outgrowth in the
presence of different concentrations of Shh (0.25, 0.50, 1, 2, 4 and 6
µg/ml) was determined in DN and VT microexplants plated on glass coverslips
coated with poly-D-lysine (10 µg/ml; Sigma) and laminin (10
µg/ml; Invitrogen) and grown for 24 hours as above. To determine Shh
transport, VT and DN explants were cultured for 24 hours on glass coverslips
(as above) and immunostained with the 5E1 antibody. Dorsal root ganglia and
floor plate explants were used as negative and positive controls,
respectively, for the immunostaining. For collapse assays, retinal explants
established as above were treated after 24 hours with the following agents:
Shh (1 µg/ml); SAG, a Smo agonist (0.3 µM); cyclopamine (5-10 µM); or
the anti-Shh blocking antibody (5E1; 1:500, 1:1000). After 30 minutes,
explants were fixed in 4% PFA and 11% sucrose at 37°C for 30 minutes, then
stained with phalloidin. Cycloheximide (25 µM; Sigma), an inhibitor of
protein synthesis, or actinomycin D (1 µg/ml), a transcription inhibitor,
was bath-applied 10 minutes before the addition of the above agents. Three
independent experiments performed in quintuplicate were analysed for each
condition.
Statistical analysis
Data were collected using the image analysis software AIS 6.0 (Analytical
Imaging Station, Imaging Research, Ontario, Canada) and quantified using SPSS
v15.0 (SPSS, Chicago, IL). To determine the extent of neurite outgrowth in
collagen gel experiments, the area of the explant was subtracted from the
total area occupied by Tuj1-immunopositive staining and normalised to explant
size. Average neurite outgrowth was expressed as the mean ± s.e.m. in
pixels. Statistical significance was calculated using an unpaired Student's
t-test. For explants grown on glass coverslips, the area of outgrowth
was determined using one-way ANOVA plus posthoc test (Dunnet test). For
collapse assays, the area (µm2) and number of filopodia in
growth cone confocal images (taken using a Leica TCS LS; Leica, Heidelberg,
Germany) were evaluated using ImageJ 1.38 (NIH). Statistical significance was
determined using the one-way ANOVA plus posthoc test (Dunnet test). Growth
cones were classified into two different categories according to morphology:
(1) `spread' morphology, when the core of the growth cone was wide with
lamellipodia and three or more filopodia; (2) `collapsed' morphology, when the
core of the growth cone was reduced with three or fewer filopodia. For each
condition, the percentage of the growth cone belonging to one or other
category was determined in three independent experiments performed in
quintuplicate. Statistical differences were established using a contingency
table with Fisher's exact test (treatment versus type of growth cone,
significance level 
0.05).
In vivo interference with Shh activity
Hybridomas secreting the anti-Shh 5E1 IgG, or control hybridomas [IgGs
against the GAG viral capsid protein (DSHB)], were prepared as described
(Merchan et al., 2007).
Timed-pregnant E13.5 C57/BL6 mice were anesthetised by inhalation (isoflurane
1-1.5% O2) and 5-10 µl of a 2x106 cells/ml
suspension were injected directly into the amniotic sac. After 5 days, embryos
were fixed and processed for immunostaining or DiI labelling. In other
experiments, axons were visualised by retinal electroporation of a pCAGGS-EGFP
construct (see below) after injection of the hybridoma into the amniotic
sac.
DiI labelling of retinal projections
For unilateral anterograde labelling of retinal projections, small DiI
crystals (Molecular Probes) were placed onto the optic nerve head of the right
eye from fixed E18.5 embryos as described
(Godement et al., 1987). For
retrograde labelling, the preoptic area was exposed and a crystal of DiI,
briefly dipped in Triton X-100, was inserted unilaterally in the initial
portion of the optic tract. After 7-15 days at 23-37°C in PFA/PBS, the
proximal visual pathway was analysed. The relative width of the optic chiasm
was determined by measuring the distance from the rostralmost to the
caudalmost DiI-labelled retinal axons at the chiasm midline using Instat 3.0
(GraphPad Software, San Diego, CA) and one-way ANOVA. In the case of
retrograde labelling, contralateral and ipsilateral retinas were dissected and
flat mounted. The extent of labelling was determined as the percentage of the
total area that was occupied by retrograde DiI-labelled retinal axons.
In utero electroporation
Shh transduction in the mouse retina was blocked by in utero
electroporation of a bicistronic vector containing
Ptc1
loop2 and a nuclear-targeted GFP
(Ptc1loop2-IRES-GFPnls), a
construct that abrogates Shh-dependent activation of the signalling cascade
(Briscoe et al., 2001). Nuclear
GFP expression allowed determination of the position of electroporated cells.
The trajectory of targeted axons was visualised by co-electroporation of
pCAGGS-EGFP. E13.5 pregnant mice were anesthetised and electroporated as
described (Garcia-Frigola et al.,
2007). Embryos were allowed to develop until E16.5.
Quantitative PCR
E16.5 retinas electroporated as described above were dissected and only
GFP-positive tissue collected. Total RNA was extracted and purified following
standard protocols (RNeasy Mini Kit, Qiagen). RNA was reverse-transcribed
using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems).
The quantitative PCR was performed using Power SYBR Green Master Mix following
the manufacturer's protocol (Applied Biosystems) in a 25 µl reaction
mixture that contained 2 µl of cDNA. Primers were designed using
PrimerExpress (Applied Biosystems) with a melting temperature of 60°C. In
all cases, the length of the amplicons was between 100 and 150 bp. Primer
sequences are available upon request. The Ptc1 3' UTR primers
were designed to detect only endogenous Ptc1 levels after
Ptc1loop2 overexpression. The threshold
cycle (Ct) values were measured by the ABI Prism 7500 Sequence
Detection System. After an initial 2 minutes at 50°C and 10 minutes at
95°C, the thermal profile consisted of 40 cycles of 15 seconds at 95°C
and 1 minute at 60°C. Triplicate samples from two independent experiments
were measured and averaged. Expression levels were normalised to 18S rRNA
levels and analysed by the unpaired Student's t-test.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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), and Boc (biregional Cdon-binding protein), a Robo-related
Shh-binding protein (Tenzen et al.,
2006). Although expression of some of these components has been
reported in the mouse retina (Wallace and
Raff, 1999; Wang et al.,
2005), it is not clear whether they are distributed differently
among the I- and C-RGCs.
Zic2-positive I-RGCs are generated at E14.5 in the ventrotemporal
(VT) crescent of the retina (Herrera et
al., 2003). Isl2-positive C-RGCs instead occupy the
entire retina (Pak et al.,
2004). Double labelling with GFP and Zic2 antibodies of E14.5
retinal sections from Shh::GFPCre/+ embryos, in which GFP expression
mimics endogenous Shh mRNA distribution
(Harfe et al., 2004), showed
that the entire population of differentiated C-RGCs expresses Shh, in
agreement with Shh mRNA distribution, whereas the vast majority of
I-RGCs do not (Fig. 1A,C-E).
This difference was still observed at E16.5 (not shown), indicating that a
lack of Shh expression does not imply ongoing I-RGC differentiation.
Conversely, Boc, which together with Smo mediates Shh chemoattraction on
precrossing commissural axons (Okada et
al., 2006), was expressed in the I-RGCs but not in the C-RGCs
(Fig. 1G,I). Low Boc expression
was also detected in the remaining retinal neuroepithelium. Similarly,
Ptc2 mRNA was detected in a subpopulation of RGCs in the VT crescent
and in neuroblasts dispersed throughout the retinal neuroepithelium
(Fig. 1F,H). In contrast to
this differential distribution, Ptc1 and Hip1, which seems
to participate in the Shh-mediated chemorepulsion on postcrossing commissural
axons (Bourikas et al., 2005),
were homogeneously distributed in all RGCs and in the remainder of the retinal
neuroepithelium (Fig. 1B,J).
Gli1-3 expression was either restricted to the neuroblast layer
(Gli1; not shown) or homogeneously expressed in all RGCs
(Gli2, Fig. 1K;
Gli3, not shown).
Shh inhibits neurite outgrowth from C-RGCs but promotes that from I-RGCs
The differential expression of Shh receptors in the mouse I- and C-RGCs and
their different trajectory at the chiasm midline suggested that their axons
might respond differently to Shh exposure. To test this, explants from E14.5
VT (I-RGC) and dorsonasal (DN) (a C-RGC source) retinas were grown on laminin
for 24 hours in the presence or absence of Shh (1 µg/ml). Under control
conditions, VT and DN explants extended numerous neurites that occupied
similar areas of outgrowth (Fig.
2). Upon Shh addition, VT explants significantly increased their
growth area, whereas DN explants did not
(Fig. 2). Similar results were
obtained when retinal explants were grown in collagen gels for 48 hours (see
Fig. S1A in the supplementary material).
Shh activity on chick retinal axons may depend on protein concentration
(Kolpak et al., 2005). Our
data could therefore reflect a differential sensitivity of the VT and DN
retinal explants to Shh concentration. To test this, VT and DN explants were
exposed to a range of Shh doses (0.25-6 µg/ml). Notably, the extent of
outgrowth from VT explants was significantly increased at high concentrations
(Fig. 2A,C), whereas that from
DN explants was reduced at low Shh concentrations
(Fig. 2A,B), indicating a
differential activity of Shh on I-versus C-RGCs. These differences were not
due to changes in RGC differentiation because Shh treatment did not modify the
levels of Zic2 and Isl2 (real-time PCR, not shown).
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). If mouse C-RGC neurites behave as chick RGC axons, a
similar behaviour should be expected in the presence of a focal source of Shh.
Indeed, Shh-soaked beads (1 µg/µl) placed at 
100 µm from the DN
explants did not deflect the growth of the fibres (not shown), but
considerably reduced neurite length as compared with controls (see Fig. S1B in
the supplementary material). This effect was reversed by the addition of
cyclopamine, a Smo activity inhibitor
(Frank-Kamenetsky et al.,
2002). Similarly, the direction of outgrowth from VT explants was
not modified by the presence of Shh-soaked beads but, in contrast to DN
explants, the length of their neurites was not affected (see Fig. S1B in the
supplementary material), possibly owing to an insufficient local concentration
of the morphogen.
Blockade of Shh alters the distribution of RGC axons at the optic chiasm
We have previously hypothesised that Shh present at the chiasm borders
might serve to anteroposteriorly restrict the midline growth of contralateral
projecting axons (Trousse et al.,
2001). If Shh plays a similar function in mice, we would expect
that neutralising Shh activity would scatter contralateral projections at the
optic chiasm without interfering with I-RGC fibres, which never cross the
SSEA-1-positive midline cells (Mason and
Sretavan, 1997), thus causing them to turn into the ipsilateral
optic tract at a distance from the Shh source
(Fig. 3E).
To test this prediction, we injected hybridoma cells producing anti-Shh
blocking antibodies directly into the amniotic sac of mouse embryos, a proven
method to interfere with endogenous Shh activity
(Merchan et al., 2007).
Embryos were injected at E13.5, when retinal fibres just approach the optic
chiasm (Bovolenta and Mason,
1987), and analysed at E18.5, when the majority of fibres should
have reached their targets. Complete unilateral Dil-filling of the optic nerve
head of uninjected embryos (n=6) or control hybridoma-injected
embryos (n=7) revealed a normal trajectory: retinal axons extended
toward the midline, where the majority crossed and entered the contralateral
optic tract, while only a small proportion projected into the ipsilateral
tract (Fig. 3A,C,E). A few
fibres were observed extending into the contralateral optic nerve, as reported
previously (Plump et al.,
2002). Notably, significant differences were observed in
anti-Shh-injected embryos (n=11): the overall region occupied by
DiI-labelled fibres at the midline was enlarged, fibres appeared less
fasciculated and many wandered, extending in an aberrant posterior direction
(Fig. 3B,E). Indeed, there was
a 30% increase in the midline scattering of the fibres in anti-Shh-treated
embryos compared with untreated or control-treated embryos, as determined by
measuring the region occupied by DiI-labelled axons at the level of the chiasm
(Fig. 3D). Thus, Shh activity
is normally needed to constrain and fasciculate the growth of contralateral
projecting visual fibres at the chiasm.
Furthermore, the number of axons that invaded the contralateral optic nerve and that grew ipsilaterally was significantly increased (Fig. 3B,E). Owing to the considerable number of axons that projected ectopically, it was difficult to quantify the precise number of fibres that took one of the abnormal trajectories in these preparations. To assess the origin of the fibres that invaded the ipsilateral optic tract and thereby determine possible alterations in the behaviour of I-RGC axons, we performed retrograde unilateral labelling from the optic tract and analysed the distribution of DiI-labelled fibres in both the ipsilateral and contralateral retinas. If the growth of I-RGC axons was affected by anti-Shh treatment (slower growth or misprojections) then a differential labelling in the VT quadrant of the ipsilateral retina was expected. However, no differences were observed in this quadrant when control (n=4) and treated (n=7) ipsilateral retinas were compared (Fig. 3F,G and see Fig. S2G,H in the supplementary material). By contrast, an abnormal number of backfilled fibres was present, particularly in the DT quadrant of the ipsilateral retina (Fig. 3G and see Fig. S2G,H in the supplementary material), indicating that a number of axons deriving from C-RGCs were aberrantly projecting to the ipsilateral optic tract in anti-Shh-treated embryos. Together, these data explain the apparent increase in the number of ipsilateral projecting fibres observed with anterograde labelling and suggest that interference with Shh mostly affects the behaviour of C-RGC axons at the midline.
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). However, at E13.5, when the hybridomas were injected, Shh
activity should no longer be required in the preoptic-hypothalamic region
(Manning et al., 2006).
Accordingly, treatment with Shh-neutralising antibodies did not perturb the
morphology of the chiasm as shown by immunostaining of midline glial and
neuronal cells with Gfap and SSEA-1 antibodies (see Fig. S2C-F in the
supplementary material), supporting a direct link between the interference
with Shh activity and the observed alterations in the RGC axon trajectory.
Alterations in the levels of Shh signalling cause transcription-dependent changes in RGC growth cone morphology
In addition to the defects described above, we observed an abnormally
frequent number of growth cones accumulating in the chiasm and both optic
tracts (Fig. 3B; see Fig. S2A,B
in the supplementary material). Furthermore, fewer fibres appeared to reach
the optic tract in the anti-Shh-treated embryos than in the controls
(Fig. 3A-C), suggesting slower
growth of the fibres. Because C-RGCs express Shh
(Fig. 1A), this source of
morphogen could contribute to the advance of the growth cones even before they
reach the midline. If this were the case, Shh should be transported and
released by the RGC growth cones, as previously suggested
(Wallace and Raff, 1999;
Traiffort et al., 2001).
Accordingly, floor plate cells (a positive control) as well as axons and
growth cones from DN explants, but not those from VT and dorsal root ganglia
(a negative control) explants, showed a clear accumulation of Shh
immunolabelling (Fig. 4A,B; see
Fig. S3 in the supplementary material). Furthermore, if RGC-derived Shh
contributes to the movement of growth cones, neutralisation of Shh activity
should modify their morphology. To test this, we used the growth cone collapse
assay, which uses growth cone morphology as a readout of the treatment
activity (Kapfhammer and Raper,
1987). VT and DN explants were cultured on laminin for 24 hours
and then treated with different reagents for 30 minutes, fixed and analysed.
Under these conditions, the majority of untreated retinal growth cones
exhibited a widespread morphology, whereas 24.7% (in VT, n=409) to
31.5% (in DN, n=426) presented a `collapsed' morphology (see
Materials and methods for definition) (Fig.
4C). Incubation with cyclopamine alone significantly increased the
number of collapsed growth cones (VT, n=121; DN, n=110)
compared with vehicle-treated explants (not shown). This effect was evident in
both DN and VT explants, whereas Shh neutralisation with antibodies was
effective only in VT explants (Fig.
4D,E), possibly reflecting a more efficient neutralisation in VT
explants, in which the endogenous levels are expected to be lower owing to the
coexistence of Shh-negative (I-) and Shh-positive (C-) RGCs. Conversely,
treatment of the growth cones with exogenous Shh (VT, n=146; DN,
n=163) or with the Smo agonist SAG (VT, n=208; DN,
n=135) decreased the number of growth cones with collapsed
morphology, although this effect was more evident in the growth cones from DN
explants (Fig. 4D,E).
Activation of the Shh signalling cascade ultimately leads to gene
transcription. However, it is unclear whether Shh activity on growth cones
requires this nuclear event or whether it might simply rely on local changes
in secondary messengers, such as cAMP, the growth cone levels of which are
modified upon Shh exposure (Trousse et
al., 2001). To address this, we tested whether the addition of
actinomycin D, a transcription inhibitor, modified the response to Shh
signalling activation in the collapse assay. In both DN and VT explants the
addition of actinomycin D alone, 10 minutes prior to ligand addition, slightly
increased the number of collapsed growth cones (by 10%) as compared with
untreated explants. Addition of SAG increased whereas exogenous Shh did not
significantly modify this level of collapse, implying that the presence of
actinomycin D abrogated the ability of Shh/SAG to reduce growth cone collapse
(Fig. 4D,E). Addition of
cycloheximide, a translation inhibitor, had similar or stronger effects
(Fig. 4D,E), a possible
reflection of protein synthesis being locally active in response to a number
of guidance cues (VanHorck and Holt,
2008).
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loop2, which dominantly represses Shh signal
transduction (Briscoe et al.,
2001). If RGC-derived Shh is cell-autonomously required to direct
axon extension, abrogation of signal transduction should cause defects in RGC
axon outgrowth before growth cones reach the chiasm.
pCAGGS-Ptc1loop2-IRES-GFPnls
together with pCAGGS-EGFP (n=8) or pCAGGS-EGFP
alone (n=5) were electroporated in the eye of E13.5 embryos, which
were then analysed at E16.5. In all embryos, EGFP-positive transfected cells
occupied the central portion of the retina and corresponded to
Isl2+ C-RGCs (Fig.
5C), whereas Zic2+ I-RGCs never showed EGFP expression
(Fig. 5A,B), indicating that
their capacity to sense Shh was not affected. In the
Ptc1loop2-EGFP electroporated
tissue, the endogenous mRNA levels of Ptc1, Ptc2 and Hip1,
which are transcriptional targets of Shh signalling
(Chuang and McMahon, 1999;
Goodrich et al., 1996;
Pearse et al., 2001), were
decreased compared with controls, indicating that the transgene effectively
blocked signal transduction in the retina
(Fig. 5D).
Normally, RGC axons grow in the fibre layer with a radially oriented,
organised pattern that converges at the optic disc, where a specialised group
of Pax2+ glial cells provide guidance information to the axons
(Dakubo et al., 2003;
de la Torre et al., 1997;
Morcillo et al., 2006). This
pattern was clearly visible in flat-mount or frontal sections of EGFP-control
retinas at E16.5 (Fig.
6A,B,D,E), when most of the growth cones from the transfected RGCs
have long passed through the disc. In
Ptc1loop2-transfected retinas, the
centripetal orientation of the axons at the optic disc was lost despite a
normal organisation of the Pax2+ glial cells
(Fig. 6F). In all the
Ptc1loop2 embryos analysed, many
EGFP-positive axons wandered, either growing parallel to the disc
(Fig. 6C,F) or away from it,
invading the peripheral retina (Fig.
6G-I). Several growth cones were still visible within the optic
disc area (Fig. 6C), suggesting
slower growth of the fibres. Similar alterations were observed in
EGFP-electroporated fibres when Shh activity was neutralised by the injection
of 5E1 (anti-Shh) hybridomas (see Fig. S4 in the supplementary material).
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loop2 embryos analysed, a few fibres
turned sharply in the mid-optic nerve to grow dorsally, a route never observed
in control embryos (Fig. 7A-C).
In spite of these alterations, the axons of a large number of the
Ptc1loop2-EGFP transfected RGCs
reached the chiasm area. Here, a number of axons turned into the ipsilateral
side of the brain (Fig. 7D),
although the electroporations hit only the Isl2+ C-RGCs. The
remaining fibres reached the midline, but wriggled or turned back in a
disorganised growth pattern (Fig.
7E,F). Together, these observations strongly support the notion that transduction of Shh signalling is continuously required along the proximal visual pathway to foster and direct axon growth (Fig. 7G,H).
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Deshpande et al.,
2001; Gering and Patient,
2005; Kato et al.,
2004; Kolpak et al.,
2005; Merchan et al.,
2007; Testaz et al.,
2001; Vokes et al.,
2004). In most of these examples, cells respond to Shh secreted by
an adjacent signalling tissue. Here, we provide evidence that, in addition to
this mechanism, proper navigation of mammalian C-RGC growth cones in the
proximal visual pathway depends on Shh that is endogenously produced by the
C-RGCs themselves, highlighting a novel aspect of Shh-mediated motility
control. We also show that, in contrast to C-RGCs, I-RGCs do not express Shh
and, at least in vitro, respond differently to this morphogen. Because this
behaviour correlates with the specific expression of Boc and
Ptc2 in I- but not C-RGCs, we hypothesise that differential
activation of Shh signalling components might contribute to the divergence of
I- from C-RGCs in the embryonic mouse retina.
|
).
Downregulation of Shh expression in the optic recess area coincides with the
arrival of pioneer RGC axons at the chiasm midline
(Trousse et al., 2001).
Conversely, abnormally expanded expression of Shh in this region correlates
with aberrant retinal axon growth
(Macdonald et al., 1995;
Torres et al., 1996;
Trousse et al., 2001). Shh
represses the growth of chick RGC axons in vitro and induces growth cone
collapse (Trousse et al.,
2001).
Confirming and extending these studies, we show here that neutralisation of
Shh function causes abnormal growth of mouse C-RGC axons at the ventral
forebrain. Defasciculation, aberrant projection to the contralateral optic
nerve and ectopic caudal extension of visual fibres at the midline are the
most prominent abnormalities, providing strong evidence that Shh present at
the chiasm borders constrains retinal fibre growth to the contralateral side
of the brain, as we previously hypothesised
(Trousse et al., 2001).
In zebrafish mutants in which components of the Hh signalling pathway are
affected, RGC axons fail to cross the midline
(Culverwell and Karlstrom,
2002). Detailed analysis of these mutants and the treatment of
wild-type zebrafish embryos with cyclopamine demonstrated that axon guidance
defects were secondary to the Shh-mediated specification of midline glial
cells as well as to the control of Slit expression
(Barresi et al., 2005;
Culverwell and Karlstrom,
2002), a potent repellent for RGC axons
(Erskine et al., 2000;
Niclou et al., 2000;
Ringstedt et al., 2000).
Although in our experiments anti-Shh-producing cells were injected when Shh
expression is already downregulated and no longer needed in the preoptic area
(Manning et al., 2006;
Trousse et al., 2001), we
cannot entirely exclude the possibility that some of the observed defects
might be secondary to subtle patterning alterations. However, we strongly
favour a direct effect of midline-derived Shh on mouse visual fibre growth,
for a number of reasons. Shh addition changes growth cone morphology in
short-term in vitro assays causing net differences in the overall outgrowth
after 24 or 48 hours. In anti-Shh-treated embryos, cells positive for Gfap and
SSEA-1, which are necessary for visual fibre sorting at the chiasm
(Mason and Sretavan, 1997),
are formed normally. In addition, Slit1- or Slit2-null mice
have no apparent visual phenotype, whereas double-knockout mice are
characterised by an additional anteriorly positioned ectopic chiasm
(Plump et al., 2002), a
phenotype clearly different from that observed in the anti-Shh-treated
embryos. Similarly, the genetic inactivation or perturbation of a number of
other molecules known to influence midline crossing or sorting of visual
fibres, including ephrin B2, CD44 and Nrcam
(Lin and Chan, 2003;
Nakagawa et al., 2000;
Williams et al., 2006;
Williams et al., 2003), causes
defects that do not mimic those observed in the anti-Shh-treated embryos. This
argues that Shh inactivation does not act by altering the expression of these
factors, although we cannot formally rule out the possibility that Shh
signalling might be needed to maintain the expression of other, as yet
unidentified molecules. Alterations similar to those observed in
anti-Shh-treated embryos, including abnormal turning in the contralateral
optic nerve, were observed upon genetic or enzymatic elimination or
modification of proteoglycan sugar moieties
(Chung et al., 2000;
Inatani et al., 2003;
Pratt et al., 2006), which are
important for Shh diffusion (Guerrero and
Chiang, 2007), supporting a direct relationship between Shh
activity and axon growth. Furthermore, interference with Shh activity in brain
slices appears to cause visual axon routing abnormalities similar to those we
observed in vivo (Hao et al.,
2006).
Consistent with the presence of additional inhibitory cues responsible for
channelling retinal axon growth across the chiasm, the defects observed in
anti-Shh-treated embryos were not extensive. Thus, the rostral expression of
Slit2 (Erskine et al., 2000)
may restrain retinal axons from entering the preoptic area in the absence of
Shh activity, whereas Slit1 together with other components expressed by
SSEA-1-positive cells (Marcus and Mason,
1995) may partially counterbalance the loss of Shh activity,
limiting the number of axons that abnormally invade the hypothalamic area in
anti-Shh-treated embryos (Fig.
3E).
In addition to defasciculation at the chiasm, anti-Shh antibodies caused an
abnormal accumulation of growth cones along the proximal visual pathway,
suggesting that Shh might also modulate axon extension. Because it is unlikely
that midline-derived Shh can diffuse as far as the optic nerves, we
hypothesised that Shh produced by the RGCs themselves could be responsible for
this effect. Indeed, Shh is transported along the axons as previously
suggested (Traiffort et al.,
2001; Wallace and Raff,
1999) and is strongly present in the C-RGC growth cones.
Modifications to growth cone behaviour observed after the addition of
cyclopamine to retinal explants, as well as axon misprojections in the
proximal visual pathway after inhibition of Shh transduction in C-RGCs,
confirmed that Shh influences axon growth by a cell-autonomous mechanism (Shh
produced by the population of RGCs and acting over the population itself).
This is particularly intriguing because there are only a few examples of such
a mechanism in axon guidance. The activity of Bdnf on pyramidal
dendritogenesis (Wirth et al.,
2003) and that of ephrin A on RGCs represent such cases
(Hornberger et al., 1999).
Owing to the size of the eye and the lack of transparency of the uterus, it
was very difficult to successfully perform targeted electroporation before
E13.5. Given that the transgene may need several hours to reach full
expression, inhibition of Shh transduction in the targeted cells should be
effective only from E14-14.5. By that time, many of the axons of the
electroporated C-RGCs have reached or passed the chiasm, which explains why
several GFP-positive axons in the
Ptc1loop2 embryos showed a normal initial
trajectory. The misprojection of a significant number of fibres at different
points along the proximal trajectory is instead likely to reflect the position
reached by the individual growth cones when their ability to sense the
presence of Shh was abolished, underscoring a continuous effect of Shh on
fibre extension along the entire proximal visual pathway.
Cell-autonomous (RGC-derived) Shh appeared to have a positive effect on
C-RGC axon growth, whereas the effect of the non-cell-autonomous
(midline-derived) Shh appeared to be negative. This discrepancy can be easily
reconciled in the following model. From their initial extension, C-RGC axons
grow in the presence of a low-level Shh gradient created by secretion from the
axons themselves as they progress along the proximal path. Because the
cellular response to Hh gradients is determined by the ratio of occupied to
unoccupied Ptc receptor (Casali and
Struhl, 2004), low occupancy may lead to a positive effect in the
growth cone, maintaining cytoskeletal organisation and possibly also
transcriptionally regulated levels of guidance cue receptors. Continuous Shh
presence along the nerve could lead to adaptation, an important mechanism that
controls the response of growth cones to guidance cues
(Ming et al., 2002;
Piper et al., 2005;
Rosentreter et al., 1998).
Adaptation changes the threshold of ligand concentration needed to induce a
response (Piper et al., 2005)
such that a critical concentration might then be required to obtain a response
(Rosentreter et al., 1998).
This abrupt change in Shh concentration must occur at the chiasm region where
an additional Shh source is present. Thus, a sharp change in the number of
occupied receptors causes a different and negative response of the C-RGC
growth cones to Shh, funnelling axon growth to the contralateral side of the
brain.
Differential effects of Shh on I-RGC versus C-RGC axons
The above model explains the behaviour of the C-RGC axons as observed in
the different experimental paradigms employed in this study. However, whether
it applies to I-RGCs is unclear. Technical limitations prevented
electroporation of the Ptc1loop2 construct
in the VT crescent of the retina, so we could not clearly establish the in
vivo behaviour of I-RGCs following interference with Shh signalling. Although
retrograde labelling experiments suggest that midline-derived Shh mostly
affects C-RGC axons, we cannot exclude the possibility that the difference
observed in vitro between I- and C-RGC axons might reflect an effect that Shh
secreted by C-RGCs may exert on I-RGCs as they grow in the initial portion of
the visual trajectory. Thus, in contrast to C-RGCs, high concentrations of Shh
consistently promoted I-RGC axon outgrowth and the spreading of their growth
cones. Notably, this response correlates with the specific expression of the
Shh receptors Boc and Ptc2 in I- but not C-RGCs. Although
the functions of Ptc2 have hardly been studied
(Pearse et al., 2001), Boc
activity has recently been associated with the Shh-induced attraction of
commissural axons (Okada et al.,
2006), which interestingly show a dual response to Shh depending
on the activated receptor: Smo and Boc mediate chemoattraction of precrossing
commissural axons (Charron et al.,
2003; Okada et al.,
2006), whereas Hip1 seems to be involved in repulsion of
postcrossing axons (Bourikas et al.,
2005). The growth-promoting effect of Shh on I-RGCs might involve
a similar Boc activation.
An additional interesting difference is that most I-RGCs do not express
Shh. It is unclear what impact this might have on axon extension, as I-RGC
axons grow intermixed with C-RGC axons that release Shh. The difference is
more likely to relate to cell specification. Diffusion of ventrally located
Shh appears to control Zic2 expression in the dorsal telencephalic
midline (Hayhurst et al.,
2008), suggesting that Zic2 activation responds to a low
level of the morphogen. In the retina, Shh diffusing from the adjacent C-RGCs
might activate Zic2 in I-RGCs, which in a feedback loop may repress
Shh, given that Zic2 mRNA is absent from the optic vesicles of
Shh-null mice (Brown et al.,
2003). This regulatory loop would reinforce the divergence of the
two RGC cell types and their specific projections, as has been proposed for a
possible Isl2 and Zic2 cross-repression
(Butler and Tear, 2007;
Pak et al., 2004).
Molecular mechanisms underlying Shh-mediated effects on growth cones
A general question relating to the axon guidance function of cell
signalling pathways, including Shh, BMP and Wnt, is whether their activation
leads simply to local changes in cytoskeletal components within the growth
cones or whether it also involves the control of gene transcription
(Bovolenta et al., 2006). At
least for the Shh pathway, the two mechanisms are not mutually exclusive and
are likely to coexist, although additional studies are needed to obtain a
detailed picture of this. In the chick embryo, decreased intracellular levels
of cAMP may underlie the effect of Shh on RGC growth cone movement
(Trousse et al., 2001),
whereas a novel transcription-independent mechanism involving arachidonic acid
metabolism seems to mediate the Shh-induced migration of mesenchymal
fibroblasts (Bijlsma et al.,
2007). Here we have shown that incubation with actinomycin D or
cycloheximide abrogates the Shh-induced spreading of the growth cone,
indicating that transcription and translation are need for these changes.
Possible targets of these activities are proteins involved in actin
organisation, such as Lasp1 or Mim [missing in metastasis; also known as basal
cell carcinoma-enriched gene 4 (Beg4)], which are regulated by Shh signalling
in other contexts (Gonzalez-Quevedo et
al., 2005; Ingram et al.,
2002). Nevertheless, it has yet to be established whether
transcription/translation in response to Shh occurs only in the cell body or
whether local protein synthesis, an important mechanism to control the growth
cone response to guidance cues (VanHorck
and Holt, 2008), is also needed.
In conclusion, we have shown that Shh signalling plays a prominent role in controlling axon extension along the proximal visual pathway and in funnelling C-RGC axons to the contralateral side of the brain. A cell-autonomous and a non-cell-autonomous mechanism, with apparently opposite effects, cooperate to achieve these functions. The key to the differential response may lie in a concentration gradient of ligand, thus providing a good example of true morphogenetic activity in axon guidance. Whether Shh signalling contributes to the functional differences between I- and C-RGC projections, as suggested by expression analyses and in vitro studies, awaits further confirmation in vivo.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/20/3531/DC1
|
ACKNOWLEDGMENTS |
|---|
loop2-IRES-GFPnls construct,
anti-Isl2 antiserum, purified N-Shh and Shh::GFPCre/+ embryos,
respectively. This work was supported by grants from the Spanish MEC
(BFU-2004-01585), Fundación la Caixa (BM04-77-0), Fundación
Mutual Madrileña (2006-0916) and Comunidad Autonoma de Madrid (CAM,
P-SAL-0190-2006) to P.B. C.S.-C. held a contract from the 'Juan de la Cierva'
Program from the Spanish MEC and is now supported by the CSIC JAE program. |
REFERENCES |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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