Department of Developmental Neurobiology, University of Heidelberg, 69120 Heidelberg, Im Neuenheimer Feld 232, Germany
* Author for correspondence (e-mail: g.e.pollerberg{at}urz.uni-heidelberg.de)
Accepted 9 June 2005
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SUMMARY |
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Key words: NrCAM, Axonal CAMs, L1, Axon navigation, Embryonic retina, Central nervous system development, Time-lapse, 3D reconstruction
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Introduction |
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RGC axons are the first to be formed during retina development, and are the
only ones to leave the eye and project to the optic tectum following
stereotype pathways (Halfter and Deiss,
1986; Stahl et al.,
1990
). All RGC axons strictly extend towards the optic fissure
(OF) in the central retina, fasciculating with other RGC axons and gradually
building up the optic fibre layer (OFL). RGC axons then have to turn to grow
towards the optic nerve head and into the optic nerve to leave the eye.
Together with the differentiation wave spreading across the retina, RGC
axonogenesis proceeds in a spatiotemporally controlled manner from the centre
to the periphery.
Here, we report the impact of NrCAM on RGC axon functions. NrCAM as a substrate affects growth cone shape and axon advance, and is able to guide RGC axons when offered as substrate stripes. Three-dimensional (3D) reconstructions of RGC growth cones in the retina reveal the importance of NrCAM for growth cone size, complexity and directed shape. Time-lapse studies in retina flat-mounts show that NrCAM is required for straight and steady RGC axon routing to the optic fissure. In eyes organ cultured under NrCAM inhibition, RGC axons fail to leave the eye. Together, these findings demonstrate a crucial role of NrCAM for RGC axonal growth and pathfinding in the developing retina.
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Materials and methods |
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Antibodies
Monoclonal antibody (mAb) 2B3 and rabbit sera against NrCAM were produced
as described (de la Rosa et al.,
1990; Suter et al.,
1995
). Secondary antibodies were purchased from Jackson
ImmunoResearch Laboratories; serum against Laminin (L9393) was purchased from
Sigma. F(ab) fragments were generated according to Mage
(Mage, 1980
), non-specific
F(ab) fragments were generated from immunoglobulins (IgGs) purified from
rabbit pre-immune serum. Immunohistochemistry was performed as described
(Avci et al., 2004
).
Affinity purification of NrCAM
NrCAM was immunoaffinity-purified from the brains of newly hatched chicks
using a mAb 2B3 column as described before
(de la Rosa et al., 1990). The
purity of NrCAM was analysed by SDS-PAGE.
Axon growth assay
Single-cell cultures of embryonic day (E) 6 retinal cells were prepared as
described (Avci et al., 2004;
Halfter et al., 1983
).
Poly-L-lysine (PLL)-coated glass coverslips were incubated with either Laminin
(5 µg/ml; Invitrogen) or a NrCAM/Laminin mixture (1.5 and 5 µg/ml,
respectively). Only unipolar neurons with a process longer than 10 µm and a
proper growth cone were counted as axon-forming. RGCs were identified in
phase-contrast microscopy by the following morphological criteria: large,
elongated soma (13 µm length, 8 µm width) compared with other
neurons/neuroblasts (round, 7 µm diameter); and a single, strong axonal
process (this was confirmed by staining with mAb RA4; a kind gift of S.
McLoon, Minneapolis). Growth cone area and perimeter were measured using
ImageJ (NIH). As a measure for the formation of protrusions/indentations, the
ratio of the perimeter and square root of area was calculated for each growth
cone. Significance of differences between mean values was determined by
t-tests.
Axon preference assay
Preference assays were performed as described
(Avci et al., 2004;
Vielmetter et al., 1990
),
except that NrCAM (3 µg/ml) was employed. A substrate lane was counted as
exerting axonal preference when it contained axons/axon bundles and at the
same time was neighboured by lanes containing no axons (with the exception of
a very few). A retinal explant strip was considered to be showing preference
if its RGC axons respected the borders of at least 50% of the lanes of a given
substrate.
Eye organ culture
Eyes of E4.5 embryos were isolated (pigment epithelium removed) and
cultured for 24 hours as described (Avci et
al., 2004). Eyes were incubated with NrCAM F(ab) fragments or
non-specific F(ab) fragments (1 mg/ml). Retinae were then flat-mounted and DiO
crystals (Sigma) were placed at a distance of 400-500 µm away from the OF
to visualise groups of RGC axons. Retinae were evaluated using an inverted
microscope (Axiovert 200M, Zeiss) equipped with a digital camera (AxioCam,
Zeiss).
Retina flat-mount culture
Retinae of E4.5 embryos were spread out flat on nitrocellulose filters and
pre-cultured in 200 µl Neurobasal medium (Invitrogen) for 1 hour in
presence of NrCAM F(ab) fragments or non-specific F(ab) fragments (1 mg/ml).
For live imaging, RGC axons were labelled by small DiO crystals (placed on the
vitreal side of the retina) and monitored for up to 8 hours using a
climate-controlled inverted microscope (37°C, 5% CO2; Axiovert
200M, Zeiss) equipped with a digital camera (AxioCam, Zeiss). Growth cone
kinetics were computed using the Track-function of ImageJ (NIH) and were
statistically analysed by t-test.
For 3D analysis of growth cone morphology, flat-mounted retinae were fixed
after 1 hour in culture in the presence or absence of F(ab) fragments and RGC
axons were labelled by DiI crystals. Images of growth cones were captured
using a laser scanning confocal microscope (TCS SP2, Leica). At least 10
growth cones per retina (in at least four retinae) were examined in each
experiment. 3D reconstructions were performed and the volume/surface of growth
cones quantified using the Volocity system (Improvision, USA). Two categories
with respect to growth cone shape were employed
(Mason and Wang, 1997).
`Simple growth cones' are defined by an elongated or torpedo-like shape, and
only occasional formation of short lamellipodial or filopodial protrusions.
`Complex growth cones' have a form that is approximately as broad as it is
long with an irregular outline formed by abundant lamellipodia and filopodia.
Significance of difference between frequencies of the two growth cone forms
was determined by Chi-squared test.
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Results |
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Together, the localisation studies show that NrCAM is present at the right time (phase of axon extension) and place (axon and growth cone) to play a role for growth and navigation of RGC axons.
NrCAM enhances axon advance and the formation of growth cone protrusions
Because in vivo RGC axons extend in contact with both the NrCAM-containing
OFL and the Laminin-rich basal lamina, we investigated the impact of NrCAM,
offered as substrate, on RGC axons in retinal single-cell cultures
(Fig. 3). On glass coverslips
coated with poly-L-lysine (PLL) only, RGCs merely form short axons
(26±13 µm, n=94) within 1 day in vitro (div;
Fig. 3A). Coating coverslips
with PLL and NrCAM increases the overall axon length by 27% (33±7
µm, n=187, P<0.0006;
Fig. 3A). The proportion of
axons longer than 50 µm is increased threefold
(Fig. 3B). Laminin-coating
results in a high overall axon length (75±47 µm, n=236);
if, in addition, coverslips are coated with NrCAM, overall axon length is
increased by 18% (88±68 µm; n=298, P<0.017;
Fig. 3A). The proportion of RGC
axons longer than 100 µm is increased by 44% on NrCAM/Laminin when compared
with axons on Laminin (Fig.
3B).
The data show that NrCAM is capable of enhancing axon extension by itself, causing an average increase in length of 7 µm. Moreover, axons extending on Laminin, which are considerably longer than those on PLL, gain an extra 13 µm in length if NrCAM is also present.
In contrast to its impact on axon extension, NrCAM does not have the capacity to promote axon formation. In E6 retinal single cells cultured on coverslips coated with PLL alone or with PLL and NrCAM, identical proportions of cells [6.6% (n=1309) and 6.4% (n=2868), respectively] form an axon within 1 div. Coating with Laminin in addition to PLL increases the number of axon-forming cells considerably; this proportion is not affected by additional coating with NrCAM [15% (n=1521) and 16% (n=1812), respectively]. These data are in concordance with RGC axon outgrowth in the retina taking place on the basal lamina, which provides Laminin but not NrCAM.
We also evaluated whether NrCAM as a substrate affects the morphology of growth cones. The average RGC growth cone size (area covered) is not significantly changed whether retinal cells are cultured on NrCAM/Laminin or on Laminin (111±45 µm2 and 106±39 µm2, respectively; Fig. 3C). By contrast, the growth cone perimeter is increased by 14% on NrCAM/Laminin when compared with Laminin [79±22 µm (n=121) and 69±22 µm (n=110), respectively; P<0.001; Fig. 3D]. The ratio of perimeter and square root of area as a measure of protrusion formation is significantly increased on NrCAM/Laminin compared with Laminin, by 16% (7.9±2.2 and 6.8±1.4, respectively; P<0.00002; Fig. 3E).
Together, these data show that NrCAM enhances RGC axon length and the formation of protrusions, indicating that NrCAM as a substrate not only has an impact on axon advance but also on form and explorative behaviour of growth cones.
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NrCAM is crucial for slim and correctly directed growth cones in the retina
To investigate the impact of NrCAM on RGC axons and growth cones in vivo,
we performed confocal microscopy studies on RGC axons in retina flat-mounts,
which allow the 3D reconstruction of the growth cone morphology in the
histotypic context (Fig. 5). In
retinae kept in the presence of non-specific F(ab) fragments, RGC growth cones
show a slim `streamlined' morphology, focussed towards the optic fissure
(Fig. 5A). Typically, one or
very few filopodia are formed at the tip of the growth cone, which extend in
contact with other axons/axon bundles and point in the growth direction
(Fig. 5B). Only very rarely is
a process extending away from the growth direction formed
(Fig. 5B). In the presence of
NrCAM F(ab) fragments, growth cones display a complex morphology with an
irregular shape: numerous lateral lamellipodia and filopodia are formed, which
make the growth cones about as wide as they are long
(Fig. 5C,D). The protrusions
typically extend perpendicular to the growth direction, exploring the growth
cone environment away from the correct pathway. Some of these protrusions,
which can extend up to 10 µm, reach through the forming OFL into the GCL.
Occasionally the entire growth cone was found to be bending away from the
original growth direction (towards the fissure); this is never observed under
control conditions (Fig. 5D).
These growth cones explore the environment to the left and right of the
correct pathway, yet still stay in contact with the basal membrane. Entire
growth cones diving into deeper retinal layers are not observed.
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NrCAM is crucial for efficient advance and direct routing of RGC axons towards the optic fissure
To analyse the role of NrCAM for growth cone dynamics and axonal
orientation in vivo, time-lapse studies of RGC axons navigating in retina
flat-mounts were performed. In the presence of non-specific F(ab) fragments,
RGC axons (n=25) grow towards the optic fissure in a straight
fashion, no axon was observed to deviate from the correct growth direction
(see Movie 3 in the supplementary material). Moreover, the axons strictly
extend on RGC axons/axon bundles and do not stray away. By contrast, under
NrCAM inhibition 20% of the axons (n=30) detach from the axons/axon
bundles and turn away at various angles from the direct route to the optic
fissure (Fig. 6A-F; see Movie 4
in the supplementary material); some even turn towards the periphery. Half of
these axons did not correct their deviations, the other half re-orientated
towards the OF. Such misrouting or wandering of RGC axons was never observed
in controls. Under NrCAM inhibition axons proceed slower and show a less
steady forward movement with long pauses
(Fig. 6E,F). Short stops,
however, are also observed under control conditions. In the pause phases,
growth cones show a strong explorative behaviour, with numerous filopodia
being rapidly formed and retracted, both in controls and under NrCAM
inhibition.
Phases of axon growth and pausing alternate (Fig. 7A,B). The overall advance speed in control retinae is 81±19 µm/hour; the elongation rate measured selectively during the advance phase is 117±32 µm/hour (n=25). When NrCAM is inhibited, the overall advance speed of RGC axons is reduced by 17% (67±25 µm/hour, P<0.025, n=30; Fig. 7C). The elongation rate in the growth phases (125±48 µm/hour), however, is not significantly changed (Fig. 7C). Inhibition of NrCAM does not make axons slower but increases the length of pauses by 34% when compared with controls (6.0±2.5 minutes and 4.4±1.6 minutes, respectively; P<0.013; Fig. 7D). Concomitantly, advance phases are 25% shorter than in controls (6.2±2.2 and 8.3±3.9 minutes, respectively; P<0.017; Fig. 7D). As a result, a 23% shorter distance is covered per advance phase under NrCAM inhibition. The frequency of pauses (4.9±1.6/hour) and advance phases (5.6±1.4/hour) under control conditions is not altered when NrCAM is inhibited (5.2±1.6/hour and 5.6±1.6/hour, respectively). Retractions are rare events under control conditions; only one of the observed axons (n=25) showed such a behaviour (Fig. 7A). By contrast, when NrCAM is inhibited, 50% of the axons (n=30) exhibited one or more retractions. The average frequency of retraction events is only 0.7/hour and therefore the average loss of axon length is merely 5 µm (Fig. 7A,B). Thus, the less efficient advance of axons under NrCAM inhibition is mainly due to longer pauses and shorter advance phases, only to a minor degree is it due to the more frequent retractions.
Taken together, the data show that, in the retina, NrCAM keeps advance phases long, pauses short, and the frequency of RGC axon retractions low. Presence of NrCAM also limits growth cone exploration and axonal deviations from the direct pathway to the optic fissure. This indicates that NrCAM has an important impact on the axonal and growth cone behaviour underlying successful and effective navigation.
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Discussion |
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`Classical' receptor/ligand systems also contribute to RGC axon guidance.
In retinae deficient for the Ephrin receptors EphB2/EphB3, axons split away
from the direct trajectory to the optic nerve head and fail to reach it
(Birgbauer et al., 2000).
Deficiency for Netrin or its receptor DCC results in a failure of RGC axons to
exit into the optic nerve (Deiner et al.,
1997
). Netrin lines the optic nerve head, forming a cuff around
the RGC axons, probably acting in a repulsive manner (together with Laminin)
(Hopker et al., 1999
).
Suppression of Slit1, a ligand that acts positively on RGC axons, causes
abnormal axon trajectories towards the optic nerve head
(Jin et al., 2003
).
Overexpression/inhibition of Sonic hedgehog (Shh), a released protein that
promotes and guides RGC axons in vitro, leads to a loss of centrally directed
projections of RGCs (Kolpak et al.,
2005
). Chondroitin sulphate proteoglycan, an inhibitory/repellent
ECM component located in the peripheral retina, is necessary to prevent RGC
axons from growing into the periphery
(Brittis et al., 1992
).
The proteins mentioned above do not form gradients across the developing
retina and therefore cannot serve as peripheral-central cues for RGC axon
navigation. The chemokine SDF1 is expressed by optic stalk cells (of zebrafish
embryos), and conceivably might spread over the retina as an attractive
central-peripheral gradient (Li et al.,
2005). SDF1 does not attract (chick) RGC axons; however, it does
reduce their repulsion by Slit2 (Chalasani
et al., 2003
). The transcription factor Zic3 is expressed in a
gradient (high in periphery) and might induce the expression of inhibitory
factors for RGC axons; its overexpression induces abnormalities in RGC
projections towards the optic nerve head
(Zhang et al., 2004
).
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The enrichment of NrCAM in the distal axon portion and growth cone suggests
that NrCAM acts as a membrane receptor probing the environment. NrCAM could,
however, play a dual role, as RGC axons extend on older RGC axons, which
present NrCAM as a growth substrate, i.e. as a ligand. In vitro, in
alternating stripe assays or homogeneous substrate assays for example, the
selective role of substrate-NrCAM for the RGC axons can be studied. Inhibition
of NrCAM in vivo affects both the NrCAM on growth cones (receptor-NrCAM) and
on axons (substrate-NrCAM); the two functions cannot be separated. NrCAM could
interact homophilically (Mauro et al.,
1992) and/or heterophilically with TAG1/Axonin 1
(Suter et al., 1995
),
F3/F11/Contactin (Morales et al.,
1993
), and Neurofascin
(Volkmer et al., 1996
), which
are known interaction partners present in the OFL at this stage.
Impact of NrCAM on axons and growth cones
NrCAM offered as a substrate is by itself not able to promote axon
formation in single RGCs. Initial axon outgrowth therefore seems to be
independent of NrCAM. This is in concordance with the observation that, in the
retina, initial outgrowth takes place in contact with the basal lamina in an
environment devoid of NrCAM. By contrast, once the RGC axon is extending,
NrCAM by itself is capable of increasing axon advance as a substrate. This
could reflect the phase of axonal growth when the RGC axon has contacted the
first NrCAM-displaying (older) RGC axon, conceivably increasing axon
elongation. This is probably enhanced by other IgSF-CAMs. L1 is capable of
promoting RGC axon growth by itself
(Morales et al., 1996).
DM-GRASP and F3/F11/Contactin promote axon growth of other neuron types, but
not of RGCs (DeBernardo and Chang,
1995
; Treubert and
Brummendorf, 1998
), indicating a differential use of receptors or
intracellular signalling pathways in various neuron types.
RGC axons extend in the retina with their growth cones in contact with both
the basal lamina, which is strongly positive for Laminin, and, as soon as they
have reached a RGC axon, with older RGC axons carrying NrCAM. We therefore
tested the impact of NrCAM as co-substrate of Laminin on RGC growth cones. In
vitro, NrCAM substrate induces RGC growth cones to form more protrusions;
however, growth cone size is not influenced. By contrast, L1 and DM-GRASP lead
to enlarged growth cones as co-substrates
(Avci et al., 2004;
Burden-Gulley et al., 1995
).
The specific effect of NrCAM on growth cone shape but not on size is
suggestive of an instructive role of this CAM rather than a merely adhesive
function.
In contrast to the in vitro assays, in the developing retina NrCAM is only presented as a growth substrate by RGC axons/axon bundles. If NrCAM is undisturbed, the pre-existing axons focus the growth cones to extend on them and thereby guide them towards the OF. Under inhibition of NrCAM, more growth cone protrusions are formed, extending in all directions, including away from the substrate axons. This is the first study revealing such details of growth cone morphology in the histotypic context in 3D. These findings do not contradict the observation of increased protrusion formation caused by substrate-NrCAM in vitro. In both cases, substrate-NrCAM induces the formation of protrusions; in vitro, NrCAM is ubiquitously present in the growth cone environment and therefore causes an undirected formation of protrusions all around the growth cone. In vivo, the focussing impact of the selective occurrence of NrCAM on RGC axons/axon bundles is lost when NrCAM is inhibited: growth cones and substrate-axons are not able to interact effectively, thus, growth cone protrusions extend away from the substrate axons. This could be the first step initiating the growth cones to splay off the axon bundles and, ultimately, leading to axonal misrouting.
To analyse the impact of NrCAM on axon extension, we offered this CAM as a
co-substrate of Laminin and observed a significant increase in RGC axon
length. An increase in axon length has been reported for the two axonal CAMs,
F3/F11/Contactin and DM-GRASP, offered as co-substrates of ECM molecules
(Avci et al., 2004;
Treubert and Brummendorf,
1998
); NrCAM was shown to increase axon growth when offered
together with NgCAM (Morales et al.,
1996
). Our data indicate that substrate-NrCAM probably triggers
intracellular signalling pathways independently of integrin signalling, which
can be assumed to be well stimulated by the Laminin concentrations offered in
vitro. The increase in axon length by NrCAM in addition to the one caused by
Laminin is in concordance with our findings that the advance of RGC axons in
their histotypic environment, which provides both Laminin and NrCAM, is
significantly reduced by NrCAM inhibition. In particular, in vivo axon advance
does not only depend on elongation rates, but also on the frequency and
duration of the pauses (filled with exploration) and retractions. The less
efficient advance of RGC axons in the retina in absence of functional NrCAM is
due to longer pauses and shorter advance phases, and not to a decreased
elongation rate. The strong increase of retractions under NrCAM inhibition
does not severely affect axon advance. Inhibition of other CAMs (L1 in rat or
Neurolin in fish retina) does not seem to increase retractions
(Brittis et al., 1995
;
Leppert et al., 1999
). This is
the first study providing insight in the differential impact of a CAM on
axonal advance, pause, exploration and retraction in a histotypic context.
Role of NrCAM in axonal pathfinding
The role of NrCAM in axonal pathfinding has been studied in a histotypic
context for two other neuron types: proprioceptive and commissural axons in
the spinal cord are no longer able to grow under NrCAM inhibition
(Perrin et al., 2001;
Stoeckli and Landmesser,
1995
). Here, the IgSF-CAMs TAG1/Axonin 1 and F3/F11/Contactin in
the axonal membrane interact with NrCAM, which is present on cells in the
environment. Inhibition of NrCAM turns the growth cone environment and
prospective pathway (spinal cord grey matter, floor plate) from permissive
into repulsive. In NrCAM-deficient mice, however, no defects in axon
orientation at the floor plate have been observed
(More et al., 2001
). In the
developing retina, NrCAM acts differently, as inhibition of NrCAM does not
make the OFL repulsive for RGC axons. In the environment of the RGC axons,
NrCAM (on pre-existing RGC axons heading into the optic fissure) serves as a
positive guidance cue defining the correct pathway; this is the first study
demonstrating such a role for NrCAM. NrCAM lanes can indeed guide RGC axons,
as seen in our substrate stripe assays. Under inhibition of NrCAM, RGC axons
in the stripe assays leave the NrCAM lanes but, in the retina, they do not
stray away from the OFL. This is probably because of the selective presence of
other axon-specific proteins in the OFL, and the GCL acting as a `no entry
zone'.
At the OF, where RGC axons dive into the optic nerve head to leave the
retina, inhibition of NrCAM results in axons overshooting onto the opposite
side of the retina. At the OF, RGC axons have to perform rectangular turns to
dive into the optic nerve head, which conceivably requires a stronger
association of their growth cones to the pre-existing axons than just tracking
on a straight route does. In addition, a minor detachment of an axon from its
bundle caused by NrCAM inhibition results in a substantial misrouting at the
OF, as the axon is irreversibly splayed off and is not `picked up' by other
bundles running in parallel, as is the case on its way towards the OF. The
observed effects could be due to a reduction in adhesion, as homo- and
heterophilic trans-interactions between the extending axon and the
pre-existing axon bundle might be weakened by NrCAM inhibition. However, NrCAM
which is not an abundant CAM might rather have an instructive
effect on navigating RGCs, e.g. by interactions with membrane proteins
(TAG1/Axonin 1 or F3/F11/Contactin), triggering signal cascades, and/or by
acting on cytoskeletal components in the growth cone
(Davis and Bennett, 1994;
Faivre-Sarrailh et al., 1999
).
Effects caused by NrCAM inhibition cannot be (sufficiently) counterbalanced by
other mechanisms despite the presence of five other axonal IgSF-CAMs,
indicating an irreplaceable, pivotal role of NrCAM in axonal pathfinding.
It is tempting to speculate that the restricted presence of proteins
selectively on RGC axons such as NrCAM might be sufficient for
RGC pathfinding into the optic nerve. Axonogenesis advances strictly regulated
from the central to the peripheral retina: the first emerging RGC axons in the
immediate vicinity of the optic fissure could find the exit by random probing
and/or local cues, the growth cone itself already covering this short distance
(about 20 µm). Later emerging RGC axons, further away from the optic
fissure, also only have to span a very short distance to find the next RGC
axon (less than 15 µm) (McCabe et al.,
1999) (P.Z. and G.E.P., unpublished), which could be based on
random search, too, and from then on could travel on the pre-existing axons
interacting with axon-specific proteins. In this scenario, the crucial
gradient in the retina is not a guidance molecule gradient (which has not been
found until now) but the differentiation gradient, which spreads over the
retina and spatio-temporally controls RGC axon formation.
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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/132/16/3609/DC1
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