Available online at www.sciencedirect.
com
Secretion and extracellular space travel of Wnt proteins
Julia Christina Gross and Michael Boutros
Wnt signaling pathways control many processes during
development, stem cell maintenance and homeostasis, and
their aberrant regulation has been linked to diseases in man
including diabetes, neurodegeneration and cancer. Wnts are
hydrophobic proteins, however, quite paradoxically, they can
travel over distances to induce cell-type specific responses.
While there has been an initial focus on elucidating the
intracellular signaling cascade, discoveries in the past few
years have shed light on a highly complex, and regulated
secretory process that guides Wnt proteins through the
exocytic pathway. Wnt proteins are at least in portion
packaged onto extracellular carriers such as exosomes. Similar
to dysregulation of components in the Wnt receiving cell, failure
to regulate Wnt secretion has been linked to cancer. Here, we
review recent discoveries on factors and processes implicated
in Wnt secretion.
Addresses
German Cancer Research Center (DKFZ), Division of Signaling and
Functional Genomics, Heidelberg University, Department of Cell and
Molecular Biology, Medical Faculty Mannheim, Heidelberg, Germany
Corresponding author: Boutros, Michael (m.boutros@dkfz.de,
M.Boutros@Dkfz-Heidelberg.de)
Current Opinion in Genetics & Development 2013, 23:385–390
This review comes from a themed issue on Developmental
mechanisms, patterning and evolution
Edited by Hiroshi Hamada and Sally Dunwoodie
For a complete overview see the Issue and the Editorial
Available online 26th March 2013
0959-437X/$ – see front matter, # 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.gde.2013.02.017
Introduction
Wnt proteins are morphogens found in organisms as
different as Hydra, Caenorhabditis elegans, Drosophila,
mouse and human. They have a prime role in regulating
many processes during development such as body axis
formation, organ development, maintenance of stem cells
and cell type differentiation [1]. In recent years, it has
become increasingly evident that Wnt activity is not only
regulated in Wnt receiving cells but also on the level of
Wnt secretion. Current research focuses on defining the
core secretion pathway for Wnt proteins and discovering
context-specific mechanisms. Here, we will review the
latest developments in the area of Wnt secretion with
a focus on lipid modifications, extracellular packaging
forms and tissue-specific regulation of Wnt secretion —
and how these processes facilitate Wnt proteins to reach
their destinations.
www.sciencedirect.com
From the ER to the plasma membrane
Wnts are cotranslationally imported into the Endoplas-
matic Reticulum (ER) lumen where they are modified by
Porcupine (Porcn), a conserved membrane bound O-acyl
transferase (MBOAT) that can mediate the palmitoyla-
tion and secretion of Wnts [2–4]. While it was previously
unanticipated that the passage of Wnt proteins through
the early secretory pathway is regulated by ER resident
transmembrane proteins, two RNAi screens in Drosophila
identified p24 proteins to be required for the ER export of
Wg and Wnt3A (but not the nonlipidated WntD) [5,6].
As these proteins act in complexes to load GPI-anchored
cargo into COPII vesicles [7,8], depletion of several of
these family members (Opm, emp24, eca, CHOp24)
impaired Wg signaling by accumulation of Wg in the
ER in wing disc and the eye [5,6].
Secretion of Wnt proteins then depends on Evi/Wls, a
dedicated multimembrane protein that shuttles all Wnts
from the Golgi to the plasma-membrane [9–11], with the
exception of WntD in Drosophila [12]. Upon binding to
Wnt proteins, Evi shuttles its cargo to the plasma mem-
brane — and possibly further on. When Evi reaches the
plasma membrane it is internalized by Clathrin-mediated
endocytosis [13], which is dependent on a tyrosine-based
endocytosis motif identified in the intracellular loop of
Evi [13]. Mutation of this motif leads to surface accumu-
lation of Evi and Wg secretion defects in Drosophila [13].
In general, these motifs are recognized at the plasma
membrane by binding of AP-2 to phosphoinositide 4,5-
bisphosphate [14] (Table 1).
The role of the retromer complex
Retrograde transport of Evi from the plasma membrane to
the Golgi is required for continuous Wnt secretion and is
mediated by the retromer complex (VPS35/VPS26) [15–
18]. Recent studies refined the composition of the Wnt-
specific retromer complex. VPS35 colocalizes with Evi in
early endosomes and associates with SNX3, a complex
that does not require the ‘canonical’ retromer members
SNX1 and SNX6 [19,20]. In addition, myotubularin lipid
phosphatases are required for this process [21]. The
recycling of Evi, from the membrane back to the Golgi,
is conserved from C. elegans to vertebrates [15–18,22].
Wnt proteins and exosomes
How lipid-modified proteins with such strong membrane
affinity move in the extracellular space has remained a
puzzling question. Several mechanisms — ‘vehicles’ —
have been proposed to make Wnt proteins diffusible. Wg/
Wnt proteins were shown to bind to lipoprotein particles
in Drosophila and human cells [23,24]. A substantial
Current Opinion in Genetics & Development 2013, 23:385–390
386 Developmental mechanisms, patterning and evolution

Table 1

Proteins implicated in secretion of Wnt proteins

Factors Function/mechanism Cellular localization Organism Reference

Porcn Palmitoylation of Wnts Endoplasmic Reticulum (ER) Human cells Mouse, [2–4]
Drosophila
Evi Trafficking of Wnts from Golgi to Golgi, PM, endosomes, Human cells, Mouse, [9–11]
extracellular medium extracellular Xenopus, Drosophila,
C. elegans
p24 Loading of Wnts into COPII vesicles ER, ER-Golgi intermediate Drosophila human cells [5,6]
Opm, emp24, eca, CHOp24 compartment (ERGIC)
Retromer Recycling of Evi to Golgi Endosomes C. elegans, Drosophila, [15–20,22]
VPS35 VPS26 SNX3 human cells
MTM-6/MTM-9 (myotubularin Dephosphorylate Endosomes Drosophila, human cells [21]
lipid phosphatases) phosphatidylinositol-3-phosphate
Ykt6 Sorting of Evi/Wnts for Golgi, endosomes Drosophila, human cells [26]
exosomal secretion
ESCRT-0 Sorting of Evi/Wnts for Endosomes Human cells [26]
HGS exosomal secretion
Rab11 Syx1a Myo5 Secretion of exosomal Evi Endosomes Drosophila [27]
Reggie-1/flotillin2 Lipid microdomains Plasma membrane (PM), Drosophila [57]
endosomes
Sulf-1 Heparan sulfate 6-O endosulfatase PM Drosophila [42,43]
Tiki Transmembrane protease cleaves PM Xenopus, Human cells [45]
the N-terminus of Wnt
SWIM Solubilization of Wg Extracellular Drosophila [58]

fraction of active Wnt proteins were also found on extra-
cellular vesicles, called exosomes, secreted from human
and Drosophila cell lines [25,26], which can be blocked
by depletion of R-SNARE protein ykt6 [26] (Figure 1).
The release of exosomes requires the passage of secretory
cargo into endosomal compartments. Exosomes often
carry specific sets of proteins and their formation depends
on regulators that are conserved from human to Drosophila
[26,27,28]. Several lines of evidence suggest that Evi
and Wnt proteins reach the endosomal compartment
together before secretion of active Wnts. Endosomal
acidification by V-ATPases is necessary for Wnt secretion
and blockage leads to accumulation of Evi on the cell
surface [29]. In human cells, ESCRT-0 protein HGS,
involved in cargo sorting into Multivesicular Bodies
(MVBs), impaired Wnt secretion at that level [26].
In the neuromuscular junction in Drosophila Evi travels
from one cell to another on exosomes at the synaptic cleft
[30]. Exosomal secretion can be blocked by depletion of
syx1a, rab11 and myo5b [27], while rab11 appears not to
have such effect in the wing disc epithelium [25]. This is
not necessarily surprising as different Rab proteins, such
as rab11, rab27A and B, rab35 have been implicated in
exosome release depending on cell type and tissue under
investigation [31–33].
It is interesting to speculate in which tissue context Wnt
would travel on exosomes or lipoproteins. Not all tissues
produce lipoproteins, for example in the Drosophila wing
disc Hedgehog and Wg are secreted but lipoprotein
particles are only produced in the fat body [23]. In C.
elegans Hedgehog protein is secreted on exosomes from
the apical domain of polarized cells, a process mediated
by V-ATPases [34]. The emerging picture is that the
machinery required for morphogen incorporation onto
exosomes and lipoprotein particles must rely on different
proteins, further research might clarify in which tissue
context these forms dominate, for example metabolic
active versus secretory tissues/cells.
Intracellular and extracellular trafficking
The analysis of Wnt proteins has always been hindered by
their hydrophobic nature. This, as it becomes clearer now,
is key to their trafficking and signaling function. Their
hydrophobicity stems from two lipid modifications con-
served in all Wnt proteins (except Drosophila WntD).
Newer publications analyzed the secretory and functional
relevance of lipid adduct compared to each other and to
Wnt’s N-glycosylation [35–37]. Although N-glycosylation
might contribute to Wnt’s stability in the extracellular
space, they appear to be dispensable for Wnt activity [37].
The palmitoylation at a conserved cysteine residue has
been described as critical for Wnt signaling capacity [38].
Newer data show that Porcn-mediated S239 lipidation is
important for binding of Evi to Wnt3A [29], which
explains impaired secretion in the mutant construct.
Palmitoylation-dependent binding of Wnts has already
been proposed for the extracellular inhibitor, WIF-1,
which is confirmed by its crystal structure [39,40]. The
first crystal structure of a Wnt protein, Wnt8 bound to the
CRD domain of Wnt receptor Frizzled (Frz), surprisingly
revealed that one of the two interaction sites in the Frz/
Wnt complex involved the palmitoylation at a serine

Current Opinion in Genetics & Development 2013, 23:385–390 www.sciencedirect.com

 Secretion and extracellular space travel of Wnt proteins Gross and Boutros 387

Figure 1

ECS Exosomes
LPP ApoL
SWIM

Dlp Dally
Evi/Wls Tiki
apical Sulf-1 CCP

Reggie-1 Clathrin Rab5

AP2
EE
Retromer/SNX3

MTM-6/9

Myo5b MVB
Rab11
Syx1a

Golgi Ykt6

Rab1
p24
Wnt

Porcupine (Porc)
WntD

ER

Reggie-1

basolateral
Current Opinion in Genetics & Development

The core Wnt secretion pathway. Schematic representation of the Wnt secretory pathway. Wnt proteins are palmitoylated in the Endoplasmic
Reticulum (ER) by Porcn, then transported to the Golgi by sorting of p24 proteins into COPII vesicles. Evi shuttles Wnts from Golgi to the plasma
membrane (PM) from where it is recognized at a tyrosine-based sorting motif and endocytosed in Clathrin-coated pits (CCP). From early endosomes
(EE), Evi is recycled to Golgi by an SNX3-retromer-dependent step involving MTM-6/9. Alternatively, Evi and Wnts reach Multivesicular Bodies (MVBs),
where they are packaged and secreted onto exosomes into the extracellular space (ECS), a process that is regulated by rab11, syx1a, myo5b and
ykt6. Other extracellular forms of Wnts include: first, Wnts attached to lipophorin (ApoL) on lipoprotein particles (LPPs), second, Wnts solubilized by
binding to SWIM proteins or third, Wnts bound by HSPGs such as Dally and Dlp and Sulf-1, a heparan sulfate 6-O endosulfatase, which controls Wg
gradient formation. The lipid microdomain protein reggie-1 regulates Wnt gradient formation through facilitating its secretion and spreading in
Drosophila. The transmembrane protease Tiki inactivates extracellular Wnts by cleavages of an N-terminal fragment. WntD in Drosophila, the only Wnt
non-lipid-modified, is secreted independent of the core Wnt secretion pathway in a rab1-dependent manner. See also Table 1 for listing of proteins
and associated references.

www.sciencedirect.com Current Opinion in Genetics & Development 2013, 23:385–390

388 Developmental mechanisms, patterning and evolution
residue [41]. As suggested by this structure, the mutant
construct lacking the cysteine, would also fail to form a
disulfate bridge at that position, which could explain its
signaling deficiency, despite membrane targeting
attempts [35]. However, it seems that the overall hydro-
phobicity by the two lipidations is important for proper
passage through the secretory pathway [36,37]. Consider-
ing that binding of Evi to Wnt’s palmitoyl group is
required for proper secretion of Wnts, makes it interesting
to speculate how Wnts are handed over from secretory
cells/carriers to the receptor. From the current structure it
should involve flipping of Wnt’s lipid anchor from the
membrane leaflet of exosomes or the Evi interaction
pocket into the Frz interaction domain.
Glypicans as extracellular carriers
Spreading of Wnts throughout the extracellular space is
facilitated by heparan sulfate proteoglycans (HSPGs)
consisting of a core protein to which heparan sulfate
(HS) glycosaminoglycan (GAG) chains are attached. Sul-
fated 1 (Sulf1) a Drosophila homolog of vertebrate heparan
sulfate 6-O endosulfatase, regulates Wg signaling by
synergistic interaction with Dally to negatively influence
extracellular Wg levels [42,43]. In accordance, synergistic
stimulation of osteoblasts with Wnt3A and heparin
induces their osteogenic, but not mitogenic potential
[44]. During Xenopus embryogenesis, the transmem-
brane-protease Tiki specifically cleaves the N-terminus
of Wnt proteins reducing receptor binding and signal
activation [45].
In summary, the extracellular regulation of Wnt stability,
diffusion and gradient formation is controlled in several
ways: structural features of modified Wnts, interaction
with HSPGs and extracellular binding and cleavage of
Wnts. These context-specific cofactors can shape the
Wnt/Wg trafficking, fine-tuning its range and robustness
during development.
Role of Wnt secretion in development
Similar to its requirement for Wnt signaling in Drosophila,
deletion of Evi in mice results in early embryonic lethality
[46], which phenocopies Wnt3A and Porcn deletions [47].
Conditional deletion of Evi in different tissues has shown
the context and tissue regulation in hand that are controlled
by Wnt secretion. For example, deletion of Evi in the
epidermis demonstrated that epidermal Wnt ligands are
required in hair follicle initiation and regeneration balance.
The epidermis and dermis engage in a Wnt-dependent
crosstalk requiring ligand secretion and reception from
each other [48,49,50]. In the developing retina, retinal
myeloid cells secrete Wnts to suppress vessel branching for
proper angiogenesis of the retina [51]. Osteoblast-specific
deletion of Evi impaired their differentiation and mineral-
ization leading to loss of bone mass [52]. Conditional
deletion in limp mesenchym or ectoderm arrested limp
outgrowth and tissue differentiation, shedding light on the
Current Opinion in Genetics & Development 2013, 23:385–390
Wnt crosstalk between germ layers during development
[53]. Thus, tissue-specific deletion of Evi has become a
powerful tool to circumvent genetic redundancy of Wnt
ligands.
Wnt secretion in cancer and metastasis
Aberrant Wnt signaling has been implicated in many
cancers. Mutations in Wnt receiving cells, such as in
APC, Axin and b-catenin, are prototypic for mutations
leading to overactive Wnt signaling. In several tumors, it
has also been shown that Wnt ligands are overexpressed.
More recently, Wnt secretion emerges as a novel level of
deregulation. Evi is overexpressed in astrocytic gliomas,
leading to enhanced proliferation, cell migration and
tumor inducing capacity in mice [54]. In contrast, Evi
levels are reduced in melanoma compared to healthy skin,
leading to enhanced proliferation and metastasis for-
mation [55]. These tissue-dependent differences could
reflect a misbalanced equilibrium between secretion of
canonical versus noncanonical Wnts impaired by
reduction of Evi levels. Interestingly, a recent study in
mice has revealed an intercellular communication path-
way in which fibroblast-derived exosomes mobilize
Wnt11 in breast cancer cells to induce Planar Cell Polarity
(PCP) signaling in the cells’ protrusions enhancing their
invasive behavior [56]. Finally, several point mutations
in Evi were found in colon cancer that will need further
functional studies.
Conclusions and outlook
Altogether these recent studies highlight the role of Wnt
signaling in the crosstalk between different cell types and
populations in the establishment and maintenance of
tissues and organs. However, many open questions
remain to understand the cellular and physiological con-
text of Wnt secretion. For example, it remains rather
unclear where exactly Wnt proteins are loaded onto Evi?
How can cytoplasmic sorting components distinguish
between ‘loaded’ Evi/Wnt complexes and ‘empty’ Evi?
It might also be interesting for future investigations to
determine whether Evi is bound to Wnt to distinguish
anterograde and retrograde routes taken by both proteins,
which might be important to answer at which point Evi/
Wnt complexes separate.
Acknowledgements
We are grateful to K. Glaeser for comments on the manuscript and T. Rath
for help with art work. JCG was supported by a fellowship from the
Excellence Cluster CellNetworks. Research in the lab of MB is supported
by the DFG-funded Research Group 1036 on Wnt signaling.
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46. Carpenter AC, Rao S, Wells JM, Campbell K, Lang RA: Generation
of mice with a conditional null allele for Wntless. Genesis 2010,
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47. Biechele S, Cox BJ, Rossant J: Porcupine homolog is required
for canonical Wnt signaling and gastrulation in mouse
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48. Myung PS, Takeo M, Ito M, Atit RP: Epithelial Wnt Ligand
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This study along with Refs. [49,50] analyzed the role of Evi deletion in
hair follicles during hair cycle phases. Lack of Evi led to hair cycle arrest
due to reduced epithelial stem cell proliferation and lack of canonical Wnt
signaling in dermal papillae.
49. Chen D, Jarrell A, Guo C, Lang R, Atit R: Dermal-catenin activity
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proliferation and hair follicle initiation. Development 2012,
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First study along with Refs. [48,50] to show the importance of epidermal
derived Wnts in epidermis-dermis crosstalk during development. By
studying conditional ablation of epidermal Wnt secretion and dermal
Wnt signaling the authors support a reciprocal model in which epidermal
Wnt ligands instruct dermal fibroblast to generate the signal for patterned
expression of epidermal b-catenin in hair follicle formation.
50. Fu J, Hsu W: Epidermal Wnt Controls Hair Follicle Induction by
 Orchestrating Dynamic Signaling Crosstalk between the
Epidermis and Dermis. J Invest Dermatol 2012.
These authors along with Refs. [48,49] also support the epidermis-
dermis cross-talk model. However, by conditional KO of Evi in epidermis
and dermis, they define two groups of epidermal Wnts required for hair
follicle formation. The first group (Wnt3, 4 and 6) depend on Evi for
secretion and induces epidermal patterned b-catenin signaling which
initiates expression of the second group of epidermal Wnts (Wnt 2, 7b,
10a and b) instructing the dermal papilla to condensate.
51. Iii JAS, Lewkowich I, Rao S, Mariggi G, Carpenter AC, Burr AR,
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angiogenesis by a non-canonical Wnt-Flt1 pathway in myeloid
cells. Nature 2011:1-6.
This study demonstrates that retinal myeloid cells (RMCs) secrete non-
canonical Wnts to regulate angiogenesis in mouse retina. By inducing
expression and secretion of Flt1, a VEGF inhibitory protein, Wnts sup-
press blood vessel branching.
52. Zhong Z, Zylstra-Diegel CR, Schumacher CA, Baker JJ,
Carpenter AC, Rao S, Yao W, Guan M, Helms JA, Lane NE et al.:
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53. Zhu X, Zhu H, Zhang L, Huang S, Cao J, Ma G, Feng G, He L,
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54. Augustin I, Goidts V, Bongers A, Kerr G, Vollert G, Radlwimmer B,
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First results demonstrating a deregulation at the level of Wnt secretion
due to overexpression of Evi in glioma that promotes cell proliferation and
migration in vitro and in vivo.
55. Yang PT, Anastas JN, Toroni RA, Shinohara MM, Goodson JM,
 Bosserhoff AK, Chien AJ, Moon RT: WLS inhibits melanoma cell
proliferation through the beta-catenin signalling pathway and
induces spontaneous metastasis. EMBO Mol Med 2012,
4:1294-1307.
This study shows that in melanoma cell lines and patients Evi/Wls levels
are reduced, which in a animal craft leads to enhanced lung metastasis.
56. Luga V, Zhang L, Viloria-Petit AM, Ogunjimi AA, Inanlou MR,
 Chiu E, Buchanan M, Hosein AN, Basik M, Wrana JL: Exosomes
mediate stromal mobilization of autocrine Wnt-PCP signaling
in breast cancer cell migration. Cell 2012, 151:1542-1556.
The authors show that stromal derived exosomes facilitate secretion of
Wnt11 from breast cancer cells, activating PCP signaling and leading to
enhanced invasiveness and metastasis.
57. Katanaev VL, Solis GP, Hausmann G, Buestorf S, Katanayeva N,
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secretion of the long-range signalling forms of Wingless and
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58. Mulligan KA, Fuerer C, Ching W, Fish M, Willert K, Nusse R:
Secreted Wingless-interacting molecule (Swim) promotes
long-range signaling by maintaining Wingless solubility. Proc
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