Trans-Synaptic Transmission of Vesicular
Wnt Signals through Evi/Wntless
Ceren Korkut,1,2Bulent Ataman,1,2Preethi Ramachandran,1James Ashley,1Romina Barria,1Norberto Gherbesi,1
and Vivian Budnik1,*
1Department of Neurobiology, University of Massachusetts Medical School, Worcester, MA 01655, USA
2These authors contributed equally to this work
Wnts play pivotal roles during development and in the
mature nervous system. However, the mechanism by
Here we demonstrate a mechanism of Wnt transmis-
ing the Wnt-binding protein Evenness Interrupted/
Wntless/Sprinter (Evi/Wls/Srt). We show that at the
Drosophila larval neuromuscular junction (NMJ),
presynaptic vesicular release of Evi is required for the
secretion of the Wnt, Wingless (Wg). We also show
that Evi acts cell-autonomously in the postsynaptic
Wnt-receiving cell to target dGRIP, a Wg-receptor-in-
teracting protein, to postsynaptic sites. Upon Evi loss
of function,dGRIP is not properly targeted tosynaptic
sites, interfering with postsynaptic Wnt signal trans-
cellular mechanism by which a secreted Wnt is trans-
ported across synapses by Evi-containing vesicles
and reveal trafficking functions of Evi in both the
Wnt-producing and the Wnt-receiving cells.
For a video summary of this article, see the Paper-
Flick file with the Supplemental Data available online.
Members of the Wnt family of morphogens orchestrate a myriad
of developmental processes in all metazoan organisms studied
to date (Siegfried and Perrimon, 1994). These include the estab-
lishment of cell identity during pattern formation, control of cell
proliferation and migration, and cytoskeletal remodeling. Wnts
are also known to coordinate major aspects of the nervous
system from early development to adult function, in which they
differentiation and plasticity, as well as learning (Ataman et al.,
2008; Salinas and Zou, 2008; Speese and Budnik, 2007; Zhao
et al., 2005). Not surprisingly, alterations in Wnt signaling in
humans have been linked to a number of cognitive disorders,
such as schizophrenia and Alzheimer’s disease (De Ferrari and
Wnts activate a variety of intracellular signal transduction
zation events (Gordon and Nusse, 2006; Salinas and Zou, 2008).
The best understood signaling pathway is the canonical Wnt
pathway, in which Wnt ligands bind to the Frizzled (Fz) family
of serpentine receptors. Receptor activation in turn stabilizes
cytoplasmic b-catenin, which enters the nucleus and regulates
gene expression. In a divergent canonical pathway, GSK3-b
operates through a nongenomic mechanism, by phosphory-
microtubule stability. Alternative signal transduction mecha-
nisms activated by Wnt ligands include the planar cell polarity
(PCP) pathway and the Wnt/Ca2+pathway. Recent studies at
the Drosophila neuromuscular junction (NMJ) and in the devel-
oping mammalian nervous system have uncovered a novel
transduction mechanism in which Wnt receptors themselves
are cleaved and translocated into the nucleus (Lyu et al., 2008;
provide alternative mechanisms for cells to regulate diverse
processes in different spatiotemporal contexts.
Whereas considerable progress has been made in eluci-
dating the signaling pathways activated by Wnts, much less
is known about how Wnts are secreted and transported to
distant locales. At the Drosophila imaginal wing disc, the Wnt-
1 homolog Wingless (Wg) is secreted by a discrete row of
Wg-producing cells. Secreted Wg forms a long-range gradient
expanding many cell diameters away from the source of Wg
secretion (Neumann and Cohen, 1997). The mechanisms by
which Wg is transported from its site of secretion to distant
target cells have remained poorly understood. Wnt proteins
membranes owing to palmitoyl modifications essential for bio-
logical activity (Willert et al., 2003). Thus, unescorted Wnt mole-
cules are not easily diffusible in the extracellular milieu. Several
mechanisms have been proposed to explain the movement of
Wnt molecules from their site of secretion, including their asso-
ciation with glycosaminoglycan-modified proteins at the extra-
cellular matrix (Baeg et al., 2001), the formation of exosome-
like vesicles called argosomes (Greco et al., 2001), extracellular
lipoprotein particles (Panakova et al., 2005), transcytosis (Cou-
dreuse et al., 2006), or a combination of the above. However,
the exact mechanism employed during intercellular Wnt trans-
port has remained elusive.
Recent studies have identified a type II multipass transmem-
braneproteincalled EvennessInterrupted/Wntless/Sprinter (Evi/
Wls/Srt),whichappears tobespecifically requiredinvivoforWnt
secretion in epithelial cells of flies and human cultured cells
proteins, thereby regulating
tightly associatedto cell
Cell 139, 393–404, October 16, 2009 ª2009 Elsevier Inc. 393
(Banziger et al., 2006; Bartscherer et al., 2006; Goodman et al.,
2006). In the wing epithelium of Drosophila, Wg cannot be
secreted from the evi mutant cells, and this leads to the accumu-
lation of Wg within these cells. In contrast, the secretion of other
morphogens, such as Hedgehog (Hh), remains unaffected, sug-
gesting that Evi is dedicated to the secretion of Wnt proteins.
Further analysis has suggested that Evi functions as a Wnt cargo
receptor during trafficking from the Golgi to the plasma mem-
brane and is recycled back to the Golgi through the retromer
complex (Belenkaya et al., 2008; Franch-Marro et al., 2008;
Pan et al., 2008; Port et al., 2008; Yang et al., 2008).
In the nervous system, Wnts are released by pre- or postsyn-
aptic cells and function in either a retrograde or anterograde
between synaptic compartments are principally unexplored.
Considering that Wnt-1 is released from synapses in an activity-
dependent manner (Ataman et al., 2008), and the substantial
short- and long-term effects of Wnt signaling on neurons, eluci-
dating the mechanisms by which Wnt secretion/transport is
regulated in the nervous system remains an important problem.
Here we have addressed this key question by using the gluta-
matergic synapses of the Drosophila larval NMJ, where Wnt-1/
Wg is secreted from motorneurons. We report that Evi is local-
ized at these synapses and its function is indispensable for
proper Wg secretion and signaling. We also demonstrate a novel
mechanism for transport of the Wg signal across the synapse
through the release of Evi-containing exosome-like vesicles.
Further, we show that Evi is required for the proper trafficking
of the Wg receptor DFrizzled-2 (DFz2), through actions that
involve the DFz2-interacting protein dGRIP, a PDZ protein
required for the transport of internalized DFz2 vesicles toward
the nucleus (Ataman et al., 2006; Mathew et al., 2005).
Evi Is Required for Wg Secretion at the Neuromuscular
Previous studies have suggested that Evi is required for Wg
secretion in non-neuronal cells (Banziger et al., 2006; Bartsch-
erer et al., 2006). Because Wg is secreted from motorneurons
at the fly NMJ (Ataman et al., 2008; Packard et al., 2002), we first
examined the distribution of Wg at the NMJ of evi null mutants,
which survive to the third instar larval stage (Bartscherer et al.,
2006). We found that secreted Wg levels were substantially
reduced at postsynaptic muscles in evi mutants (Figures 1A,
1C, and 1E). However, this reduction was not limited to the post-
synaptic compartment but was also observed in the presynaptic
boutons as determined by volumetric quantifications of the Wg
signal inside the presynaptic bouton demarcated by anti-HRP
staining (Figures 1A, 1C, and 1E). A similar decrease was
expressed in neurons using the elav-Gal4 driver (Figures S1A
and S1B available online). These results could indicate that Evi
might be required for the stability or synthesis of Wg in motor-
neurons. However, we did not observe any changes in Wg levels
Figure 1. Wg Localization at the Neuromuscular Junction Is Regulated by Presynaptic Evi
(C) an evi mutant, and (D) an evi mutant expressing transgenic Evi in muscles (evi, Evi-post).
(E) Normalized Wg levels inside synaptic boutons (pre-) and at the postsynaptic region (post-).
(F) Western blot of larval brain extracts. Numbers at the right of the blot represent molecular weight in kDa. Calibration bar is 7 mm.
394 Cell 139, 393–404, October 16, 2009 ª2009 Elsevier Inc.
in the nervous system (Figure 1F). Thus, at the NMJ, Evi is
required for the transport and/or secretion of Wg in presynaptic
terminals. Aprediction of thishypothesis isthatWgshould accu-
mulate in the cell bodies or axons of motorneurons in evi
mutants. We found that there was a substantial increase in Wg
immunoreactivity levels in motorneuron cell bodies and longitu-
dinal axons within the neuropil (Figures S1C–S1E).
The above model was further tested by rescue experiments.
Expressing an Evi-GFP transgene in the motorneurons of evi
mutants, by using the Gal4 driver C380, completely rescued
the low levels of Wg in both the pre- and postsynaptic compart-
ments (Figures 1B and 1E). Notably, however, expression of the
Evi-GFP transgene in postsynaptic muscles, by using the Gal4
strain BG487, did not (Figures 1D and 1E). Thus, Evi is required
in motorneurons for normal Wg transport and/or secretion.
We also observed that mutations in evi mimicked synaptic
phenotypes previously observed in mutations affecting Wg
Figure 2. Mutations in evi Mimic Abnormal
(A–D) Confocal images of NMJs labeled with anti-
HRP and anti-DLG in (A and C) wild-type and (B
and D) evi. (A and B) Projections of entire NMJs.
(C and D) Single confocal slices of NMJ branches
(arrowheads in C and D = abnormal boutons;
arrows = ghost boutons).
(E–G) Number of (E and G) boutons and (F) ghost
Calibration bars are 30 mm for (A) and (B) and
13 mm for (C) and (D).
signaling (Ataman et al., 2006; Mathew
et al., 2005; Packard et al., 2002). As
muscle fibers grow in size, the Drosophila
larval NMJ continuously expands by
expansion is critically dependent on Wg
signaling (Packard et al., 2002). Wg
and suppressing Wg secretion substan-
tially reduces synaptic bouton prolifera-
tion. Further, in wg mutants many bou-
tons are misshapen, and some remain in
an undifferentiated state (ghost boutons),
lacking active zones and postsynaptic
apparatus. Conversely, increasing Wg
motorneurons enhances formation of
synaptic boutons. In the presynaptic cell
(Ataman et al., 2008; Franco et al., 2004;
Miech et al., 2008). In the postsynaptic
muscle cell Wg initiates an atypical
pathway in which the DFz2 receptor itself
is cleaved and a fragment imported to the
nucleus (Ataman et al., 2006, 2008;
Mathew et al., 2005). In evi mutants the total number of synaptic
boutons was decreased by over 50%, without any change in
muscle size, and the boutons had an aberrant morphology being
large and deformed (Figures 2A–2E and S2). In addition, evi
NMJs had a significantly higher number of ghost boutons
(Figures 2D, arrows and 2F). The decrease in bouton number
was only partially rescued by expressing Evi in either the pre-
or postsynaptic cell (Figure 2E). However, it was completely
rescued by simultaneously expressing Evi in both cells
(Figure 2E). In the case of ghost boutons, expressing the Evi
transgene in motorneurons or in both motorneurons and
muscles completely rescued the abnormal increase in ghost
boutons in evi mutants (Figure 2F). Expressing Evi in muscles
using the weaker Gal4 driver BG487-Gal4 did not rescue the
increase in ghost boutons, but this phenotype was completely
rescued by using the stronger muscle Gal4 driver C57-Gal4
(Figure 2F). Thus, although Evi is required only in motorneurons
Cell 139, 393–404, October 16, 2009 ª2009 Elsevier Inc. 395
for proper Wg transport and/or secretion, Evi is needed both in
neurons and muscles for normal synaptic growth.
The similarity in the synaptic phenotypes between evi and wg
mutants at the NMJ, together with previous evidence suggesting
that both proteins establish biochemical interactions (Banziger
et al., 2006), raised the question of whether there were genetic
interactions between evi and wg during NMJ growth. This was
addressed by analysis of transheterozygotes. The number of
boutons was normal in heterozygotes, but there was a supra-
additive reduction in the number of boutons in the transhetero-
zygotes (expected decrease in bouton number by simple
additivity in wg/+; evi/+ is 14.2% versus 27% observed in wg/+;
evi/+ transheterozygotes; Figure 2G), suggesting that evi and wg
genetically interact during synaptic bouton proliferation.
Evi Is Localized Both Pre- and Postsynaptically
and Is Transferred Trans-Synaptically from the Pre-
to the Postsynaptic Compartment
To examine the synaptic localization of Evi, we generated two
antibodies directed to a predicted either extracellular (Evi-Nex)
or intracellular (Evi-Cin) region of the Evi protein (Figures 3A,
S3A, and S3B). Both antibodies strongly labeled the NMJ in
similar patterns (Figures 3B and 3C). This immunoreactivity
was specific, as it was severely decreased in evi mutants (Fig-
ures 3D and 3E). Immunoreactive Evi label was observed both
in pre- and postsynaptic compartments at the NMJ, as deter-
mined by double labeling with anti-HRP, which defines the
boundary of the presynaptic compartment. However, Evi was
particularly enriched at the postsynaptic junctional region
Figure 3. Evi Is Localized Pre- and Postsynaptically at the Neuromuscular Junction, and It Is Transported Trans-Synaptically as an Intact
(A) Predicted structure of Evi and the regions (underlined) used for generation of the Evi-Nex and Evi-Cin antibodies.
(B–E) Single confocal slices of NMJs double stained with anti-HRP and antibodies to (B and D) Evi-Nex and (C and E) Evi-Cin in (B and C) wild-type and
(D and E) evi.
GFP-Pre stained with anti-GFP and anti-HRP, or (K) Evi-RNAi-post with anti-Evi-Nex and anti-HRP are shown.
(I) Normalized pre- and postsynaptic Evi levels.
(J) Single confocal slices of a bouton at (J1–J3) low and (J4–J6) high magnification in Evi-GFP-pre stained with anti-GFP, anti-Wg, and anti-HRP.
(L and M) Images of NMJs from evi;Evi-GFP-pre triple stained with anti-GFP, anti-HRP, and antibodies to (L2 and L3) Evi-Nex or (M2 and M3) Evi-Cin.
(N and O) Models on thepotential mode of Evi trans-synaptic transfer.In (N) an extracellular region of Eviis cleaved and transported to the postsynaptic compart-
ment. In (O) Evi is transferred as an intact protein through the use of vesicular compartments.
Calibration bars are 2 mm for panels J1–J3 and 6 mm for the rest of the panels.
396 Cell 139, 393–404, October 16, 2009 ª2009 Elsevier Inc.
(Figures 3B and 3C), the same region occupied by secreted Wg
and its receptor DFz2 at the NMJ (Packard et al., 2002). In this
region Evi immunoreactivity was present in a punctate pattern
presumably reflecting vesicular structures (Figures 3B and 3C).
and postsynaptically during synaptic bouton proliferation, but
that it is required solely presynaptically for Wg transport and/or
secretion. To further determine the requirement of Evi in the
pre- and postsynaptic side, we expressed Evi-RNAi with the
cell-specific Gal4 drivers. Surprisingly, expressing Evi-RNAi in
the motorneurons (Evi-RNAi-pre) not only led to a reduction in
Evi immunoreactivity inside presynaptic terminals but also
3F and 3I). The observation that presynaptic knockdown of Evi
pected. Such a phenomenon is not observed when knocking
also notdue to a leaky Gal4driver, as C380-Gal4 expressesGal4
in motorneurons and not in muscles (Budnik et al., 1996; Sanyal
et al., 2003). Further, expressing a nuclear LacZ (UAS-LacZ-
NLS) with C380-Gal4 resulted in strong labeling of neuronal but
not muscle nuclei (Figures S3D and S3E), and expressing myris-
synaptic myr-mRFP signal (Figure S3C). These observations
suggest that postsynaptic Evi is at least partly derived from the
presynaptic motorneurons. The possibility that Evi could be
transferred from the pre- to the postsynaptic compartment was
tested by expressing Evi-GFP in motorneurons (Evi-GFP-pre).
Notably, GFP was observed both in presynaptic boutons as
well asatthepostsynapticjunctional region(Figure3G),support-
ing the notion that Evi could be transferred from pre- to postsyn-
aptic compartments. This transfer was unlikely to result from Evi
overexpression, as when the Evi-GFP transgene was expressed
presynaptically in an evi mutant background, at levels similar to
endogenous levels (Figure S3F), a similar distribution of the GFP
label in the postsynaptic side was observed (Figures 3H and
S3F). This transfer of presynaptic Evi was also clearly observed
in vivo in samples expressing myr-mRFP and Evi-GFP in motor-
neurons and imaged live (Figure S3C).
Given that Wg is secreted by presynaptic boutons and that Evi
is required for normal Wg secretion, we next examined if presyn-
aptically derived Evi colocalized with endogenous secreted Wg
observed at postsynaptic sites. For these experiments we
expressed Evi-GFP in motorneurons and examined both the
Wg and Evi-GFP labels at the postsynaptic compartment. We
found that there was substantial colocalization between Wg
and Evi-GFP distal to the bouton rim, right outside the HRP label
(Figure 3J), consistent with the idea that secreted Evi vesicles
In contrast to the expression of Evi-RNAi in motorneurons,
expressing Evi-RNAi in the muscles (Evi-RNAi-post), although
significantly reducing the levels of Evi protein in the postsynaptic
compartment, did not change the levels of Evi in presynaptic
boutons (Figures 3K and 3I). These results demonstrate that
Evi is expressed by both motorneurons and muscles, but that
there is a unidirectional transfer of Evi from presynaptic boutons
to the postsynaptic region.
Considering that Evi is a multipass transmembrane protein,
two possible scenarios might account for the above transfer of
Evi from the pre- to the postsynaptic region. One possibility is
that an extracellular region of Evi is cleaved, as is the case for
other membrane receptors (Selkoe et al., 1996) (Figure 3N).
However, this possibility is highly unlikely, as in the Evi-GFP
transgene the GFP tag is fused to the intracellular C-terminal
region of Evi, and thus the transfer must include the intracellular
domain. An alternative possibility is that the entire Evi protein
could be transported in the form of a vesicle from the pre- to
the postsynaptic compartment (Figure 3O), as has been previ-
ously suggested with argosomes, vesicular structures that can
transport Wg from cell to cell (Greco et al., 2001). To address
this possibility, we took advantage of the Evi-Nex antibody,
which recognizes an epitope localized at the first extracellular
loop of Evi (Figure 3A; red region in Figures 3N and 3O), and
which is separated from the C-terminal GFP tag by seven trans-
membrane domains. For these experiments, we expressed Evi-
GFP in motorneurons in an evi null mutant background and
determined whether the postsynaptic GFP signal colocalized
with the Evi-Nex and Evi-Cin immunoreactivity. We found that
anti-Evi-Nex and anti-Evi-Cin immunoreactivities were exactly
colocalized with Evi-GFP at the postsynaptic region (Figures
3L, 3M, S3G, and S3H). Thus, these results support the notion
We also examined Drosophila Schneider-2 (S2) cells trans-
fected with the Evi-GFP construct. We found that untransfected
S2 cells in contact with Evi-GFP-transfected cells often con-
tained Evi-GFP-positive puncta within their cytoplasm (Fig-
ure 4A, arrowheads). To verify that this was due to transfer of
Evi-GFP from transfected to nontransfected cells, Evi-untrans-
fected cells were separately transfected with mCherry and
mixed with the Evi-GFP-transfected cells. Again, we found that
mCherry-positive (Evi-untransfected) cells had GFP puncta
within their cytoplasm (Figure 4B), suggesting that Evi-trans-
fected cells transferred Evi to nearby cells.
We also found that Evi-GFP puncta were observed in the
medium, suggesting the secretion of Evi vesicles into the
medium (Figure 4A, arrow). To determine if the Evi vesicles that
were transferred to adjacent cells contained Wg, we cotrans-
fected S2 cells with Evi-GFP and Wg. We found that the Evi vesi-
cles transferred to adjacent cells or to the medium contained Wg
(Figures 4C and 4D, arrowhead). Interestingly, in these double-
transfected cells Wg localized to varicosities within filopodia
(arrows in Figure 4D). These filopodia were also present in
untransfected cells as seen with phalloidin staining to label
endogenous F-actin (Figure S4C). Two other membrane
proteins, DFz2 and rCD2-mRFP, which also become localized
to filopodia, were not observed to be secreted (Figures S4A
and S4B). We also carried out a western blot analysis of the S2
cells and the culture medium. We found that indeed the culture
medium contained full-length Evi protein, suggesting that Evi
was secreted to the medium (Figure 4E). The above observation
was directly visualized by time-lapse imaging of the Evi-GFP
fluorescence. We found that Evi-GFP puncta trafficked within
highly dynamic filopodia-like structures in the S2 cells and that
some of these puncta were secreted to the media in a time frame
of several minutes (Figure 4F; Movies S1 and S2, arrows). Thus,
Cell 139, 393–404, October 16, 2009 ª2009 Elsevier Inc. 397
release and transcellular transfer of Evi vesicles to adjacent cells
isa commonbiological mechanism utilized byboth neuronal and
Evi Is Present in Multiple Compartments
at the Neuromuscular Junction
To determine the subcellular localization of Evi within pre- and
postsynaptic compartments we next carried out immunoelec-
iments 1.4 nm gold-conjugated secondary antibodies followed
by silver intensification were used to mark sites of Evi-antibody
binding using the pre-embedding technique. Consistent with
our immunofluorescence studies, Evi was found to be localized
in several pre- and postsynaptic structures.
At the postsynaptic junctional region, Evi was found within the
subsynaptic reticulum (SSR), a system of muscle-derived mem-
brane folds that completely surrounds synaptic boutons (Fig-
ure 5A; the presynaptic bouton highlighted in pink overlay).
Within the SSR, silver-intensified gold particles were observed
Notably, gold particles were also found inside approximately
200 nm in diameter membranous vesicles within the SSR (Fig-
ures 5A and 5B; arrows and insets). In summary, at the postsyn-
aptic region, Evi is present in association with SSR membranes
and with novel postsynaptic vesicles.
Evi was also associated with the pre- and postsynaptic
membrane (Figure 5F, arrows) and sometimes the signal was
Figure 4. Evi Is Transferred from Cell to Cell
and to the Medium
(A and B) Single confocal slices of S2 cells (A)
either untransfected (outlined by white circles) or
transfected with Evi-GFP and (B) either trans-
fected with mCherry or Evi-GFP (arrowheads =
Evi-negative cells; arrows = Evi in the media).
(C) Evi-GFP and Wg are transferred together into
an untransfected cell (arrowheads).
(D) Wg localizes with Evi into punctuate structures
within filopodia (arrows), as well as in the medium
(E) Western blot of lysates and media from Evi-
GFP transfected S2 cells.
(F) Time-lapse imaging of an S2 cell transfected
with Evi-GFP showing the shedding of an Evi-
GFP vesicle to the medium (arrows).
Calibration bars are 3 mm for (D) and 8 mm for the
rest of the panels. Time points in (F) are in min.
observed at the synaptic bouton cleft
(Figure 5H, arrow and inset). Within the
presynaptic bouton, Evi was observed in
large multimembrane structures (Fig-
ure 5G, arrowhead). Thus, Evi is present
in multiple structures
including pre-and postsynaptic vesicular
structures, the SSR, and synaptic mem-
To determine if these vesicles were
endocytosed from the muscle surface,
we next conducted an internalization assay. These experiments
were facilitated by the finding that the Evi-Nex antibody can bind
to surface Evi in vivo (Figure S3A). For these studies, unfixed and
unpermeabilized bodywallmuscleswereincubated withthe Evi-
Nex antibodies in the cold, washed, and brought to room
temperature for 30 min prior to fixation. Then, samples were per-
meabilized and incubated with the gold-conjugated secondary
antibody, followed by preparation for electron microscopy
(EM). Interestingly we found that the Evi label was found at
SSR membranes as well as inside the large SSR vesicles
(Figure 5C, arrows and insets), suggesting that at least a subset
of these postsynaptic vesicles are derived from the endocytosis
of postsynaptic surface Evi.
To verify that Evi was transferred from presynaptic boutons to
the postsynaptic SSR at the ultrastructural level, GFP-tagged
Evi was expressed in motorneurons using the C380-Gal4 driver,
and the NMJ was examined by immunoelectron microscopy
using an anti-GFP antibody. We found that the GFP label was
found not only within synaptic boutons (Figure 5D, arrowhead)
We also expressed Evi-GFP in the motorneurons of evi mutants
and immunolabeled Evi with the Evi-Cin antibody using the post-
embedding technique. Again, we found the label in the presyn-
cleft (Figure 5J, arrowhead), as well as in the postsynaptic SSR
region (Figures 5I and 5K, arrows). Thus, Evi is transferred trans-
synaptically as expected from the observations at the light level.
398 Cell 139, 393–404, October 16, 2009 ª2009 Elsevier Inc.
Postsynaptic Evi Is Required for the Trafficking of DFz2
through the DFz2-Interacting Protein dGRIP
Given that presynaptic Evi alone is not sufficient for normal NMJ
development, we predicted that Evi was also endogenously
expressed in muscle. To test this prediction we carried out real-
time PCR experiments from body wall muscle mRNA. We found
that there were significant levels of Evi-mRNA in muscles and
that these levels were substantially decreased upon expressing
Evi-RNAi (Figure S5). What is the cell-autonomous role of Evi in
the postsynaptic target cell? To address this issue we examined
postsynaptic Wg signaling while downregulating Evi selectively
in the muscle using Evi-RNAi. Previous studies suggested that
Wg is secreted by presynaptic boutons (Packard et al., 2002)
and unraveled a novel postsynaptic Wg signal transduction
pathway in the postsynaptic muscles, the frizzled nuclear import
Figure 5. Evi Is Localized to Pre- and Postsyn-
aptic Vesicular Structures as well as Pre- and
(A–K) Electron micrographs of synaptic bouton
regions in preparations stained with antibodies to
Evi-Nex or GFP, labeled with silver intensified 1.4 nm
gold secondaries, or antibodies to Evi-Cin labeled
with 18 nm gold secondaries. The presynaptic com-
partment has been overlayed in pink, and insets are
high-magnification views of the structures indicated
by the arrows. In (A), (B), and (D)–(H) samples were
stained pre-embedding with anti-Evi-Nex or anti-
GFP. In (C) samples were processedfor an internaliza-
tion assay. (I–K) Samples were stained post-embed-
ding with anti-Evi-Cin. (A and B) Immunoreactive
vesicles found at the SSR region. (C) Internalized Evi
isfound inpostsynaptic SSR vesicles. (Dand K)Local-
ization of label at SSR membranes. (E–H) Evi label at
the perisynaptic region of pre- and postsynaptic
membranes. Arrowheads in (F) mark the active zone.
(I) Evi localization at a presynaptic multimembrane
body. (J) Evi-immunoreactive gold particles at the
presynaptic region and the synaptic cleft. Calibration
bar is 0.6 mm in (A), (C), (D), (K); 0.3 mm in (B) and (E)–
(H); 0.2 mm in the insets of (A); 0.15 mm in the inset of
(B); and 0.1 mm in the inset of (C).
(FNI) pathway (Speese and Budnik, 2007),
which is also shared by other Wnt receptors
(Lyu et al., 2008). In this pathway, the Wg
receptor, DFz2, is internalized from the post-
synaptic muscle membrane and back-trans-
ported from the synapse to the nucleus
through a mechanism that requires an inter-
action between the PDZ-binding C-terminal
tail of DFz2 and the PDZ4-5 domain of the
7-PDZ protein dGRIP (Ataman et al., 2006).
The entire cytoplasmic domain of DFz2
(DFz2-C) is then cleaved and imported into
the nucleus (Mathew et al., 2005).
In muscles expressing Evi-RNAi we found
thatDFz2was localized normally at thepost-
synaptic region of the NMJ. However, the
postsynaptic levels of DFz2 were substan-
tially increased (Figures 6A, 6B, and 6E). In contrast, no such
increase in DFz2 levels was observed in the presynaptic cell
upon expression of Evi-RNAi in motorneurons (normalized
presynaptic DFz2 intensity in wild-type is 1.0 ± 0.07 versus
0.93 ± 0.07 in Evi-RNAi-pre). The same phenotype has been
previously observed when the transport of DFz2 from the
synapse to the nucleus is prevented by interfering with dGRIP
function in muscles (Ataman et al., 2006). Interestingly, a similar
accumulation of Wg at the postsynaptic region was observed
upon downregulating Evi in muscle (Figures 6C–6E), consistent
with the notion that Wg is trafficked with its receptor (Gagliardi
et al., 2008). To determine if the increase in DFz2 at synapses
of Evi-RNAi-post larvae was due to a defect in the internalization
and/or trafficking of DFz2, we carried out DFz2 internalization
assays. In these experiments we used an anti-DFz2-N antibody
Cell 139, 393–404, October 16, 2009 ª2009 Elsevier Inc. 399
that binds to the extracellular domain of the receptor in vivo,
instar body wall muscles were incubated with the DFz2-N anti-
body at 4?C in vivo, and after washing the excess antibody,
samples were brought to room temperature and fixed at 5 and
60 min after the antibody-binding step. To determine the fraction
of DFz2 that remained at the surface, samples were then incu-
bated with Alexa 647-conjugated secondary antibody in the
absence of detergent permeabilization as previously reported
(Ataman et al., 2006; Mathew et al., 2005). To determine the
amount of internalized DFz2, the above procedure was followed
by permeabilization and incubation with a FITC-conjugated
As in previous studies (Ataman et al., 2006; Mathew et al.,
2005), in wild-type samples, surface DFz2 was internalized and
observed near synaptic boutons at 5 min after the antibody-
binding step (Figures 6G2 and 6F). However, at 60 min after
the antibody-binding step, internalized DFz2 was significantly
reduced at the NMJ as a result of its trafficking away from the
synapse (Figures 6H2 and 6F; Ataman et al., 2006; Mathew
et al., 2005). In contrast, upon expressing Evi-RNAi in muscles,
no decrease in internalized synaptic DFz2 was observed at 60
min (Figures 6I2, 6J2, and 6F). No significant changes were
observed in surface DFz2 in both genotypes (Figures 6G1–6J1
and 6F), suggesting that only a small pool of the DFz2-antibody
complexes become internalized. Thus, similar to alterations in
dGRIP, a decrease in Evi function in muscles appears to inter-
fere with the trafficking of DFz2 away from the synapse. This
conclusion was further supported by examination of the levels
of DFz2-C imported into the muscle nuclei. Previous studies
show that the C-terminal region of DFz2 is cleaved and imported
into the nucleus, where it is observed in the form of discrete
immunofluorescent puncta (Figure 6K; Mathew et al., 2005). In
evi mutants and upon expressing Evi-RNAi in muscles alone,
nuclear DFz2-C puncta were almost completely abolished (Fig-
ures 6K–6M), in agreement with the model that in the absence of
Evi function, DFz2 is not properly transported to the muscle
nucleus. Furthermore, a complete rescue of the DFz2-C nuclear
spots to wild-type levels was observed in the evi mutant by
expressing the Evi transgene in the muscle alone. In contrast,
expressing Evi in motorneurons provided only a partial rescue
of the nuclear DFz2-C foci (Figure 6M). Therefore, we conclude
that muscle Evi is involved in the trafficking of DFz2 to the
Figure 6. Evi Downregulation in Muscle Results in Postsynaptic Wg and DFz2 Accumulation and Alterations in the Frizzled Nuclear Import
(E) Wg and DFz2 immunoreactivity levels at the postsynaptic region.
(F) Intensity of surface and internalized DFz2 at 5 and 60 min after the antibody-binding step in wild-type and Evi-RNAi-post.
(G–J) Single confocal slices of NMJs subjected to the internalization assay, showing (G1–J1) surface DFz2 and (G2–J2) internalized DFz2 (G and I) at 5 min and
(H and J) 60 min after the antibody-binding step in (G and H) wild-type and (I and J) Evi-RNAi-post.
(K and L) Confocal slices of muscle nuclei in preparations stained with anti-DFz2-C and Hoechst in (K) wild-type and (L) evi mutants.
(M) Normalized number of DFz2-C nuclear spots.
Calibration bars are 10 mm for (A)–(H2) and (I)–(L); 5 mm for (A)–(D4).
400 Cell 139, 393–404, October 16, 2009 ª2009 Elsevier Inc.
Given the substantial similarities between the phenotypes
observed upon knocking down evi and dgrip in postsynaptic
DFz2 trafficking as well as in the synaptic morphology and
NMJ growth (Ataman et al., 2006), we next examined whether
interfering with Evi function could be disrupting the postsynaptic
function of dGRIP. For these studies we examined the localiza-
tion of dGRIP in larvae expressing Evi-RNAi in muscles. In
wild-type, dGRIP is present in small trafficking vesicles highly
concentrated at postsynaptic sites (Figure 7A, arrows; Ataman
Figure 7. Downregulating Evi in Postsyn-
aptic Muscles Alters the Localization of
dGRIP and Proposed Function of Evi in the
Pre- and Postsynaptic Compartment
(A and B) Single confocal slices of NMJs triple
labeled with antibodies to HRP, dGRIP, and Lva
in (A) wild-type and (B) Evi-RNAi-post (arrows in
A = synaptic dGRIP; arrowheads in A and B =
(C) dGRIP levels at the postsynaptic junctional
region, Golgi bodies, and muscle cortex in wild-
type and Evi-RNAi-post.
(D) Postsynaptic DFz2 levels in wild-type, Evi-
RNAi-post, and Evi-RNAi-post, dGRIP-post.
(E) Bouton number in wild-type, evi mutants, and
evi mutants expressing dGRIP-post.
(F) DFz2C spots in wild-type, evi mutants, and evi
mutants expressing dGRIP-post.
(G) Proposedmodel for Evi function inthe pre- and
postsynaptic compartment (see text).
Calibration bars are6mmfor panels A1–A3, B1–B3
and 3 mm for panels A4–A6, B4–B6.
et al., 2006), as well as in Golgi bodies in
juxtaposition to the cis-Golgi marker
Lavalamp (Lva; Figure 7A, arrowheads;
Ataman et al., 2006). Notably, we found
that upon knocking down Evi specifically
in muscles, dGRIP was substantially
reduced from postsynaptic sites as well
as from Golgi bodies in muscle (Figures
7B and 7C). In addition, dGRIP was local-
ized throughout the muscle submem-
brane region in a diffuse manner (Figures
7B and 7C). Thus, Evi controls dGRIP
localization at the postsynaptic muscle
region, and in the absence of Evi function
dGRIP is not normally localized to the
Golgi and synapses, likely disrupting
postsynaptic DFz2 trafficking. A predic-
tion of this model is that overexpressing
dGRIP in muscles should overcome
some of the defects arising from the
lack of Evi in muscles. To test this model
we overexpressed dGRIP in muscles
while downregulating Evi in these cells.
We found that the DFz2 accumulation
at the postsynaptic region of Evi-RNAi-
indeed the postsynaptic levels of DFz2
became significantly lower than wild-type (Figure 7D). In addi-
tion, both the number of synaptic boutons and nuclear DFz2-C
spots were partially rescued by overexpressing dGRIP in evi
mutants (Figures 7E and 7F).
An additional prediction is that a population of Evi vesicles
should traffic with dGRIP vesicles. To determine if this was the
case we performed time-lapse imaging of muscles expressing
both Evi-GFP and dGRIP-mRFP. We found that in many
instances Evi and dGRIP vesicles colocalized and followed the
Cell 139, 393–404, October 16, 2009 ª2009 Elsevier Inc. 401
same trajectory (Movies S3 and S4). These results demonstrate
that Evi, in addition to its important role in the Wg-secreting cell,
has a critical function in Wg-target cells, as it mediates the trans-
port of the downstream Wg signaling component, dGRIP.
Here we show that the multipass transmembrane protein Evi has
a critical role in trans-synaptic Wnt-1/Wg transport through
vesicular structures. To our knowledge, this is the first report to
identify trans-synaptic communication through a vesicular
structure. Further, our studies identify a mechanism by which
secreted factorscan betransmitted fromcell tocell.Wepropose
that presynaptic Evi is required for trafficking Wg from the cell
body to the presynaptic terminals, and across the synaptic cleft,
to present Wg to postsynaptic DFz2 receptors (Figure 7G). On
the other hand, postsynaptic Evi is required to transport dGRIP
to postsynapticsites.Atthepostsynaptic regiondGRIPinteracts
with postsynaptic DFz2 receptors and participates in the traf-
ficking of DFz2tothe nucleus,whereits C-terminal tailiscleaved
and imported to the nucleus (Figure 7G).
Previous studies had implicated Evi only in the secretion of
Wnts in Wnt-expressing cells (e.g., Banziger et al., 2006). How-
ever, endogenous Evi was also found in Wnt-target cells (Port
here identify an unprecedented role for Evi in Wg-receiving cells
in trafficking the Wg receptor DFz2 through the regulation of the
synaptic targeting of the DFz2-interacting protein dGRIP, which
was previously shown to function in transporting DFz2 receptors
et al., 2006). These studies unravel new processes and cellular
mechanisms by which Evi functions as an essential component
of synaptic Wnt signaling.
Trans-Synaptic Signaling in the Nervous System: Role
Intercellular communication in the brain is primarily accom-
plished through the exocytosis of neurotransmitter-laden vesi-
cles or by direct current conduction through gap junctions. Pre-
and postsynaptic partners also release factors important for cell
survival, synapse development, synapse maintenance, and
synaptic plasticity (reviewed in Lu and Figurov, 1997; reviewed
in Marques, 2005). Among these are neurotrophins such as
bone-derived neurotrophic factor (BDNF) and nerve growth
factor (NGF), members of the bone morphogenetic protein
(BMP) family, and Wnts. These molecules are released from
pre- or postsynaptic terminals and they function in retrograde
or anterograde manners to influence synaptic growth, function,
tion of synaptic growth in relationship to muscle size requires the
synaptic growth is also controlled by the release of Wg, which is
thought to act on DFz2 receptors in both the pre- and postsyn-
aptic cells where it initiates alternative transduction pathways
(Ataman et al., 2008; Franco et al., 2004; Miech et al., 2008).
A major gap in our understanding of how Wnts function is the
mechanism by which they reach their destination once released.
Despite the presence of charged amino acid residues in the
primary sequence of Wnts, Wnts are hydrophobic molecules
tightly bound to cell membranes due to the addition of palmitate
moieties during maturation (Willert et al., 2003; Zhai et al., 2004).
of passive diffusion in the extracellular milieu. The studies pre-
sented here suggest that one mechanism for this transport is the
from presynaptic boutons and become localized to postsynaptic
sites. This model is supported by several lines of evidence. (1)
Downregulating Evi in the presynaptic motorneurons (Evi-RNAi-
pre) not only led to a reduction in Evi immunoreactivity inside
presynaptic terminals but also substantially reduced the label at
the postsynaptic region. (2) Expressing Evi-GFP in motorneurons
(Evi-GFP-pre) led to the localization of the GFP label in the post-
synaptic junctional region in the form of puncta that colocalized
with both the Evi-Cin and Evi-Nex antibodies and that showed
substantial colocalization with secreted Wg at the postsynapse.
S2 cells, and S2 cell-culture medium contained full-length Evi
protein, suggesting that Evi was also secreted in cultured cells.
In addition, the secreted and transferred Evi vesicles contained
Wg. This trans-synaptic transfer of a synaptogenic signal through
specialized vesicles containing a dedicated membrane protein is
a novel signaling mechanism in the nervous system that might
be used for a number of secreted signaling factors.
The release of endosomal vesicles, called exosomes, has
been reported in a variety of tissues, including cultured neurons
(Faure et al., 2006; Fevrier and Raposo, 2004; van Niel et al.,
2006). These exosomes are released by the fusion of multivesic-
ular bodies (MVBs) with the plasma membrane and are thought
to be involved both in the removal of cellular debris as well as
in intercellular communication. For example, in the immune
system integrin- and MHC-containing exosomes are used for
antigen presentation, and they are able to prime T lymphocytes
in vivo (van Niel et al., 2006). In cultured cortical neurons, the
release of exosomes containing the cell adhesion molecule L1,
the GPI-anchored prion protein, and the GluR2/3 subunit of
glutamate receptors has been reported in a process that is regu-
lated by membrane depolarization (Faure et al., 2006). Our
finding that exosome-like vesicles containing a synaptogenic
factor are released at synapses provides a previously unidenti-
fied mechanism for trans-synaptic communication.
The mechanism by which Evi-containing vesicles are released
from the presynaptic cell is not known, but a few potential possi-
ported within the presynaptic cell in MVBs that fuse with the
plasma membrane thus releasing the Evi vesicle. In turn, after
presentation of Wg to DFz2 receptors, the vesicle might fuse
with the postsynaptic membrane. Interestingly, we found that
Eviinpresynapticterminals waspresentinmultimembrane com-
partments. Similarly, in Wg-secreting wing disc epithelial cells,
Evi (Franch-Marro et al., 2008) and Wg (van den Heuvel et al.,
1989) have been shown to be localized within MVBs. We also
found that Evi label was found in association with postsynaptic
SSR membranes and in the form of approximately 200 nm vesi-
cles in the SSR. Our internalization assays suggest that these
vesicles are endocytosed from the postsynaptic membrane.
402 Cell 139, 393–404, October 16, 2009 ª2009 Elsevier Inc.
Cell-Autonomous Role of Evi in Wg-Target Muscles
Besides its involvement in transporting the Wg signal across the
synapse, we also found that Evi had a cell-autonomous function
in the postsynaptic target cell, as revealed by specifically down-
regulating Evi in muscle. Inthiscase both Wg and DFz2accumu-
lated at the postsynaptic region. In addition, DFz2 did not traffic
normally from the NMJ and the nuclear import of DFz2-C was
largely abolished. These findings suggest that Evi, beyond the
regulation of Wnt secretion, organizes further downstream
signaling events in the Wg-target cell.
The phenotypes observed upon downregulating Evi in muscle
cells were highly reminiscent of those observed upon loss of
dGRIP function. Further, decreasing Evi levels led to the virtual
elimination of synaptic and Golgi dGRIP. The evidence relating
Evi to dGRIP function is further supported by the findings that
Evi and dGRIP are often observed trafficking in the same vesi-
cles, and that overexpressing dGRIP in either evi mutants or
Evi-RNAi-post elicited partial rescue of phenotypes resulting
from evi loss of function. Thus, Evi appears to be required for
the trafficking of dGRIP to synaptic sites where dGRIP binds
DFz2 receptors and functions to traffic them toward the nucleus.
The elimination of dGRIP from the Golgi complex might arise
from a defect in its recycling to the Golgi due to its abnormal tar-
geting to synapses. In the absence of postsynaptic dGRIP, DFz2
postsynaptic sites. Interestingly, Evi has been demonstrated to
be involved in trafficking Wg from the Golgi to the plasma
membrane in Wg-secreting cells (Belenkaya et al., 2008;
Franch-Marro et al., 2008; Pan et al., 2008; Port et al., 2008;
Yang et al., 2008). Our studies showing that Evi is required for
the trafficking of dGRIP to postsynaptic sites suggest that Evi
might have a role not solely in transporting Wnts, but also in traf-
ficking components associated with Wnt pathways.
Although this study identifies a pre- and postsynaptic role for
Evi, it is clear that the roles are not completely independent.
For example, we found that restoring Evi levels only in the motor-
neurons of evi mutants was sufficient for a complete rescue of
the ghost bouton phenotype and resulted in a partial rescue in
the number of nuclear DFz2C spots. These results were sur-
prising given that in the absence of postsynaptic Evi, dGRIP
does not traffic normally and thus interferes with postsynaptic
Wnt signaling. A potential explanation is that the transferred
presynaptic Evi can partially compensate for the lack of Evi in
the postsynaptic cell.
In conclusion, our studies identify a mechanism by which the
Wnt-1/Wg signal is transmitted across the synapse, through
the use of an Evi vesicle, and find an additional cell-autonomous
role of Evi in Wnt-receiving cells, the synaptic recruitment of
dGRIP, which functions in transporting the signal to the muscle
Flies were reared in standard Drosophila media at 25?C unless otherwise
stated. (See Supplemental Experimental Procedures for fly strains.) RNAi
crosses and controls were performed at 29?C. The wgtsflies were tested at
the restrictive temperature (25?C).
Third instar larvae were dissected in Ca2++-free saline and fixed in either 4%
paraformaldehyde or non-alcoholic Bouin’s fixative (see Supplemental Exper-
imental Procedures for antibodies and Hoechst conditions).
Confocal images were acquired using a Zeiss Pascal Confocal Microscope.
Preparations from different genotypes were processed simultaneously and
imaged using identical confocal acquisition parameters. Fluorescence signal
intensity was quantified by volumetric measurements of confocal stacks using
Volocity 4.0 Software (see Supplemental Experimental Procedures). Measure-
ments were taken from muscles 6 and 7, abdominal segment 3. A Student’s
t test was performed for pair-wise comparisons between each genotype and
controls. Error bars in the histograms represent mean ± standard error of the
mean (SEM), where *** = p < 0.0001; ** = p < 0.001; * = p < 0.05.
Schneider-2 Cell Cultures
Schneider-2 (S2) cells were transfected as described in Supplemental Exper-
imental Procedures. For Evi-GFP transfer experiments, pAc-Evi-EGFP
(Bartscherer et al., 2006) transfected S2 cells were washed 24 hr after trans-
fection and mixed with pAc-mCherry transfected S2 cells. For cotransfection
experiments we used pAc-Wg (Bartscherer et al., 2006), pAc-Evi-EGFP, pAc-
rCD2-RFP, and pAc-DFz2-myc (Mathew et al., 2005). Cells were then grown
for 24–48 hr and processed for immunocytochemistry.
Live imaging of transfected S2 cells and body wall muscles was performed
using an Improvision Spinning Disk confocal microscope as described in
Supplemental Experimental Procedures.
tion of Evi in S2 cells and the culture medium, transfected cells were washed
were harvested for immunoblotting (Supplemental Experimental Procedures).
For the pre-embedding technique, third instar body wall muscles were fixed
and incubated with anti-Evi-Nex (1:100) or anti-GFP (1:300) followed by anti-
rabbit IgG-1.4 nm nanogold (1:50; Nanoprobes) and intensification using HQ
silver reagents (Nanoprobes). The EM Internalization assay was performed
as above, except that 1.4 nm nanogold secondary antibody was used after
permeabilization. For the post-embedding technique, samples were fixed
and then embedded in LR White resin followed by antibody staining on grids
with secondaries conjugated to 18 nm gold (1:75; Jackson). Transmission
electron microscopy analysis was performed as described by Torroja et al.
(1999). See Supplemental Experimental Procedures for further details.
Supplemental Data include Supplemental Experimental Procedures, five
figures, four movies, and a video summary and can be found with this article
online at http://www.cell.com/supplemental/S0092-8674(09)01047-2.
We thank Drs. Marc Freeman, Michael Francis, and Motojiro Yoshihara as well
as members of the Budnik lab for helpful comments and critical discussion on
the manuscript. We especially thank John Nunnari for great help with immu-
noelectron microscopy, Yuly Fuentes for providing the images with UAS-
LacZ-NLS, and Dr. Sean Speese for help with the qPCR. We also thank
Dr. Michael Boutros for providing the evi2and Evi-EGFP fly lines as well as
the Evi-EGFP and Wg plasmids for studies in S2 cells. This work was sup-
ported by NIH grant RO1 MH070000 to V.B. Core resources supported by
the Diabetes Endocrinology Research Center grant DK32520 were also
used. This paper is dedicated to Susan Cumberledge (3/5/1956–7/28/2008).
Cell 139, 393–404, October 16, 2009 ª2009 Elsevier Inc. 403
Received: March 9, 2009
Revised: June 3, 2009
Accepted: July 31, 2009
Published: October 15, 2009
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