|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online May 16, 2007
doi: 10.1242/10.1242/dev.002345
1 Department of Anatomy, University of Cambridge, Downing Street, Cambridge CB2
3DY, UK.
2 Department of Biology, Massachusetts Institute of Technology, Cambridge, MA
02139, USA.
3 Department of Genetics, Cell Biology and Development, University of Minnesota,
Minneapolis, MN 55455, USA.
4 Département de Biologie Cellulaire et de Morphologie, University of
Lausanne, Rue de Bugnon, 9, 1005 Lausanne, Switzerland.
* Author for correspondence (e-mail: cecile.lebrand{at}unil.ch)
Accepted 18 March 2007
 |
SUMMARY |
|---|
 TOP SUMMARY IntroductionMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key words: Ena/VASP, Filopodia, Branching, Guidance, Axon, Retina, Xenopus
|
Introduction |
|---|
|
TOPSUMMARYIntroductionMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
;
Davenport et al., 1993;
Holt, 1989;
Isbister and O'Connor, 2000;
Mason and Wang, 1997;
Myers and Bastiani, 1993;
O'Connor et al., 1990;
Zheng et al., 1996). The
elimination of filopodia by treatment with cytochalasin, a drug that prevents
actin filament elongation from barbed ends, causes misdirected axon growth in
vivo in the developing vertebrate visual system
(Chien et al., 1993) and in
the developing grasshopper limb (Bentley
and O'Connor, 1994).
The formation of filopodia and growth cone dynamics require continuous
rearrangement of the actin and microtubule cytoskeleton
(Lin and Forscher, 1995;
Mallavarapu and Mitchison,
1999; Schaefer et al.,
2002; Tanaka and Sabry,
1995; Zhou et al.,
2002). Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP)
proteins are enriched at the tips of neuronal growth cone filopodia
(Lanier et al., 1999) and have
been shown to regulate the formation and the elongation of filopodia via actin
dynamics (Lebrand et al.,
2004). This family of proteins has been shown to contribute to
axon guidance signaling in different species. Drosophila ena mutants
have mild CNS defects and a `bypass' phenotype of the intersegmental nerve
(ISNb), which fails to branch in the appropriate regions
(Gertler et al., 1995;
Wills et al., 1999). Ena acts
downstream of the Slit repulsive guidance factor by regulating the signal
transduction of its receptor Robo (Bashaw
et al., 2000). Similarly, genetic analysis in Caenorhabditis
elegans identifies a function for the Ena homolog UNC-34 downstream of
the attractive guidance receptor UNC-40/DCC for UNC-6/Netrin 1 as well as
downstream of the repulsive guidance receptors UNC-5 for UNC-6/Netrin 1 and
SAX-3/Robo for SLT-1/Slit (Colavita and
Culotti, 1998; Gitai et al.,
2003; Yu et al.,
2002). In vertebrates, three Ena/VASP proteins exist: Mena, EVL
(Ena/VASP-like) and VASP (Gertler et al.,
1996). In mice, they are necessary for the appropriate guidance of
commissural and sensory axons along the visual path
(Lanier et al., 1999;
Menzies et al., 2004).
Netrin-1-induced filopodium formation is dependent upon the function of the
Ena/VASP proteins and is directly correlated with their phosphorylation at a
regulatory protein kinase A (PKA) site
(Lebrand et al., 2004). These
findings implicate a key role for the Ena/VASP proteins in the chemotropic
responses of growth cones to guidance cues.
To investigate fully the role of the Ena/VASP proteins in growth cone
dynamics and axon pathfinding in vivo, we used a sequestration strategy to
block the function of all Ena/VASP family members - Xena, XVASP and XEVL - in
the retinotectal system of Xenopus embryos. In brief, these proteins
need to be localized adjacent to the plasma membrane in order to function in
actin polymerization and have been rendered non-functional using an approach
that instead targets them to the mitochondrial membrane
(Bear et al., 2000). In
contrast to previous genetic approaches that affect all cells, we have
modified the activity of the Ena/VASP proteins in a subset of neurons, the
retinal ganglion cells (RGCs) of the Xenopus retina. Using this
approach, we confirmed that Xena/XVASP proteins play a fundamental role in the
formation of growth cone filopodia in vivo. The lack of filopodia induced by
the depletion of the Xena/XVASP proteins did not affect retinal axon
pathfinding in vivo. However, depleted axons showed a reduced growth rate and
longer pauses along the visual path. Strikingly, within the tectum, Ena/VASP
protein knock-down drastically affected the branching of growing retinal
axons. We thus propose that Xena/XVASP proteins control axonal growth cone
dynamics, the speed of outgrowth and axonal branching by remodeling the actin
network in response to guidance signals.
|
MATERIALS AND METHODS |
|---|
|
TOPSUMMARYIntroductionMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Sequestration strategy specificity
We used the specific binding of the EVH1 domain of Ena/VASP proteins with
the ligand motif DFPPPPXDE (abbreviated FP4) to sequester the Ena/VASP
proteins (Bear et al., 2000).
FP4-Mito expression in Rat2 fibroblasts and primary neurons removes all
detectable Ena/VASP proteins from their normal site of localization at the
cell plasma membrane and sequesters the proteins on the surface of
mitochondria (Bear et al.,
2000; Bear et al.,
2002; Goh et al.,
2002; Lebrand et al.,
2004). In fibroblasts, it causes a phenotype identical to that of
fibroblast lines that lack detectable Ena/VASP proteins. Expression of
FP4-Mito induces no additional phenotypes in Ena/VASP-deficient fibroblasts,
indicating that FP4-Mito-induced phenotypes in other cells most likely result
from the inhibition of the function of the Ena/VASP proteins.
Plasmids and in vivo cDNA lipofection
Plasmid subcloning and PCR were performed using standard methods. The
FP4-Mito-Green fluorescent protein (GFP) and FP4-Mito-Red
fluorescent protein (RFP) constructs (Bear
et al., 2000) were cloned into pCS2R plasmids for in vivo
expression in Xenopus. Xena and XEVL have been cloned and described
in great detail (Wanner et al.,
2005; Xanthos et al.,
2005). The RFP (Campbell et
al., 2002) and GAP-GFP were cloned into pCS2R and used as generic
controls and as markers to highlight the morphology of the retinal axons
expressing the Mito constructs.
The constructs were introduced into all retinal precursor cells by pressure
injecting the left eye primordia of stage 19 embryos with a mixture of the DNA
construct and the lipofecting reagent DOTAP (Boehringer, Mannheim) at 1:3
weight:volume (w/v) (Holt et al.,
1990; Lilienbaum et al.,
1995). The embryos were transferred to 0.1x MBS and were
allowed to grow for 44 hours (stage 33/34), 56-66 hours (stage 39/40) or 78-82
hours (41/42), depending on the experiments. RGCs were identified on the basis
of layer position and cellular morphology. Retinas lipofected with the
Mito constructs exhibited a similar organization to retinas
expressing control constructs. Cells expressing the Mito constructs
exhibited the entire range of differentiated retinal cell types and were not
pycnotic, indicating that expression of the FP4-Mito construct does
not interfere with the specification, migration, differentiation or short-term
survival of retinal cells.
Cell culture
Retinal explant cultures were prepared from 33/34 embryos as described
(Mann et al., 2002). After 40
hours, cells were fixed in 4% paraformaldehyde/phosphate-buffered saline (PBS)
with 15% sucrose.
Imaging
Fixed and living samples were analyzed with a Nikon fluorescence microscope
equipped with 10x, 20x or 40x Plan-NEOFLUAR objectives and
63x, 100x Plan-NEOFLUAR oil immersion objectives. Images were
captured with an Orca-ER cooled CCD camera (Hammamatsu). All peripherals were
controlled with Openlab software (Improvision). Z stacks of 15 images
at 1 µm intervals, comprising the entire section thickness, were processed
for deconvolution in Metamorph (Universal Imaging) and transferred to
Photoshop software.
In vivo time-lapse video
At stage 39 or 42, embryos were anesthetized and the right optic tract and
tectal area were exposed by removing carefully the right eye and skin as
described (Chien et al.,
1993). The heads of the embryos were mounted in a closed chamber
containing freshly made 1x MBS, pH 7.4-7.5 containing 0.3 mM MS222.
Heads were maintained at room temperature during the observation and were
viable for as long as required (up to 12 hours).
Axons were observed with a 20x lens (Nikon Plan Apo, 0.75 NA), using adapted-epifluorescences filters connected to a standard 100W mercury lamp as well as neutral-density filters to reduce the illumination. To locate the position of the axon relative to brain landmarks, we captured low power (10x) bright-field and fluorescence pictures. For all movies, images were collected at 1 µm intervals in the z axis every 5, 15 or 30 minutes, depending on the experiment. Images from z-stack series of 15 images taken at the indicated intervals were de-convolved, compiled, aligned and converted into Quick-time movies with Metamorph.
Analysis
In vivo analysis of filopodial dynamics, elongation rate and stalling duration of retinal axon growth cones
A total of 15 GAP-GFP- and 15 GAP-GFP+FP4-Mito-RFP-expressing growth cones
were analyzed at stage 39/40. Filopodial morphometric parameters were
determined by measuring filopodial number and length every 30 minutes while
the axons were growing from the ventral (VOT) to the dorsal optic tract (DOT)
and at the tectal border. The average rate of axonal elongation was calculated
from the time taken by axons to travel from the VOT to the DOT and to cross
the tectal border. The average rate of axonal elongation represents a
combination of the actual motion speed of intermittent displacement (basal
rate of extension), and the frequency and duration of the intervening pauses.
Growth cones were considered to be making a pause when they grew less than 10
µm in 20 minutes.
Analysis of axon elongation and pathfinding along the visual path
Analysis was made on fixed specimens because they allow measurement of a
large number of axons. Trajectories of control and FP4-Mito-expressing RGC
axons were analyzed at stage 33/34 in whole-mount retina, which permits us to
follow the entire axons inside the eye
(Holt, 1989). At stages 39 and
42, the trajectories of control and FP4-Mito-expressing axons were studied in
12 µm serial coronal cryostat sections from their origin in the retina to
their tips in the tectum as well as in whole-mount brain preparations, as
described (Holt, 1989;
Riehl et al., 1996;
Ruchhoeft et al., 1999).
Different parameters were analyzed: polarization of the RGC axons in the
retina as well as trajectories and the terminal point of each individual axon.
In 12 µm serial sections of the retina, the number of ganglion cells
expressing the RFP or FP4-Mito-GFP+RFP proteins was counted to give a total
number of positive RGCs per retina. The point along the pathway where each
positive-expressing axon ended (i.e. inside the eye, the brain entry point,
the chiasm, the optic tract, tectum) was determined and then expressed as a
percentage of the total number of positive RGCs. The percentages were an
average of the counts made in ten embryos per condition and analyzed from
three separate sets of experiments. Using appropriate statistical tests, the
percentage values for FP4-Mito-GFP+RFP-expressing axons were compared to
percentage values for RFP-expressing axons within the same animal, as well as
from a different group of animals lipofected only with the RFP.
Branching analysis of retinal axons in vivo
The extent of axonal branching of stage 39/40 embryos was determined by
measuring the branching of 20 control GAP-GFP- and 20
FP4-Mito-RFP+GAP-GFP-expressing retinal axons. Labeled axons were imaged at 5
or 15 minute intervals for a total observation period of at least 3 hours
after the retinal axons had penetrated the tectum. All measurements were
performed on images of reconstructed arbors taken every 30 minutes using
Neurolucida software. The number and the length of the processes were defined
as filopodia when they were less than 8 µm and as branches when they were
greater than 8 µm. Branching development for each condition was analyzed by
comparing parameters at time 0, 1, 2 and 3 hours after axons had entered the
tectum. In addition, branching parameters of FP4-Mito-expressing axons was
compared to branching parameters of control axons at similar times after
tectal entry.
Statistical analysis
For all analysis, control and FP4-MITO values from at least three separate
experiments were first tested for normality. Values that followed a normal
distribution were compared using Student's t-test or one-way ANOVA
and Fisher's t-tests. Values that did not follow a normal
distribution were compared using Mann-Whitney and Kolmogorov-Smirnov non
parametric tests.
|
|
RESULTS |
|---|
|
TOPSUMMARYIntroductionMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
;
Xanthos et al., 2005). To
address the role of this family of proteins in RCG axonal pathfinding, we used
a sequestering strategy that has been shown to neutralize specifically all
three Ena/VASP proteins (Bear et al.,
2000; Bear et al.,
2002; Goh et al.,
2002; Lebrand et al.,
2004) (see Materials and methods). In brief, we used the very
specific binding of the EVH1 domain of Ena/VASP proteins with the FP4 motif to
deplete Ena/VASP proteins from the cell surface
(Bear et al., 2000). By
coupling four copies of the FP4 motif with a mitochondrial-targeting sequence
(`FP4-Mito') all the endogenous Ena/VASP proteins were sequestered at
the mitochondrial surface. A fusion of these constructs with the green
fluorescent protein (GFP) sequence (`FP4-Mito-GFP') or with
the red fluorescent protein (RFP) sequence (`FP4-Mito-RFP')
allows a direct visualization of the targeted cells. In addition, simple
vectors expressing the RFP or the GFP sequence fused to a
plasma membrane-targeting motif (GAP-GFP) were used as lipofection
controls and to visualize the morphology of cells and axons.
We first verified that the FP4-Mito construct is targeted to mitochondrial
membrane (Fig. 1A-C) and that
the sequestrating strategy previously tested in mouse neurons operates in
Xenopus RGCs. To this aim, we performed retinal co-transfection of
the FP4-Mito-RFP plasmid with a construct encoding a fusion of the
Xena protein gene sequence and GFP (Xena-GFP), and analyzed
the cellular localization of the Xena-GFP protein in vivo
(Fig. 1D-F) and in explants
(Fig. 1G-L). Xena-GFP normally
localized on the cell surface of RCGs. At growth cones, Xena-GFP punctate
labeling was observed at the surface and was enriched at the tips of filopodia
(Fig. 1G). By contrast,
co-transfection of Xena-GFP with the FP4-Mito-RFP construct
in retinal cells, from stage 34 to stage 42, showed Xena-GFP-positive staining
typical of mitochondrial localization (Fig.
1D-F) (Bear et al.,
2000; Lebrand et al.,
2004). This RFP mitochondrial labeling correlates with a severe
reduction of cell surface Xena-GFP staining, thereby confirming that the
sequestering strategy efficiently depletes Xena-GFP proteins from the growth
cones (Fig. 1J-L). Similar
observations were made using XEVL-GFP (data not shown), indicating that all
Xenopus Ena/VASP proteins present in retina are similarly sequestered
by FP4-Mito on the mitochondria.
Xena/XVASP proteins regulate the morphology and dynamics of growth cone filopodia in vitro and in vivo
We next analyzed the effects of Xena/XVASP protein plasma membrane (PM)
depletion on retinal axon growth cone morphology in vitro. Whereas control
growth cones exhibited typical morphology, including the presence of
lamellipodia and numerous filopodia (Fig.
1M,N), FP4-Mito-positive growth cones showed a drastic reduction
in the number of filopodia (Fig.
1O). To investigate further how Xena/XVASP proteins regulate
growth cone dynamics, we analyzed the behavior of individual retinal growth
cones in vivo using time-lapse video microscopy in live Xenopus
brains (Chien et al., 1993;
Harris et al., 1987).
GFP-positive control growth cones along the optic tract exhibited
lamellipodial dynamics and rapid filopodial extension, exploration and
withdrawal (Fig. 2A and see
Movie 1 in the supplementary material). By contrast, the PM depletion of
Xena/XVASP proteins induced, in vivo, a severe reduction in growth cone
filopodial number, as observed previously in vitro. All along the optic tract
and inside the tectum, retinal growth cones with reduced Xena/XVASP activity
almost entirely lost their ability to form filopodia and frequently generated
lamellipodia and ruffles (Fig.
2B and see Movie 2 in the supplementary material; and
quantification Fig. 2C,D).
Moreover, the few filopodia generated by Xena/XVASP-depleted growth cones were
shorter than in control conditions. Therefore, Xena/XVASP function controls
growth cone and filopodia dynamics in vitro and, more importantly, in
vivo.
|
). In the retina, from stage 33/34 to stage 42,
FP4-Mito-expressing axons exhibited normal trajectories along the vitreal
surface as well as in the optic nerve head (compare
Fig. 3E,F with 3A,B). In
control brains, at stage 39, numerous RGC axons had grown dorsally through the
optic chiasm and the contralateral optic tract to innervate the optic tectum
(Fig. 3A-D and
Fig. 4A,B). By contrast, very
few axons of neurons expressing the FP4-Mito construct were detected at stage
39 along the chiasm, the optic tract or in the tectum
(Fig. 3G,H and
Fig. 4C). However, the
FP4-Mito-positive axons that did grow followed the normal visual pathway and
did not stray into aberrant territories (compare
Fig. 3G,H with 3C,D and
Fig. 4C with 4B). The
percentage of axons displaying guidance errors (axons with abrupt orientation
changes or with extreme ventral or dorsal positions in the diencephalon) was
identical in FP4-Mito-expressing and control retinal neurons (Figs
3,
4). With time-lapse video
microscopy, we observed that growth cones of neurons expressing FP4-Mito
followed normal paths and reached the tectum, their final target
(Fig. 2B and see Movie 2 in the
supplementary material). Thus, the absence of filopodia in neurons with
depleted Xena/XVASP function does not interfere with the ability of growth
cones to follow a normal route along the retinotectal pathway.
Xena/XVASP function interferes with axon elongation in the retina
In each control and FP4-Mito retina, on average 11 RGCs expressed the
plasmid and the majority had an axon that could be followed at various
distances along the visual pathway from the retina to the tectum. At stage 39,
the majority of the RGCs expressing control constructs had initiated an axon
(79.8±5.5%), many of which had reached the optic tract and the tectum
(59.6±8.8%) (Fig. 3A,
Fig. 5A). Similarly, nearly all
FP4-Mito-expressing RGCs had sent out an axon (79.5±5.6%)
(Fig. 3E). However, the
distribution of their axonal tips along the visual pathways was significantly
different from controls, because the majority of FP4-Mito-expressing axons
remained inside the retina (65.8±4.8% compared with 33.9±9.4%
for controls, P<0.001; Fig.
5A) and only few axons extended in the optic tract
(Fig. 3H,
Fig. 4C and
Fig. 5A). Therefore, at stage
39, the majority of the axons of control neurons had already reached the
tectum, whereas most of the FP4-Mito-expressing axons were still at the level
of the optic nerve head in the retina. These results indicated that the PM
depletion of Xena/XVASP proteins does not interfere with axon initiation but
affects their extension, particularly within the eye.
|
|
Xena/XVASP function regulates the speed of axonal elongation in the retina and along the optic tract
To test the consequences of Xena/XVASP protein PM deletion on axonal
outgrowth speed, we measured, in vivo, the rate of axon elongation as well as
the duration of growth cone pauses by time-lapse video microscopy. Between the
ventral and the dorsal optic tract, control growth cones exhibited filopodial
dynamics and the axonal growth rate was 54 µm per hour
(Fig. 5B). At the tectum
entrance, axons significantly slowed down to an average speed of 30 µm per
hour and became more complex, with filopodia-like processes extending in all
directions, similar to previous findings
(Harris et al., 1987).
By contrast, in FP4-Mito-expressing neurons, correlated to the drastic decrease of growth cone filopodia number, the average elongation rate of growth cones was significantly reduced compared with controls along the optic tract and at the tectal border (Fig. 5B). Instead of the steady forward motility observed in control axons, growth cones of FP4-Mito-expressing retinal neurons exhibited a significant decrease of their basal rate of extension and a significant increase in the duration of pauses in all regions of the optic tract and tectal border. The frequency of pauses was also increased in FP4-Mito-positive axons in the optic tract, but was about the same as controls at the border of the tectum. Some retinal growth cones that were observed to pause for several hours continued to exhibit active lamellipodial movement without forward advance (see Fig. S1 in the supplementary material). These results indicate that the function of Xena/XVASP proteins controls the speed of axonal elongation and the pause time of growth cones.
|
). By
contrast, pausing FP4-Mito-expressing retinal axons exhibited a drastic
reduction in filopodial activity, as well as in branch formation, after they
penetrated the tectum, and this lasted for the whole recording period of 12
hours (Fig. 6B and see Movie 4
in the supplementary material). Unlike control axons, which made sharp turns,
these axons followed a straight path within the tectum. To analyze and quantify the observed branching defects, axonal terminal arborizations were reconstructed using images collected every 30 minutes during the 3 hours following the entry of retinal axons into the tectum (Fig. 6C,D). In controls, the numbers of primary, or secondary, branch segments and of total branch segments, the number of nodes and the length of the total arborization all increased during the 3 hours of recording (Fig. 6C and Fig. 7). The significant increase of total arborization during the first hour was due to an increase in the number of branches, because the average length of the branch segments did not change significantly during the recording period. During the last two hours, although new primary and secondary branches were generated, the total length of the arbors remained relatively constant, because branch formation was counterbalanced by branch retraction.
By contrast, after Xena/XVASP protein depletion, the number of primary or secondary branch segments, of total branch segments, of nodes and the total length of the axon terminal arborizations did not significantly increase compared to the starting point (Fig. 6D and Fig. 7). At all time points, the expression of FP4-Mito significantly impaired all these parameters compared with controls. These neurons lost the capacity to branch, because they only generated very few primary branches and never formed secondary nor tertiary branches. However, the average length of branch segments of FP4-Mito-expressing axons, at all times, was unchanged as compared with the length of control branches. This indicated that Xena/XVASP activity regulated the elaboration of new branches and not the elongation of existing branches. These observations underscore the involvement of Xena/XVASP proteins in regulating the building of retinotectal axon arborizations.
|
DISCUSSION |
|---|
|
TOPSUMMARYIntroductionMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Ena/VASP proteins regulate the actin cytoskeleton dynamics of growth cones
Our current study in Xenopus embryos shows that Ena/VASP activity
is directly correlated with the capacity of retinal axon growth cones to
generate and extend filopodia. This in vivo study confirms our previous work
on cultured primary hippocampal neurons showing that growth cones with
impaired Ena/VASP function lost almost all their ability to form filopodia but
frequently developed large lamellipodia with ruffles
(Lebrand et al., 2004). These
effects were found to result principally from the reduced capacity of growth
cones, in the absence of Ena/VASP proteins, to form the actin filament bundles
that comprise filopodia, as well as from the increase in the density of
networks of short actin filament at the lamellipodia leading edge
(Lebrand et al., 2004).
|
;
Bear et al., 2002). After
binding at the barbed ends of growing actin filaments, Ena/VASP proteins
facilitate continued elongation of these filaments by protecting them from the
activity of capping proteins, which stop the polymerization. The anti-capping
activity of the Ena/VASP proteins could facilitate the elongation of the
privileged filaments that constitute nascent filopodia. In addition, Ena/VASP
proteins may play a direct role in actin filament elongation via their ability
to reduce the branching of actin filaments as well as their action on the
bundling of actin filaments (Barzik et al.,
2005; Gertler et al.,
1996; Huttelmaier et al.,
1999; Krause et al.,
2003; Lambrechts et al.,
2000; Niebuhr et al.,
1997; Reinhard et al.,
1992; Skoble et al.,
2001; Walders-Harbeck et al.,
2002).
Ena/VASP and axonal guidance
Whereas Ena/VASP inhibition induces a drastic reduction of growth cone
filopodial dynamics, it does not cause any major pathfinding defects of
retinal axons along the visual pathway. The absence of pathfinding defects is
surprising, because numerous in vitro and in vivo studies have previously
indicated a major contribution of growth cone filopodia to axon guidance.
Filopodia are considered as sensory structures at the tips of growth cones
that coordinate the first responses to guidance signals
(Bovolenta and Mason, 1987;
Davenport et al., 1993;
Dingwell et al., 2000;
Holt, 1989;
Isbister and O'Connor, 2000;
Mason and Wang, 1997;
Myers and Bastiani, 1993;
O'Connor et al., 1990;
Sabry et al., 1991;
Zheng et al., 1996). The use
of cytochalasin, a drug that disrupts the actin filaments of filopodia, shows
that they are not required for growth cone advance but are indispensable for
growth cone steering in vitro (Marsh and
Letourneau, 1984; Zheng et
al., 1996). More importantly, when filopodial formation is
inhibited by cytochalasin treatment in vivo, the axons do not orient properly
and follow abnormal pathways (Bentley and
O'Connor, 1994; Chien et al.,
1993). Time-lapse experiments in Xenopus indicate that
cytochalasin-treated retinal growth cones lacking filopodia slow down and fail
to reach the tectum (Chien et al.,
1993).
Why do the filopodia-less growth cones in the current study fail to show
this phenotype? The experiments with cytochalasin treatment must be
interpreted with caution because this drug interferes with the organization of
the actin cytoskeleton of all cell compartments. Therefore, cellular functions
other than just filopodial dynamics may be affected. This includes mRNA
trafficking and translation as well as membrane trafficking, endocytosis and
degradation, processes that are important for directed axon outgrowth
(Campbell and Holt, 2001;
Ellis and Mellor, 2000;
Lee and De Camilli, 2002;
Merrifield et al., 1999;
Murphy et al., 1996;
Ochoa et al., 2000;
Qualmann et al., 2000;
Qualmann and Kessels, 2002;
Salazar et al., 2003;
Taunton et al., 2000;
van Horck et al., 2004). In
the current study, only filopodial dynamics were affected while other
functions remained unchanged.
Our results are consistent with a recent study reporting that interfering
genetically with Ena (UNC-34) function in C. elegans affects the
dynamics of lamellipodia and filopodia but does not result in axonal
pathfinding defects (Chang et al.,
2006). In addition, in Xenopus embryos, expression of a
dominant-negative Cdc42 construct by retinal axons reduces the number and
length of filopodia on growth cones, but does not cause targeting errors
(Ruchhoeft et al., 1999). In
Drosophila, in vivo time-lapse microscopy suggests that growth cone
pathfinding and filopodial dynamics are independently regulated by Cdc42
activation (Kim et al.,
2002).
Retinal growth cones without filopodia might find their appropriate
pathways by using other strategies for axonal guidance. In the
Xenopus visual system, growth cones respond to long-range cues acting
via a diffusion gradient or to local short-range cues that include cell-cell
interactions (Brittis et al.,
1995; Dingwell et al.,
2000; Tessier-Lavigne and
Goodman, 1996). Thus, retinal axon growth cones without filopodia
after impaired Ena/VASP function could use mechanisms of contact adhesion or
contact repulsion in order to follow their appropriate paths. In our work,
unlike in cytochalasin experiments, not all RGCs express the
Ena/VASP-interfering construct. Therefore, some pioneer axons are preserved
and, because they elongate at higher rate, they will lead in front of the
axons with reduced Ena/VASP function. The retinal axon growth cones of the
latter could also use adhesion mechanisms to follow the intact pioneer
axons.
|
;
Menzies et al., 2004). Because
Ena/VASP proteins are extensively expressed in all neurons as well as glial
cells of the CNS, the axonal pathway defects observed in mutant mice may be
caused by the absence of Ena/VASP activity in neurons, in glia, or in both. In
our work, at variance to the genetic approach, the activity of Ena/VASP
proteins is impaired in a subset of retinal ganglion cells and not in the
cells of the entire animal. This strategy, as well as differences between
species, may explain the absence of the mis-targeting phenotype observed in
our experimental conditions compared with knock-out mice.
Ena/VASP proteins, axonal outgrowth and branching
The present study indicates that Ena/VASP depletion autonomously regulates
the rate of retinal axon elongation, as well as growth cone pauses at the
retina exit and along the optic tract. These results indicate that, whereas
filopodia are not required for the retinal growth cones to find their
appropriate paths, they are necessary for the axon to grow at the appropriate
speed. Our observations of retinal growth cones that exhibit active
lamellipodial movement without forward advance corroborate with the study by
D. Goldberg's group showing that, as compared to laminin substrate, growth
cones on polysine have a predominant lamellar morphology and slower motion
(Rivas et al., 1992). The
capacity of growth cones to sense with their filopodia long-range cues such as
gradients of Netrin 1, Slit1, Slit2 and Ephrins localized along the visual
paths may constitute an additional mechanism for the axons to respond quickly
and appropriately at decision points (de
la Torre et al., 1997; Deiner
et al., 1997; Dingwell et al.,
2000; Hutson and Chien,
2002; Plump et al.,
2002). Ena/VASP are known to act downstream of Abl and the axon
guidance receptors Robo, DCC and UNC-5 in vertebrates, Drosophila and
C. elegans (Bashaw et al.,
2000; Colavita and Culotti,
1998; Gertler et al.,
1995; Gitai et al.,
2003; Wills et al.,
1999; Yu and Bargmann,
2001). Thus, in the absence activity of the Ena/VASP proteins, the
guidance signaling responses to these cues may be blocked, resulting in longer
pauses and forcing the growth cones to find their route using less-efficient
compensatory mechanisms.
This study in Xenopus embryos has highlighted the crucial function
of Ena/VASP proteins in regulating retinal axon terminal branching inside the
tectum. The development of the Xenopus retinotectal arborization is
dependent on different cellular mechanisms that regulate the formation of new
branches or the equilibrium between extension, retraction and elimination of
existing branches (Ruthazer and Cline,
2004; Sin et al.,
2002; Witte et al.,
1996). Our data indicate that Ena/VASP proteins promote the
addition of new branches but do not regulate the length of existing branches.
These results are in accordance with previous observations made on primary
dissociated hippocampal neurons (Lebrand
et al., 2004). Studies on the branching of cortical neurons or
dendritic trees of Purkinje cells have shown that a branch often begins as a
single filopodium and that initiation of axon branching requires both dynamic
microtubules and actin filaments (Berry and
Bradley, 1976a; Berry and
Bradley, 1976b; Dent et al.,
1999; Dent et al.,
2003; Dent and Kalil,
2001). Thus, Ena/VASP proteins, by regulating actin filament
elongation in filopodia localized at the growth cone or along the axonal
shaft, are good candidates for the regulation of the elaboration of new
branches. These results suggest strongly that, after Ena/VASP inactivation,
the final targeting of retinal axons inside the tectum may be impaired, a
topic of major interest for future investigations.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/11/2137/DC1
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPSUMMARYIntroductionMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Barzik, M., Kotova, T. I., Higgs, H. N., Hazelwood, L., Hanein,
D., Gertler, F. B. and Schafer, D. A. (2005). Ena/VASP
proteins enhance actin polymerization in the presence of barbed end capping
proteins. J. Biol. Chem.
280,28653
-28662.
Bashaw, G. J., Kidd, T., Murray, D., Pawson, T. and Goodman, C. S. (2000). Repulsive axon guidance: Abelson and Enabled play opposing roles downstream of the roundabout receptor. Cell 101,703 -715.[CrossRef][Medline]
Bear, J. E., Loureiro, J. J., Libova, I., Fassler, R., Wehland, J. and Gertler, F. B. (2000). Negative regulation of fibroblast motility by Ena/VASP proteins. Cell 101,717 -728.[CrossRef][Medline]
Bear, J. E., Svitkina, T. M., Krause, M., Schafer, D. A., Loureiro, J. J., Strasser, G. A., Maly, I. V., Chaga, O. Y., Cooper, J. A., Borisy, G. G. et al. (2002). Antagonism between Ena/VASP proteins and actin filament capping regulates fibroblast motility. Cell 109,509 -521.[CrossRef][Medline]
Bentley, D. and O'Connor, T. P. (1994). Cytoskeletal events in growth cone steering. Curr. Opin. Neurobiol. 4,43 -48.[CrossRef][Medline]
Berry, M. and Bradley, P. (1976a). The growth of the dendritic trees of Purkinje cells in irradiated agranular cerebellar cortex. Brain Res. 116,361 -387.[CrossRef][Medline]
Berry, M. and Bradley, P. (1976b). The growth of the dendritic trees of Purkinje cells in the cerebellum of the rat. Brain Res. 112,1 -35.[CrossRef][Medline]
Bovolenta, P. and Mason, C. (1987). Growth cone morphology varies with position in the developing mouse visual pathway from retina to first targets. J. Neurosci. 7,1447 -1460.[Abstract]
Brittis, P. A., Lemmon, V., Rutishauser, U. and Silver, J. (1995). Unique changes of ganglion cell growth cone behavior following cell adhesion molecule perturbations: a time-lapse study of the living retina. Mol. Cell. Neurosci. 6, 433-449.[CrossRef][Medline]
Campbell, D. S. and Holt, C. E. (2001). Chemotropic responses of retinal growth cones mediated by rapid local protein synthesis and degradation. Neuron 32,1013 -1026.[CrossRef][Medline]
Campbell, R. E., Tour, O., Palmer, A. E., Steinbach, P. A.,
Baird, G. S., Zacharias, D. A. and Tsien, R. Y. (2002). A
monomeric red fluorescent protein. Proc. Natl. Acad. Sci.
USA 99,7877
-7882.
Chang, C., Adler, C. E., Krause, M., Clark, S. G., Gertler, F. B., Tessier-Lavigne, M. and Bargmann, C. I. (2006). MIG-10/lamellipodin and AGE-1/PI3K promote axon guidance and outgrowth in response to slit and netrin. Curr. Biol. 16,854 -862.[CrossRef][Medline]
Chien, C. B., Rosenthal, D. E., Harris, W. A. and Holt, C. E. (1993). Navigational errors made by growth cones without filopodia in the embryonic Xenopus brain. Neuron 11,237 -251.[CrossRef][Medline]
Colavita, A. and Culotti, J. G. (1998). Suppressors of ectopic UNC-5 growth cone steering identify eight genes involved in axon guidance in Caenorhabditis elegans. Dev. Biol. 194,72 -85.[CrossRef][Medline]
Davenport, R. W., Dou, P., Rehder, V. and Kater, S. B. (1993). A sensory role for neuronal growth cone filopodia. Nature 361,721 -724.[CrossRef][Medline]
de la Torre, J. R., Hopker, V. H., Ming, G. L., Poo, M. M., Tessier-Lavigne, M., Hemmati-Brivanlou, A. and Holt, C. E. (1997). Turning of retinal growth cones in a netrin-1 gradient mediated by the netrin receptor DCC. Neuron 19,1211 -1224.[CrossRef][Medline]
Deiner, M. S., Kennedy, T. E., Fazeli, A., Serafini, T., Tessier-Lavigne, M. and Sretavan, D. W. (1997). Netrin-1 and DCC mediate axon guidance locally at the optic disc: loss of function leads to optic nerve hypoplasia. Neuron 19,575 -589.[CrossRef][Medline]
Dent, E. W. and Kalil, K. (2001). Axon
branching requires interactions between dynamic microtubules and actin
filaments. J. Neurosci.
21,9757
-9769.
Dent, E. W., Callaway, J. L., Szebenyi, G., Baas, P. W. and
Kalil, K. (1999). Reorganization and movement of microtubules
in axonal growth cones and developing interstitial branches. J.
Neurosci. 19,8894
-8908.
Dent, E. W., Tang, F. and Kalil, K. (2003). Axon guidance by growth cones and branches: common cytoskeletal and signaling mechanisms. Neuroscientist 9, 343-353.[Abstract]
Dingwell, K. S., Holt, C. E. and Harris, W. A. (2000). The multiple decisions made by growth cones of RGCs as they navigate from the retina to the tectum in Xenopus embryos. J. Neurobiol. 44,246 -259.[CrossRef][Medline]
Ellis, S. and Mellor, H. (2000). Regulation of endocytic traffic by rho family GTPases. Trends Cell Biol. 10,85 -88.[CrossRef][Medline]
Gertler, F. B., Comer, A. R., Juang, J. L., Ahern, S. M., Clark,
M. J., Liebl, E. C. and Hoffmann, F. M. (1995). enabled, a
dosage-sensitive suppressor of mutations in the Drosophila Abl tyrosine
kinase, encodes an Abl substrate with SH3 domain-binding properties.
Genes Dev. 9,521
-533.
Gertler, F. B., Niebuhr, K., Reinhard, M., Wehland, J. and Soriano, P. (1996). Mena, a relative of VASP and Drosophila Enabled, is implicated in the control of microfilament dynamics. Cell 87,227 -239.[CrossRef][Medline]
Gitai, Z., Yu, T. W., Lundquist, E. A., Tessier-Lavigne, M. and Bargmann, C. I. (2003). The netrin receptor UNC-40/DCC stimulates axon attraction and outgrowth through enabled and, in parallel, Rac and UNC-115/AbLIM. Neuron 37, 53-65.[CrossRef][Medline]
Goh, K. L., Cai, L., Cepko, C. L. and Gertler, F. B. (2002). Ena/VASP proteins regulate cortical neuronal positioning. Curr. Biol. 12,565 -569.[CrossRef][Medline]
Harris, W. A., Holt, C. E. and Bonhoeffer, F. (1987). Retinal axons with and without their somata, growing to and arborizing in the tectum of Xenopus embryos: a time-lapse video study of single fibres in vivo. Development 101,123 -133.[Abstract]
Holt, C. E. (1989). A single-cell analysis of early retinal ganglion cell differentiation in Xenopus: from soma to axon tip. J. Neurosci. 9,3123 -3145.[Abstract]
Holt, C. E., Garlick, N. and Cornel, E. (1990). Lipofection of cDNAs in the embryonic vertebrate central nervous system. Neuron 4,203 -214.[CrossRef][Medline]
Hutson, L. D. and Chien, C. B. (2002). Pathfinding and error correction by retinal axons: the role of astray/robo2. Neuron 33,205 -217.[CrossRef][Medline]
Huttelmaier, S., Harbeck, B., Steffens, O., Messerschmidt, T., Illenberger, S. and Jockusch, B. M. (1999). Characterization of the actin binding properties of the vasodilator-stimulated phosphoprotein VASP. FEBS Lett. 451,68 -74.[CrossRef][Medline]
Isbister, C. M. and O'Connor, T. P. (2000). Mechanisms of growth cone guidance and motility in the developing grasshopper embryo. J. Neurobiol. 44,271 -280.[CrossRef][Medline]
Kim, M. D., Kolodziej, P. and Chiba, A. (2002).
Growth cone pathfinding and filopodial dynamics are mediated separately by
Cdc42 activation. J. Neurosci.
22,1794
-1806.
Krause, M., Dent, E. W., Bear, J. E., Loureiro, J. J. and Gertler, F. B. (2003). Ena/VASP proteins: regulators of the actin cytoskeleton and cell migration. Annu. Rev. Cell Dev. Biol. 19,541 -564.[CrossRef][Medline]
Lambrechts, A., Kwiatkowski, A. V., Lanier, L. M., Bear, J. E.,
Vandekerckhove, J., Ampe, C. and Gertler, F. B. (2000).
cAMP-dependent protein kinase phosphorylation of EVL, a Mena/VASP relative,
regulates its interaction with actin and SH3 domains. J. Biol.
Chem. 275,36143
-36151.
Lanier, L. M., Gates, M. A., Witke, W., Menzies, A. S., Wehman, A. M., Macklis, J. D., Kwiatkowski, D., Soriano, P. and Gertler, F. B. (1999). Mena is required for neurulation and commissure formation. Neuron 22,313 -325.[CrossRef][Medline]
Lebrand, C., Dent, E. W., Strasser, G. A., Lanier, L. M., Krause, M., Svitkina, T. M., Borisy, G. G. and Gertler, F. B. (2004). Critical role of Ena/VASP proteins for filopodia formation in neurons and in function downstream of netrin-1. Neuron 42,37 -49.[CrossRef][Medline]
Lee, E. and De Camilli, P. (2002). Dynamin at
actin tails. Proc. Natl. Acad. Sci. USA
99,161
-166.
Lilienbaum, A., Reszka, A. A., Horwitz, A. F. and Holt, C. E. (1995). Chimeric integrins expressed in retinal ganglion cells impair process outgrowth in vivo. Mol. Cell. Neurosci. 6,139 -152.[CrossRef][Medline]
Lin, C. H. and Forscher, P. (1995). Growth cone advance is inversely proportional to retrograde F-actin flow. Neuron 14,763 -771.[CrossRef][Medline]
Mallavarapu, A. and Mitchison, T. (1999).
Regulated actin cytoskeleton assembly at filopodium tips controls their
extension and retraction. J. Cell Biol.
146,1097
-1106.
Mann, F., Ray, S., Harris, W. and Holt, C. (2002). Topographic mapping in dorsoventral axis of the Xenopus retinotectal system depends on signaling through ephrin-B ligands. Neuron 35,461 -473.[CrossRef][Medline]
Marsh, L. and Letourneau, P. C. (1984). Growth
of neurites without filopodial or lamellipodial activity in the presence of
cytochalasin B. J. Cell Biol.
99,2041
-2047.
Mason, C. A. and Wang, L. C. (1997). Growth
cone form is behavior-specific and, consequently, position-specific along the
retinal axon pathway. J. Neurosci.
17,1086
-1100.
Menzies, A. S., Aszodi, A., Williams, S. E., Pfeifer, A.,
Wehman, A. M., Goh, K. L., Mason, C. A., Fassler, R. and Gertler, F. B.
(2004). Mena and vasodilator-stimulated phosphoprotein are
required for multiple actin-dependent processes that shape the vertebrate
nervous system. J. Neurosci.
24,8029
-8038.
Merrifield, C. J., Moss, S. E., Ballestrem, C., Imhof, B. A., Giese, G., Wunderlich, I. and Almers, W. (1999). Endocytic vesicles move at the tips of actin tails in cultured mast cells. Nat. Cell Biol. 1,72 -74.[CrossRef][Medline]
Murphy, C., Saffrich, R., Grummt, M., Gournier, H., Rybin, V., Rubino, M., Auvinen, P., Lutcke, A., Parton, R. G. and Zerial, M. (1996). Endosome dynamics regulated by a Rho protein. Nature 384,427 -432.[CrossRef][Medline]
Myers, P. Z. and Bastiani, M. J. (1993). Growth cone dynamics during the migration of an identified commissural growth cone. J. Neurosci. 13,127 -143.[Abstract]
Niebuhr, K., Ebel, F., Frank, R., Reinhard, M., Domann, E., Carl, U. D., Walter, U., Gertler, F. B., Wehland, J. and Chakraborty, T. (1997). A novel proline-rich motif present in ActA of Listeria monocytogenes and cytoskeletal proteins is the ligand for the EVH1 domain, a protein module present in the Ena/VASP family. EMBO J. 16,5433 -5444.[CrossRef][Medline]
Nieukoop, P. D. and Faber, J. (1967).Normal Table of Xenopus laevis (Daudin) . Amsterdam: North Holland.
O'Connor, T. P., Duerr, J. S. and Bentley, D. (1990). Pioneer growth cone steering decisions mediated by single filopodial contacts in situ. J. Neurosci. 10,3935 -3946.[Abstract]
Ochoa, G. C., Slepnev, V. I., Neff, L., Ringstad, N., Takei, K.,
Daniell, L., Kim, W., Cao, H., McNiven, M., Baron, R. et al.
(2000). A functional link between dynamin and the actin
cytoskeleton at podosomes. J. Cell Biol.
150,377
-389.
Plump, A. S., Erskine, L., Sabatier, C., Brose, K., Epstein, C. J., Goodman, C. S., Mason, C. A. and Tessier-Lavigne, M. (2002). Slit1 and Slit2 cooperate to prevent premature midline crossing of retinal axons in the mouse visual system. Neuron 33,219 -232.[CrossRef][Medline]
Qualmann, B. and Kessels, M. M. (2002). Endocytosis and the cytoskeleton. Int. Rev. Cytol. 220,93 -144.[Medline]
Qualmann, B., Kessels, M. M. and Kelly, R. B.
(2000). Molecular links between endocytosis and the actin
cytoskeleton. J. Cell Biol.
150,F111
-F116.
Reinhard, M., Halbrugge, M., Scheer, U., Wiegand, C., Jockusch, B. M. and Walter, U. (1992). The 46/50 kDa phosphoprotein VASP purified from human platelets is a novel protein associated with actin filaments and focal contacts. EMBO J. 11,2063 -2070.[Medline]
Riehl, R., Johnson, K., Bradley, R., Grunwald, G. B., Cornel, E., Lilienbaum, A. and Holt, C. E. (1996). Cadherin function is required for axon outgrowth in retinal ganglion cells in vivo. Neuron 17,837 -848.[CrossRef][Medline]
Rivas, R. J., Burmeister, D. W. and Goldberg, D. J. (1992). Rapid effects of laminin on the growth cone. Neuron 8,107 -115.[CrossRef][Medline]
Ruchhoeft, M. L., Ohnuma, S., McNeill, L., Holt, C. E. and Harris, W. A. (1999). The neuronal architecture of Xenopus retinal ganglion cells is sculpted by rho-family GTPase
