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First published online March 6, 2009
doi: 10.1242/10.1242/dev.027920
1 Department of Complexity Science and Engineering, Graduate School of Frontier
Sciences, University of Tokyo, Kashiwanoha, Kashiwa, Chiba 277-8561,
Japan.
2 Department of Physics, Graduate School of Science, University of Tokyo, Hongo,
Bunkyo-ku, Tokyo 113-0033, Japan.
* Author for correspondence (e-mail: nose{at}k.u-tokyo.ac.jp)
Accepted 23 January 2009
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SUMMARY |
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INTRODUCTION
MATERIALS AND METHODS
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DISCUSSION
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Key words: Target recognition, Postsynaptic filopodia, Capricious, Synaptogenesis
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INTRODUCTION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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; Garrity and Zipursky,
1995; Benson et al.,
2001). A traditional view is that presynaptic growth cones explore
the target region by elaborating motile filopodia, which sense and recognize
specific target-recognition molecules that are expressed on the postsynaptic
cells. The postsynaptic cell has generally been regarded as a static structure
that plays only a passive role in this synaptic matchmaking process. Recent
time-lapse studies using live-imaging techniques, however, show the dynamic
nature of postsynaptic cells during periods of targeting and synaptogenesis.
For example, dendritic filopodia of mammalian central neurons display rapid
and extensive dynamics before synaptogenesis
(Ziv and Smith, 1996;
Maletic-Savatic et al., 1999;
Yuste and Bonhoeffer, 2004).
It has also been shown that postsynaptic motoneurons in zebrafish extend
highly motile filopodia, which interact with the presynaptic Mauthner axons
(Jontes et al., 2000). These
studies led to the suggestion that postsynaptic dynamism may contribute to the
formation and plasticity of synapses
(Cohen-Cory, 2002;
Goda and Davis, 2003).
However, whether postsynaptic filopodia are able to select appropriate
presynaptic partners has not been addressed in these previous studies. Just as
the presynaptic growth cone specifically recognizes the target cells, do
postsynaptic filopodia seek out appropriate presynaptic cells?
The neuromuscular connectivity of Drosophila is well suited for
studying the molecular mechanisms of synaptic target recognition, as it is
possible to follow the behavior of individual motoneurons as they establish
contacts with the target muscles
(Keshishian et al., 1996).
Several target recognition molecules that are expressed in specific muscles
and have roles in determining synaptic specificity have previously been
identified (Nose et al., 1992;
Chiba et al., 1995;
Winberg et al., 1998;
Shishido et al., 1998). Before
the discovery of myopodia, it was generally believed that active axonal growth
cones find and recognize such target recognition cues expressed on muscles and
establish specific synaptic contacts. Muscle cells were thought to be passive
players in this process, probably due to their static appearance in fixed
embryos. The discovery of myopodia, however, challenged this view, suggesting
the possibility that neuromuscular recognition is a reciprocal process whereby
neurons and muscles seek each other out
(Ritzenthaler et al., 2000;
Ritzenthaler and Chiba, 2001).
Myopodia are dynamic filopodia of muscles that are most active before the
arrival of the motoneuronal growth cones and progressively cluster at the
nascent synaptic site. Based on their dynamic nature and on the observation
that they make intimate contacts with the presynaptic growth cones, myopodia
were proposed to play active roles in the guidance of motoneuronal growth
cones. However, although there is evidence that clustering of myopodia depends
on specific interaction between pre- and postsynaptic cells
(Ritzenthaler and Chiba,
2001), the precise role of myopodia in mediating target
specificity is unknown. Myopodia might simply function to increase the
probability of chance encounters with presynaptic growth cones by increasing
the surface area of muscles. Alternatively, protrusive activity of myopodia
might be important for a local and precise interaction with the presynaptic
cells, with possible mutual exchange of information.
In this study, we first investigated the subcellular localization of
Capricious (CAPS), in relation to the behavior of myopodia, by live imaging of
the green fluorescent protein (GFP)-tagged protein CAPS-GFP. CAPS is a
transmembrane protein with leucine-rich repeats (LRRs) that is expressed in
specific subsets of muscles and motoneurons, including ventral muscle 12 (M12)
and the motoneurons that innervate it (MN12s)
(Shishido et al., 1998).
Previous genetic analysis implicated CAPS as an attractive target recognition
molecule on M12. Here, we found that CAPS expressed in M12 localizes to the
tips of myopodia before the arrival of motoneuronal growth cones. The
distribution of CAPS suggested the possibility that neuromuscular interaction
is initiated at the tips of myopodia, at a distance from the body of the
muscles. To address this issue, we performed simultaneous live imaging of pre-
and postsynaptic cells. By following the behavior of individual myopodia as
they extend to and make contact with the approaching growth cones, we found
that initial contacts do occur at the tips of myopodia. Although some of these
contacts were stabilized to form synapses, others, including those with the
non-partner motoneurons, were retracted, suggesting that myopodia are able to
select specific presynaptic cells. In caps mutants, the number of
contacts between M12 myopodia and the presynaptic growth cones is reduced.
These findings are consistent with the notion that local, contact-mediated
signaling at the tips of postsynaptic filopodia is crucial for target
selection.
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MATERIALS AND METHODS
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DISCUSSION
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) and elav-GAL4-3E1
(Davis et al., 1997),
respectively. For vital visualization of cell morphology, we used
UAS-myristylated-GFP (a gift from Dr Chiba, University of Illinois)
(Ritzenthaler et al., 2000),
UAS-myristylated-CFP and elav-myristylated-YFP
(Kohsaka et al., 2007). For
muscle expression of intact CAPS, UAS-capsIa5
(Taniguchi et al., 2000) was
crossed with GAL4-5053A. To express an intracellular deletion form of
CAPS in muscles or in motoneurons, UAS-capsID4
(Taniguchi et al., 2000) was
crossed with GAL4-5053A or elav-GAL4-3E1, respectively. The
pros mutant allele used was prosm4
(Broadie and Bate, 1993). The
caps null mutant alleles used were capsC28fs and
capsL253fs (Sakurai et
al., 2007). Df(3L)Ly is a deficiency that lacks the
entire caps gene (Shishido et
al., 1998). The trn null mutant allele used was
trn25/4 (Chang et al.,
1993; Milan et al.,
2001). The caps, trn double mutant alleles used were
capsC28fs, trndelta17 (null for both
caps and trn) (Sakurai
et al., 2007) and caps65.2,
trnS064117 (strong hypomorphic alleles for caps and
trn) (Kurusu et al.,
2008). The capsC28fs, trndelta17
chromosome is deficient for two neighboring genes, CG33262 and CG11281, in
addition to caps and tartan. However, the involvement of
CG33262 and CG11281 is unlikely because similar phenotypes were observed in
capsC28fs, trndelta17 and in
caps65.2, trnS064117. capsC28fs,
trndelta17 embryos also display mild phenotypes in axon
guidance within the ventral nerve cord and muscle attachment site formation of
M12 (see Kurusu et al., 2008).
We therefore restricted our analyses to segments with normal motor axon
projection and muscle attachment sites.
Generation of UAS transgenic lines
The 3' end of the caps open reading frame (ORF) was fused
with the 5' end of GFP (or YFP) ORF via a six amino
acid linker. The resultant caps-GFP (or caps-YFP) cDNA was
cloned into the pUAST vector. Individual constructs were introduced
into y, w flies by germline transformation according to a standard
protocol. Two independent transformant lines, UAS-caps-GFP 25 (an
insertion on the third chromosome) and UAS-caps-YFP B1 (an insertion
on the second chromosome), were used in this study. CAPS-GFP/YFP expressed on
M12 using the two lines showed the same distribution pattern.
Immunohistochemistry
Dissection and immunohistochemistry of embryos and larvae were performed as
described previously (Nose et al.,
1997). The following antibodies were used: monoclonal antibody
(mAB) 22C10 (Fujita et al.,
1982), mAB1D4 (Van Vactor et
al., 1993), rabbit serum antibodies (sABs) against intracellular
domains of CAPS (Shishido et al.,
1998) and goat anti-HRP antibodies (Jackson Laboratories). Vital
labeling of motor axons was performed by incubating dissected embryos with
anti-HRP antibodies conjugated with Cy5 (Jackson Laboratories) for 10 minutes
before visualization (Ritzenthaler et al.,
2000). For developmental analysis of embryos, we collected eggs
laid for 30 minutes and verified the developmental stage based on both the
actual time passed before the time of observation or fixation, and the
morphology of the midgut.
Live visualization
We captured the images of myopodia and growth cones of dechorionated whole
embryos in insect saline using an LSM 510 confocal system (Zeiss) or FV1000
confocal system (Olympus). We confirmed that interruption of synaptogenesis by
photodamage was very minor if any: ISNb axons made normal T-shaped terminals
on muscles 12, 13, 6 and 7 in a normal time window under our visualization
condition. Typically, 10-20 optical sections of 0.5-1 µm were recorded over
the course of 2 hours, starting every 1-2 minutes. Dual-color time-lapse
images of a whole embryo are despeckled once by ImageJ software (NIH).
Quantification of interaction between myopodia and growth cones
To analyze the dynamics of myopodia and growth cones, we used 4D confocal
time-lapse images. We defined `contact' between myopodia and growth cones as
positions at which there was no gap in fluorescence intensity at our level of
resolution. We manually counted points of contact (or no contact) using LSM510
or FV1000 software.
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RESULTS
DISCUSSION
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;
Kohsaka et al., 2007), we
examined the subcellular localization of CAPS by live imaging of the dissected
embryos expressing CAPS-GFP. We generated a C-terminal CAPS-GFP fusion
construct, which appears to be functional according to the following criteria.
First, CAPS-GFP retains its ability to induce ectopic synapse formation by
MN12s when expressed in all muscles
(Shishido et al., 1998) (see
Fig. S1 in the supplementary material). Second, CAPS-GFP retains its ability
to induce the pathfinding defect of a motor nerve ISNb, when misexpressed in
all neurons (Taniguchi et el.,
2000) (data not shown). We expressed CAPS-GFP in M12, a CAPS-positive muscle, and investigated the subcellular distribution of CAPS-GFP during target selection. To express CAPS-GFP, we used an M12-specific driver, 5053A-GAL4, which confers expression of UAS transgenes before the arrival of MN12 growth cones. This allowed us to examine CAPS-GFP distribution within muscle 12 during the period of time in which it is normally innervated.
At 13:00 hours after egg-laying (AEL), when the growth cones of MN12s are
approaching M12, a number of myopodia protrude from the muscles
(Ritzenthaler et al., 2000).
We observed strong accumulation of CAPS-GFP at the tips of myopodia
(Fig. 1A, arrowheads). This was
in contrast to the distribution of myristylated GFP (mGFP), which is uniform
along the length of myopodia (Fig.
1B). Immunohistochemistry of intact CAPS (without GFP) expressed
on muscles also revealed its presence at the tips of short myopodia that
survived fixation, indicating that the localization is not an artifact caused
by the addition of GFP (Fig.
1C). Although endogenous caps is expressed in M12 at this
stage, CAPS protein expression is below detection by our immunohistochemical
procedures (Shishido et al.,
1998). However, the data described above suggest that endogenous
CAPS would also be localized to myopodia during this time period. When
CAPS-GFP was expressed in neurons, we did not observe concentration of
CAPS-GFP at the tips of growth cone filopodia (see Fig. S2 in the
supplementary material).
The accumulation of a target recognition molecule such as CAPS at the tips of myopodia suggests that activity of far-reaching myopodia might function to efficiently present the target markers to presynaptic motoneurons that are approaching the target. To pursue this possibility further, we examined the trajectory of myopodia in relation to the paths of motoneuronal growth cones. Concurrent visualization of CAPS-GFP by fluorescence and contours of muscles by differential interference contrast (DIC) showed that some of the myopodia of M12 extended along the interior side of a neighboring muscle 13 (M13) and reached as far as the proximal (closer to the central nervous system) edge of M13 (Fig. 1D,E). This trajectory of myopodia corresponds to the path that the growth cones of MN12s will later take (shown schematically in Fig. 1F,G). These observations are consistent with the idea that M12 can guide neuronal growth cones toward itself by extending myopodia.
The accumulation of CAPS-GFP at the tips of myopodia appears to be an
autonomous process in muscles that is independent of innervating motor axons.
First, CAPS-GFP is concentrated at the tips of myopodia more than 30 minutes
before the arrival of motoneuronal growth cones. Second, concentration of
CAPS-GFP at the tips of myopodia took place normally in prospero
(pros) mutants, in which extension of motoneuronal axons is severely
delayed (Ritzenthaler et al.,
2000; Broadie and Bate,
1993) (Fig. 2).
These observations suggest that accumulation of CAPS at the tips of myopodia
is not dependent on signals from the motoneurons.
Neuromuscular contacts at the tips of myopodia
Accumulation of CAPS-GFP at the tips implies that initial recognition
events between presynaptic motoneurons and postsynaptic muscles might occur at
the tips of myopodia rather than at the muscle fiber itself. Previous
time-lapse analysis of myopodia and immunohistochemical visualization of motor
axons at the end of the live imaging showed that myopodia extensively interact
with the presynaptic filopodia and become clustered at the future synaptic
site (Ritzenthaler et al.,
2000). However, where the initial contact between the pre- and
postsynaptic cells occurs and how these initial contacts are stabilized to
form the synaptic sites have remained unknown. To analyze the dynamics of
myopodia and presynaptic filopodia in intact embryos as they initiate their
first contact and eventually intermingle to form synapses, we conducted
time-lapse imaging of both structures by genetically expressing mGFP. We
captured time-lapse images that span the period of target selection and early
synaptogenesis (see Movie 1 in the supplementary material). By following the
behavior of individual myopodia, we could locate the contacts between myopodia
and motoneurons and trace the final fate of these initial contacts.
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We next followed the final fate of the contacts between myopodia and motoneuronal growth cones. We found that 45% (29/65) of the contacts were stabilized and contributed to the formation of synapses. The remaining 55% of contacts were eventually eliminated. For example, in the time-lapse imaging shown in Fig. 4A, we followed the behavior of two myopodia as they encountered the growth cones. The contact made by the upper myopodium was stabilized. Stabilization of the contact was accompanied by the rapid recruitment of additional myopodia and neuronal filopodia to that site, leading to much thicker bundles of microprocesses (Fig. 4A, 88 minutes). By contrast, contact made by the lower myopodium was eliminated. We analyzed the duration of such lost contacts and found that 78% (28/36) of them were transient contacts that lasted less than 10 minutes (4.0 minutes on average) and the remaining 22% were semi-stable contacts that were maintained for 10-60 minutes. Thus, myopodial contacts can be categorized into three groups: transient contacts that last less than 10 minutes, semi-stable contacts that last 10-60 minutes, and stable contacts that contribute to the formation of synapses.
Partner recognition by myopodia
The concentration of CAPS and occurrence of the initial neuromuscular
contact at the tip of the myopodium strongly suggests that it is a major site
of neuromuscular recognition. It might be the site at which myopodia present
their target marker(s) to the presynaptic cell by direct cell-cell contact. At
the same time, it might also be the site at which myopodia `sense' the
information provided by the presynaptic cells. If such mutual recognition
process takes place, then one would expect the behavior of myopodia to differ
depending on which cells they encounter. For example, myopodia of M12 might
distinguish between neurites of partner (MN12s) and non-partner motoneurons,
such as those that innervate the neighboring muscle 13 (MN13s). During the
early phase of our time-lapse imaging described above (12:45-13:30 hours), the
growth cone of MN12s and MN13s were too close to be discerned by morphology
(Fig. 5C). To determine whether
the behavior of myopodia differs depending on whether they encounter partner
or non-partner neurons, we next studied contacts made during a later phase of
the imaging period (13:30-14:00 hours), when growth cones of MN12s and MN13s
are mostly discernable because of distal movement of the MN12 growth cones
(Fig. 5C'). By this
stage, MN13s (including RP1 and RP4 neurons) had begun to arborize along the
proximal edge of M13 (Sink and Whitington,
1991; Halpern et al.,
1991). By contrast, the growth cones of MN12s (including RP5) had
moved to a more distal region. We therefore compared the behavior of myopodial
contacts with the neural processes situated on the distal half of muscle 13
(putative MN12 growth cones) to the behavior of contacts with those situated
on the proximal half of M13 (putative MN13 growth cones) (see
Fig. 5A). Although
discrimination of MN12s and MN13s is not unambiguous because these neurons
have yet to withdraw all of the ectopic filopodia projecting to non-target
muscles at this stage, we hoped to be able to detect some tendency by this
analysis. Indeed, we found that while 54% of the contacts with the putative
partner neurons were stabilized (n=26 myopodia in 21 hemisegments),
none of the contacts with the putative non-partner neurons was stabilized
(n=30 myopodia in 21 hemisegments; the difference in stabilization
rate was statistically significant, P=1.7x10-6,
Fisher's test) (Fig. 5A,B,D).
These results support the notion that myopodia are able to discriminate
between partner and non-partner motoneurons.
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). While interacting with the clustered myopodia,
growth cones transform into a bifurcated terminal-like structure
(Fig. 6C,D, arrowheads). The
nascent presynaptic terminals were often observed within the lamellipodia-like
structure of myopodia (Fig. 6C,
asterisk). These results suggest that filopodial interaction between neurons
and the target muscle is important not only for myopodia clustering but also
for the formation of presynaptic terminals. The filopodia-myopodia complex may
provide a substrate upon which the initial pre- and postsynaptic
differentiation takes place. We also studied CAPS localization during this period by triple labeling of CAPS-YFP, myopodia and growth cones (Fig. 7). As described above, CAPS is concentrated at the tip of myopodia just before contact with the growth cone (Fig. 7A). Interestingly, CAPS-YFP concentration remained at the tips even after the growth cones contacted and extended along the myopodia (Fig. 7B). When myopodia clustering is completed, strong concentration of CAPS-YFP was no longer observed in myopodia (Fig. 7C), even though CAPS-YFP continued to be expressed by M12. The specific and temporal concentration of CAPS is consistent with the idea that CAPS is involved in the initial recognition between muscles and neurons at the tip of myopodia.
Synaptogenesis in caps mutants
The existence of CAPS at the tips of myopodia suggests its possible
involvement in early recognition events mediated by myopodia. We therefore
performed live imaging to study the interaction between myopodia of M12, a
caps-expressing muscle, and the innervating motoneuronal growth
cones, in caps null mutants. There are no gross developmental defects
in the differentiation of motoneurons in caps mutants
(Shishido et al., 1998). Axon
projection of MN12s to the vicinity of the target muscle also appeared normal,
as assessed by the distance between the growth cones and M12 at the beginning
of the time-lapse imaging (13:00 hours) (see Fig. S4 in the supplementary
material). There was no overt abnormality in the differentiation of muscles
(see Fig. S5 in the supplementary material) or in the morphology of myopodia.
The number of myopodia was also normal [7.2 per muscle (n=21) in wild
type compared with 6.1 (n=24) in caps mutants;
P>0.05, two-tailed t-test]. Therefore, in caps
mutants, myopodia protrude and initiate their contacts with the growth cones
as in the wild type. However, subsequent stabilization of the contacts
appeared to be affected. At 13:30 hours, there was a reduced number of contact
points between myopodia and growth cones
(Fig. 8A,B) [4.7 in cont
(n=17); 3.0 in caps mutant (n=22);
P<0.01, two-tailed t-test]. The number of free myopodia
that do not contact the growth cones was unchanged, suggesting that formation
of myopodia was not affected [5.7 per muscle (n=17) in wild type
compared with 5.7 (n=22) in caps mutants;
P>0.05, two-tailed t-test]. These observations imply that
fewer contacts between the myopodia and growth cones are stabilized in
caps mutants. Thus, caps may be involved in the initial
recognition between myopodia and their partner motoneurons.
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As described in the Introduction, CAPS is expressed both in presynaptic
MN12 and postsynaptic M12. To assess if caps is required pre- and/or
postsynaptically, we studied the effects of expressing a putative
dominant-negative form of CAPS (CAPS-ID, a deletion mutant lacking an
intracellular domain) (Taniguchi et al.,
2000) in either motoneurons or M12s. We found that expression in
muscles but not in motoneurons causes the same terminal defects as seen in
caps mutants (Fig.
8G). These results further support the notion that
caps-mediated recognition at the tip of myopodia is important for the
formation of specific synaptic connections.
The phenotype observed in caps mutants described above is relatively mild, suggesting that there are redundant molecules that can contribute to target recognition in the absence of CAPS. The best candidate for such a redundant target recognition molecule is Tartan, which is the closest relative of CAPS in the Drosophila proteome (65% identical to CAPS within the extracellular domain) and is encoded by a nearby gene. Caps and Tartan are clearly the product of a gene duplication, suggesting that an ancestral organism may have used only one CAPS/Tartan homolog for target recognition.
Indeed, Kurusu et al. (Kurusu et al.,
2008) recently reported that double mutants for caps and
tartan have stronger axon guidance phenotypes than the single
mutants, and some segments have ISNbs with `looped' terminals, suggesting a
defect in recognition of M12 (Kurusu et
al., 2008). We therefore analyzed filopodia-myopodia interaction
in caps tartan double mutants. As in caps mutants, axon
elongation towards the target region was relatively normal in the double
mutants (see Fig. S4 in the supplementary material). However, the following
target recognition and terminal formation were severely affected. The number
of contact points between growth cones and M12 was greatly reduced
(Fig. 8A,B). The length of axon
terminals on M12 was also much shorter
(Fig. 8D,E), with some muscles
completely lacking the terminals (7 out of 51 hemisegments compared with 0 out
of 47 hemisegments in control; P=0.0084, Fisher's test) (see
Kurusu et al., 2008). These
phenotypes were qualitatively similar to those of caps single mutants
and trn single mutants (Fig.
8) but were much stronger. These results are consistent with the
idea that the function of CAPS in mediating recognition between M12 and its
presynaptic motoneurons is partially redundant with that of Tartan. In the
absence of both CAPS and Tartan, most M12s still became innervated, suggesting
that there are additional cell surface proteins that also contribute to target
recognition of M12, but the synapse on M12 is much reduced relative to wild
type. Whether the synapse could repair itself and grow to a normal size in the
absence of CAPS and Tartan could not be determined, because tartan
mutants are embryonic lethal.
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SUMMARY
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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). Sensory
function of filopodia has been best illustrated in neuronal growth cones
(Henley and Poo, 2004;
Gallo and Letourneau, 2004).
For example, a single filopodium of a neuron in culture is able to steer the
entire growth cone when it contacts a more adhesive substrate
(Letourneau, 1975). Growth
cones of grasshopper pioneer axons in vivo change their direction upon the
contact of a single filopodium with guidepost cells
(O'Connor et al., 1990).
Filopodia are also found both pre- and postsynaptically during the process of
synaptogenesis and are believed to actively participate in cellular
interactions (Cohen-Cory, 2002;
Goda and Davis, 2003).
Although there is evidence for the sensory function of presynaptic filopodia,
few studies have addressed the question of whether postsynaptic filopodia are
able to select specific partners. In this study, we took advantage of the fact
that highly specific and stereotyped synaptogenesis occurs during a relatively
short period of time at the neuromuscular junction of Drosophila. We
present several lines of evidence that support the active role of myopodia,
postsynaptic filopodia of muscles, in synaptic partner recognition.
We found a striking concentration of CAPS at the tips of myopodia. If
myopodia have a passive role and merely help increase the target area, then it
would seem to be more efficient for CAPS to be distributed uniformly along the
length of myopodia. There would be more chance for the target marker to be
presented to the presynaptic cells. Concentration of CAPS at the tips instead
supports the idea that myopodia play an active role in directing growth cones
to their targets. Filopodia and other cellular extensions have been observed
in a variety of cells that send and/or receive signals, and implicated in
contact-mediated long-distance communication
(Rørth, 2003). By
presenting molecules such as CAPS at the tips, myopodia might be able to
efficiently send signals to presynaptic motoneurons. At the same time,
myopodia might receive signals from the presynaptic cells by using molecules
such as CAPS as sensors. Such bidirectional signaling occurring at the tips of
myopodia might be a trigger for synaptic matchmaking. As CAPS is also
expressed in MN12s and can function as a homophilic cell adhesion molecule in
vitro (Shinza-Kameda et al.,
2006), the signaling might be mediated by homophilic interaction
between CAPS on pre- and postsynaptic cells.
It has been postulated that the tips of filopodia are the signaling center
that regulate filopodial extension, retraction and adhesion from the following
reasons. First, it is often the distal part of the filopodium that makes
contact with the environmental cues
(Letourneau, 1975;
O'Connor et al., 1990).
Second, actin monomers are added to the barbed ends of filaments at the tips
of filopodia (Mallavarapu and Mitchison,
1999). Third, several signaling and adhesion molecules, including
Mena, integrins and the tyrosine-phosphorylated proteins, are concentrated at
the tips of filopodia (Bear et al.,
2002; Grabham and Goldberg,
1997; Wu and Goldberg,
1993). Therefore, filopodia tips are well situated to link
information from the environment to the dynamics of filopodia. Concentration
of CAPS suggests a possibility that signaling events at the tips of myopodia
are crucial in selective synapse formation. Consistent with this idea, we
found, by live imaging of neuromuscular interaction in vivo, that many of the
initial contacts between motoneuronal growth cones and muscles do occur at the
tips of myopodia. By tracing the dynamics and final fate of individual
myopodia, we also found that some of these contacts are stabilized to form the
synaptic site, whereas others are eliminated. These results suggest that
protrusive activity of myopodia actively contributes to neuromuscular
interaction. Furthermore, we provide evidence that the behavior of myopodia
differs depending on whether or not contact was made with a partner
motoneuron. These results suggest that myopodia search for appropriate
synaptic partners. Taken together with the localization of CAPS, these
observations provide strong evidence for the sensory function of myopodia, and
also highlight the role of myopodial tips as a possible signaling center for
synaptic matchmaking. As the dynamic behavior of the postsynaptic cell is just
as important as that of the presynaptic cell, future studies of axon guidance
and target recognition during the formation of synapses should focus on the
postsynaptic cell as much as the presynaptic cell.
What kind of signaling is taking place at the tips of myopodia? How is the signal transmitted to other regions of muscles to affect the process of synaptogenesis? Recognition by cell adhesion molecules such as CAPS might allow for stabilization of specific myopodial contacts. Signaling at the tips might be transmitted to neighboring regions of the muscle to induce myopodia clustering and postsynaptic differentiation. Our data indicate that signaling in muscles through the cytoplasmic domain of CAPS is likely to be required for target recognition, as CAPS-ID, which lacks the cytoplasmic domain, acts as a dominant negative when expressed in muscle.
The reduction in the number of contacts between myopodia and growth cones
and size of the nascent synaptic sites observed in caps and caps,
tartan double mutants are consistent with this model. Myopodia-filopodia
interaction appears also to be important for the differentiation of the
presynaptic terminals. We observed that presynaptic terminals form while
interacting with the surrounding myopodia. Reduction in the size of nerve
terminals observed in caps and caps, tartan double mutants
also suggests that stabilization of contacts between growth cones and myopodia
is crucial for presynaptic differentiation. As myopodia-like structures and
their clustering have been reported in vertebrates
(Misgeld et al., 2002),
signaling events that regulate myopodia-growth-cone interaction might be a
common mechanism for neuromuscular synaptogenesis. Future studies on the
molecular events occurring at the tips of myopodia might shed light on the
very beginning of synaptogenesis.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/7/1127/DC1
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Footnotes |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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2 test. We define a
nascent terminal as a bifurcated structure formed on lamellipodia-like
myopodial cluster (as shown in