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.
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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