InteractiveFly: GeneBrief

wntless: Biological Overview | References

Gene name - wntless

Synonyms - Evi

Cytological map position - 68A9-68A9

Function - transmembrane transport

Keywords - wingless pathway, wing, transmembrane transport, recycling of wingless from endosomes to the trans-Golgi network, links retromer function to Wnt secretion pathway

Symbol - wls

FlyBase ID: FBgn0036141

Genetic map position - 3L:11,159,720..11,162,741

Classification - DUF1171, Protein of unknown function

Cellular location - transmembrane protein

NCBI links: Precomputed BLAST | EntrezGene

Recent literature
Zhang, P., Zhou, L., Pei, C., Lin, X. and Yuan, Z. (2016). Dysfunction of Wntless triggers the retrograde Golgi-to-ER transport of Wingless and induces ER stress. Sci Rep 6: 19418. PubMed ID: 26887613
Secreted Wnts play diverse roles in a non-cell-autonomous fashion. However, the cell-autonomous effect of unsecreted Wnts remains unknown. Endoplasmic reticulum (ER) stress is observed in specialized secretory cells and participates in pathophysiological processes. The correlation between Wnt secretion and ER stress remains poorly understood. This study demonstrate that Drosophila miR-307a initiates ER stress specifically in wingless (wg)-expressing cells through targeting wntless (wls/evi). This phenotype could be mimicked by retromer loss-of-function or porcupine (porc) depletion, and rescued by wg knockdown, arguing that unsecreted Wg triggers ER stress. Consistently, it was found that disrupting the secretion of human Wnt5a also induced ER stress in mammalian cells. Furthermore, it was shown that a C-terminal KKVY-motif of Wg is required for its retrograde Golgi-to-ER transport, thus inducing ER stress. Next, it was investigated if COPI, the regulator of retrograde transport, is responsible for unsecreted Wg to induce ER stress. Surprisingly, it was found that COPI acts as a novel regulator of Wg secretion. Taken together, this study reveals a previously unknown Golgi-to-ER retrograde route of Wg, and elucidates a correlation between Wnt secretion and ER stress during development.


Cell-cell communication via Wnt signals represents a fundamental means by which animal development and homeostasis are controlled. The identification of components of the Wnt pathway is reaching saturation for the transduction process in receiving cells but is incomplete concerning the events occurring in Wnt-secreting cells. This paper describes the discovery of a novel Wnt pathway component, Wntless (Wls/Evi) and shows that it is required for Wingless-dependent patterning processes in Drosophila, for MOM-2-governed polarization of blastomeres in C. elegans, and for Wnt3a-mediated communication between cultured human cells. In each of these cases, Wls is acting in the Wnt-sending cells to promote the secretion of Wnt proteins. Since loss of Wls function has no effect on other signaling pathways yet appears to impede all the Wnt signals analyzed, it is proposed that Wls represents an ancient partner for Wnts dedicated to promoting their secretion into the extracellular milieu (Bänziger, 2006).

The development of complex multicellular organisms relies to large extent on the ability of cells to communicate via extracellular signals. One family of such signaling molecules comprises the Wnt proteins. They direct growth and cell fates in processes as diverse as embryonic segmentation, limb development, and CNS patterning. Recent evidence also implicates Wnt signaling in the postembryonic regulation of stem cell number and stem cell differentiation in mammalian skin and colon epithelia as well as in the hematopoietic system. Perturbations in Wnt signaling promote both human degenerative diseases and cancer (Bänziger, 2006).

Due to the immense importance of this class of signaling proteins, there is a profound interest to understand how Wnt proteins are synthesized, transported, received, and their signal transduced. A powerful way to reach this goal and identify the components involved is to study Wnt signaling in genetic model systems, such as Drosophila melanogaster or Caenorhabditis elegans (Bänziger, 2006).

A defining member of the Wnt family is the product of the Drosophila gene wingless (wg). In the embryo, Wg acts as a short-range inducer to pattern the epidermis: it is secreted by subpopulations of cells within each segment and received by their neighbors, which respond by expressing particular target genes. Loss of Wg function causes a segment-polarity phenotype, in which the larval epidermis forms a lawn of ventral denticles and lacks naked cuticle between the segmental denticle belts. At later stages, during larval development, Wg also acts at longer distances and in a concentration-dependent manner. In the wing imaginal disc, for example, Wg is produced by cells of the prospective wing margin and spreads symmetrically along the dorsoventral axis to define nested patterns of target-gene expression (Bänziger, 2006).

Many of the components of the cellular machinery responsible for transducing Wnt signals from the cell surface to the nucleus have been identified: receptors of the Frizzled and LRP families, the cytosolic proteins Dishevelled, Glycogen Synthase Kinase 3, Axin, APC, and β-catenin, the transcription factors TCF/Lef as well as many of their nuclear cofactors. Less is known about the processes controlling the secretion of Wnt and its movement through the territory between source and target cells. Wnts are secreted as glycoproteins and contain as a hallmark 22 cysteine residues at conserved positions in their amino acid sequence. Some of these cysteine residues might be engaged in the folding of Wnt proteins by disulfide bond formation. Purification and precise biochemical characterization of a Wnt protein succeeded only recently and led to the discovery that the first conserved cysteine residue (C77 in Wnt3a) is palmitoylated. A candidate acyltransferase to mediate this lipid addition is encoded by the Drosophila porcupine (porc) and C. elegans mom-1 genes. This acyltransferase is the only molecularly known pathway component that acts in Wnt-sending cells only. Glypican members of heparan sulfate proteoglycans (Dally and Dally-like in Drosophila) also act in Wnt-sending cells, but they mainly function in transporting Wnt proteins as well as other signaling molecules toward target cells (Bänziger, 2006).

To complete the identification of components involved in Wg signaling, an in vivo screening system was established that permits the isolation of Wg pathway genes that have so far resisted isolation by genetic means. This study describes the discovery of wntless (wls), a novel segment-polarity gene in Drosophila, and shows that it is required for Wg signaling throughout development. In an accompanying report, Bartscherer (2006) refers to this gene as evenness interrupted (evi). This characterization of wls/evi revealed that it encodes a novel, yet evolutionarily and functionally conserved, transmembrane protein that, intriguingly, is only required in Wg-sending cells. Analogous to Dispatched (Disp), a protein functioning in the release of Hedgehog (Hh) proteins, Wls is necessary for the secretion of Wnt proteins. However, unlike Disp, this function of Wls is not limited to the lipid-modified form of its target signal. Wls represents only the second component dedicated to the Wnt pathway that acts exclusively in signal-sending cells. Its discovery adds an unanticipated level of complexity that must be factored into concepts for Wnt-mediated cell-cell communication (Bänziger, 2006).

Genetic experiments showing that Wls is only required in Wg-sending cells, together with the finding that Wnt3a activity requires Wls function in cultured producer but not responder cells, placed Wls upstream of the Wnt/Wg receptors LRP/Arrow and Frizzled and the downstream signal-transduction cascade. These results on their own could not discriminate between the possibilities that in the absence of Wls Wnt ligands are sent out in an inactive form or are not sent out at all. However, the accumulation of Wg protein in wls mutant cells, together with the severe reduction of Wg/Wnt levels in tissue or medium surrounding mutant cells, indicated that the prime cause of the wls mutant phenotype must be a shortage of Wg/Wnt ligands in their target field. In the absence of Wls, Wg/Wnt proteins are predominantly retained in the producing cells and are thus unable to move in adequate quantities to nearby cells (Bänziger, 2006).

Some residual Wg-dependent patterning still occurs in wls mutants, such as reduced expression of the low-threshold Wg target gene Distal-less. Hence, a small fraction of Wg protein must be escaping, below the limits of immunohistochemical detection. Consistent with this assumption, it was found that wls homozygous mutant larvae did not survive a 50% reduction of Wg expression (by removal of one wg gene copy). All these observations suggest that the function of Wls is to ensure the efficient secretion of Wnts at physiologically effective levels. It is important to emphasize that these results do not exclude an additional role of Wls in conferring activity to Wnt ligands, a role that could be masked by the secretion phenotype (Bänziger, 2006).

The identification of a multitransmembrane protein required for the secretion of Wnt signaling proteins is reminiscent of the analogous discovery of Dispatched. In the absence of Disp, Hh signals are not secreted from cells and are unable to reach neighboring cells. In both pathways, the ligands are lipid-modified and tightly associated with cell membranes, yet are able to move over considerable distances in vivo. The predominant view of how Disp functions is that it releases Hh ligands from their membrane association. In apparent contrast to Disp, Wls seems to function earlier in the pathway, as it is already required for the transport of Wnt ligands to the cell surface, not just for their release from the membrane (Bänziger, 2006).

Other proteins implicated in the release of signaling proteins are the glypicans Dally-like (Dlp) and Dally, which belong to the family of heparan sulfate proteoglycans (HSPGs). Both Dlp and Dally bind Wg protein, and if their function is perturbed (by removing Sulfateless, an enzyme required for their synthesis) Wg protein no longer accumulates at the surface of Wg-secreting cells and animals exhibit wg loss-of-function phenotypes. These phenotypes are superficially similar to those caused by the lack of Wls function, raising the possibility that Wls might function like Dlp and Dally or play an essential role in their biosynthesis. However, in embryos with perturbed glypican function, Wg protein is undetectable at the surface of secreting cells because it is not stabilized there and released too readily. By contrast, when Wls function is perturbed, Wg/Wnt protein is poorly released, and its extracellular concentration is diminished rather than increased. Moreover, glypicans play a role in the movement of Wg from cell to cell, a role that was excluded for Wls by analysis of genetic mosaics. Together, the results indicate that Wls is required for proper intracellular transport of Wg/Wnt proteins to the cell surface (Bänziger, 2006).

If Wnt proteins use the general mode of intracellular conveyance, like other proteins destined for secretion, they may need special assistance to do so. For example, it is possible that Wnt polypeptides require a molecular chaperone function to reach a particular structural state for proceeding in vesicular transport from the ER to the Golgi apparatus. Wls might provide such a chaperone function, and in its absence Wnt ligands might not be admitted to public transportation. An intriguing parallel to such a chaperone function for Wnt signals is the recently identified chaperone for Wnt receptors: Boca and its mammalian homolog Mesd (Culi, 2003; Hsieh, 2003) are specifically required for the folding and stability of a subset of the low-density lipoprotein receptor (LDLR) family, which also comprises the Wg and Wnt receptors Arrow and LRP5/6. A high degree of client specificity is not unprecedented for molecular chaperone systems, but several observations argue against the view that Wls is a Wnt chaperone. First, for most chaperone systems analyzed, unfolded client proteins are targeted for degradation, rather than accumulation, in the absence of chaperone function. Second, the movement of such improperly folded polypeptides is blocked early along the secretory pathway, at the level of the ER, a phenotype not observed in cells lacking Wls function. And finally, the predominant location of Wls in the Golgi apparatus does not support a function in the ER for this protein (Bänziger, 2006).

Wnt proteins are posttranslationally modified by the addition of a palmitate group at a conserved cysteine residue. A candidate enzyme for catalyzing this lipid modification is encoded by the Drosophila porc gene. It is possible that lipid-modified Wnt proteins require a dedicated mechanism for their secretion and that Wls is part of such a mechanism. In principle, it is also possible that Wls is required together with Porc to palmitoylate Wg protein. However, Wls and Porc localize to different compartments of the secretory pathway, and the findings in mammalian cells indicate that Wnt3aC77A, which lacks the palmitoylation site, is still subject to regulation by Wls. Therefore, although a functional overlap between Porc and Wls function cannot be ruled out, the view is favored that Wls is acting subsequent to Porc in an aspect of the secretion of Wnt proteins that does not center on their palmitate modification (Bänziger, 2006).

Two recent observations that may relate to the mechanism of Wls action are particularly interesting. One concerns the discovery that a significant fraction of Wg protein inside secreting cells of the embryo is present in endocytic vesicles and sent back to the surface of such cells (Pfeiffer, 2002). The precise function of this ligand recycling is not yet known, but it may allow Wg protein to gain access to cellular compartments from which standard secretory proteins are excluded. A role that could be envisaged for Wls is to relieve Wg from this 'holding pattern' and allow it to ride public transportation out of the cell. Hence, loss of wls function would cause an abnormally high amount of Wg to cycle in this holding pattern, accompanied by a corresponding deficit in the extracellular milieu (Bänziger, 2006).

The other observation concerns lipophorin particles in Drosophila and the notion that such particles may serve as vehicles for the movement of lipid-modified ligands such as Wg or Hh (Panakova, 2005). An intriguing possibility would be if Wls is required for loading Wg protein onto such particles in a certain cellular compartment to which lipophorin particles and Wg have common access (Bänziger, 2006).

In a variation of these themes, Wnt proteins may have to cycle between different secretory or endocytic compartments before they can be secreted as active proteins. This cycling may endow them with posttranslational modifications or permit their charging onto lipophorin particles. Wls might be required to liberate Wnt proteins from this cycle and permit their subsequent secretion as mature signals (Bänziger, 2006).

Whatever the precise molecular mechanism by which Wls allows Wnt proteins to be secreted, it is remarkable that Wls functions so specifically on Wnt proteins. In a diverse set of experiments in which Wls function was reduced, spatially restricted, or abolished, no effects of Wls on other secreted proteins or cellular processes were detected. Equally astonishing is the observation that Wls is required for all members of the Wnt family that have been analyzed in this respect. In nematode and vertebrate systems, a subset of Wnt ligands has been found to signal via 'noncanonical' routes to control the polarities of individual cells; examples for such ligands are MOM-2 and Wnt5a, respectively. The current findings suggest that both polarity Wnts, as well as Wnt signals activating canonical transduction pathways, depend on Wls function for their respective activities. This apparent 'pan-Wnt promiscuity' argues for a fundamental relationship between Wls and structural aspects, as opposed to functional properties, of Wnt proteins. The presence of a single Wls ortholog in all metazoan genomes, and its absence in unicellular phyla and plants, indicates that Wnt signaling always depended on, and hence coevolved with, Wls function (Bänziger, 2006).

Secretion of Wnt ligands requires Evi, a conserved transmembrane protein

Wnt signaling pathways are important for multiple biological processes during development and disease. Wnt proteins are secreted factors that activate target-gene expression in both a short- and long-range manner. Currently, little is known about how Wnts are released from cells and which factors facilitate their secretion. This study has identified a conserved multipass transmembrane protein, Evenness interrupted (Evi/Wls), through an RNAi survey for transmembrane proteins involved in Drosophila Wingless (Wg) signaling. During development, evi mutants have patterning defects that phenocopy wg loss-of-function alleles and fail to express Wg target genes. evi's function is evolutionarily conserved; depletion of its human homolog disrupts Wnt signaling in human cells. Epistasis experiments and clonal analysis place evi in the Wg-producing cell. These results show that Wg is retained by evi mutant cells and suggest that evi is the founding member of a gene family specifically required for Wg/Wnt secretion (Bartscherer, 2006).

How organisms control the spatial and temporal activation of signaling pathways remains an important question in biology. With the availability of whole genome sequences, one of the key challenges is the functional annotation of the encoded gene products. RNAi approaches have become a powerful genetic tool to systematically dissect cellular pathways. This study analyzed the consequences of depleting almost all known Drosophila transmembrane proteins for Wg signaling. The survey in cultured cells identified Fz, Fz4, and LRP6/Arr, transmembrane proteins known to positively regulate Wg signaling. It is interesting to note that both Fz and Fz4, but not Fz2, appear to be required in cells for Wg signal transduction in a nonredundant manner. Moreover, a previously uncharacterized locus, evi/CG6210, which is a member of an evolutionarily conserved gene family, has been identified as a novel regulator of Wnt/Wg signaling both during Drosophila development and in human cells. evi has not been found in other RNAi screens for Wg signaling components. In imaginal discs and embryos, evi is specifically required for Wg secretion, and loss of evi leads to accumulation of Wg in the producing cell (Bartscherer, 2006).

While significant progress has been made in understanding of how signals are transduced from the ligand-activated receptor to the nucleus in the signal-receiving cell, little is known about the events that lead to the secretion of a functional ligand from the producing cell. Recent studies investigating Hh signaling have identified several factors required in secreting cells, which posttranslationally modify the ligand prior to secretion (Bartscherer, 2006).

Like Hh, Wg has been demonstrated to be lipid modified, a modification that accounts for its hydrophobicity and its targeting to lipid rafts. This posttranslational modification is probably mediated by the ER-resident protein Porcupine (Por). To date, Por remains the only factor known to be specifically required for Wg secretion. Another factor implicated in the secretion of Wg is the dynamin Shibire (Shi). However, Shi has been shown to have a general function in vesicular transport rather than to be dedicated exclusively to Wg secretion (Bartscherer, 2006).

The multipass transmembrane protein Evi is specifically required for Wg/Wnt secretion across species. Three independent lines of evidence suggest that Evi has a specific role in Wnt signaling. (1) Loss-of-function alleles phenocopy wg mutations and do not show other obvious phenotypes. During early embryonic development, germline clones of evi phenocopy mutations in other core Wg pathway components. The phenotype of evi is similar to wg during wing imaginal-disc development. (2) evi knockdown does not prevent the JAK/STAT ligand Upd from activating a cell-based reporter. (3) In vivo analysis demonstrates that evi loss of function leads to neither abrogation of Hh target-gene expression in receiving cells nor Hh accumulation in producing cells. This demonstrates that Evi is not required for the secretion of either Upd or Hh ligands and supports the model that Evi is not part of a general secretory machinery. Interestingly, Evi does not seem to be involved in the PCP signaling pathway, which might indicate that Fz-dependent PCP signaling in Drosophila might not rely on a Wnt factor (Bartscherer, 2006).

While there is no evidence that evi acts in other signaling pathways, the results indicate that evi is a core Wg/Wnt signaling-pathway component in both invertebrates and vertebrates. Cell-based experiments in Drosophila and human cells show that Wg/Wnt signaling is impaired when evi is depleted by RNAi. In vivo, germline and somatic clones of evi phenocopy wg loss-of-function mutations (Bartscherer, 2006).

These results show that Evi is necessary for efficient secretion of Wg. Cell culture experiments and clonal analyses in imaginal-disc tissues demonstrate that evi is required in the Wg-producing cell. Like wg, evi shows cell-nonautonomous phenotypes on target-gene expression in somatic cell clones. It was observed that Wg accumulates in Wg-producing cells in an evi mutant background. Wg is not efficiently released into the supernatant of cultured cells treated with evi dsRNA. In addition to the embryonic phenotype, these results demonstrate an impaired Wg secretion when evi is absent. However, target genes that require only low levels of Wg, such as dll, are expressed in evi2 mutant discs. This phenotype can be explained by maternally contributed evi transcript, although it cannot be excluded that Wg is still secreted in low amounts by evi mutant cells (Bartscherer, 2006).

Sequence analysis of evi predicts that the gene encodes a transmembrane protein. Consistently, an Evi-EGFP fusion protein localizes to the plasma membrane, suggesting that Evi, unlike the ER-resident protein Por, might regulate Wg secretion at the plasma membrane. Sequence analysis does not indicate any obvious enzymatic activity, such as glycosyl- or acyltransferase activity. However, in polarized cells, such as wing imaginal-disc cells, loss of evi leads to the delocalization of Wg protein from the apical side. In mammalian cells, apical secretion of proteins has been attributed to specialized secretory pathways that include protein sorting into cholesterol/sphingolipid-rich microdomains (lipid rafts) within the trans-Golgi network. It has been shown that acylation of Wg by Por is required for incorporation of Wg in polarized vesicles that transport Wg to specific sites at the plasma membrane. Since loss of evi leads to the delocalization of Wg protein from the apical side, it is proposed that Evi might be involved in the proper localization of Wg prior to its secretion. However, in evi mutant cells, accumulation of Wg alone may account for its delocalization and may not be due to a specific involvement of Evi in apical sorting. Interestingly, apical localization of Wg-containing vesicles has been shown to be involved in Wg signaling in the Drosophila embryo. A possible function of Evi may lie in the packaging, localization, or fusion of such Wg-containing vesicles (Bartscherer, 2006).

This stuyd shows that Evi, a conserved transmembrane protein, is required for efficient secretion of Wg in embryos and from imaginal-disc cells in vivo. As Evi appears to localize to the plasma membrane, it is tempting to speculate that the biological role of Evi is downstream of the ER-resident protein Por in the secretion of Wnts. The results support a model in which the cellular release of growth factors is performed by dedicated machineries that provide an additional level of regulation for the secretion of ligands. Evi might therefore belong to a growing family of specialized factors that regulate the cellular release of specific families of growth factors (Bartscherer, 2006).

Sprinter/Wntless: a novel transmembrane protein required for Wg secretion and signaling

Wingless (Wg) is a secreted ligand that differentially activates gene expression in target tissues. It belongs to the Wnt family of secreted signaling molecules that regulate cell-to-cell interactions during development. Activation of Wg targets is dependent on the ligand concentration in the extracellular milieu; cellular mechanisms that govern the synthesis, delivery and receipt of Wg are elaborate and complex. sprinter (srt) encodes a novel, evolutionarily conserved transmembrane protein required for the transmission of the Wg signal. Mutations in srt cause the accumulation of Wg in cells that express it, and retention of the ligand prevents activation of its target genes in signal-receiving cells. In the absence of Srt activity, levels of Wg targets (including Engrailed in embryos lacking maternal and zygotic srt, and Senseless and Achaete in wing discs) are reduced. Activation of Wg targets in the receiving cells does not require srt. Hence, the function of Srt is restricted to events occurring within the Wg-producing cells. srt is not required for any aspect of Hedgehog (Hh) signal transduction, suggesting specificity of srt for the Wg pathway. It is proposed that srt encodes a protein required for Wg secretion that regulates maturation, membrane targeting or delivery of Wg. Loss of srt function in turn diminishes Wg-pathway activation in receiving cells (Goodman, 2007; full text of article).

The srt/CG6210/Wntless genomic locus is composed of three exons with two possible splice variants to encode novel proteins of 594 (isoform A) and 562 (isoform B) amino acids that include or exclude exon 2. Both splice variants are expressed in Drosophila, since the EK288129 and CK00022 ESTs exclude the second intron, whereas the srt GH01813 cDNA used in the rescue experiment includes it. Indeed, both splice variants are expressed in S2R+ cells. Analysis of the amino acid sequence suggests that Srt is composed of four to eight transmembrane domains. The signal sequence constitutes the first transmembrane domain because it does not have a good consensus-signal peptidase-cleavage site. The next four hydrophobic sequence elements all represent potential transmembrane domains, but are either too short to traverse the membrane or are weakly hydrophobic, reducing the likelihood that they are within the membrane. The next three hydrophobic regions of the Srt protein are probably transmembrane domains. Based on these observations, it is hypothesized that Srt has four transmembrane domains with a large N-terminal globular extracellular/luminal domain that has two potential N-linked glycosylation sites, although several other topologies are clearly possible. This predicted structure places the Trp492 srt7E4 nonsense mutation within the last transmembrane domain to yield either a truncated protein or one that is earmarked for degradation through nonsense-mediated decay of the message or through the breakdown of the misfolded protein. It was also noticed that, although Flybase has srt/CG6210 annotated as a multi-drug-resistance-related protein (MRP), this analysis of the Srt amino acid sequence indicates that the only commonality between these proteins is that they are multi-transmembrane-spanning proteins. Hence, the current annotation of srt/CG6210 in Flybase as MRP is incorrect (Goodman, 2007).

Sequence comparison of Srt to all protein databases reveals that its closest known relative is found in Drosophila pseudoobscura sharing 87% identity and 91% similarity along its length. In Drosophila melanogaster, the closest relative of Srt is encoded by CG13409, located at cytological region 94A, and has only 22% identity and 42% similarity. Srt shows much stronger homology to protein sequences from its evolutionarily distant relatives, suggesting that Srt is unique in Drosophila. The alignment of the Drosophila Srt isoform B relative to nematode, frog and human reveals that Drosophila Srt isoform B shares 43% identity and 62% similarity with human Srt (hSrt). Whereas some regions in the N-terminus and the majority of C-terminal regions of Srt diverge from its vertebrate relatives, there is a high level of conservation that extends throughout the central region of Srt. The most N-terminal amino acids, including the signal sequence/first putative transmembrane domain, are fairly well conserved across species, even in the absence of a good consensus peptidase cleavage site, supporting the hypothesis that this constitutes a transmembrane domain (Goodman, 2007).

It is believed that the primary function of Srt in the Wg pathway is to support the maturation of activate Wg ligand. In this capacity, it is possible that Srt acts in post-translationally processing Wg, in the targeting of Wg to the plasma membrane or in the release of active Wg from the membrane. Since porcupine mutants, as well as point mutations in the Wg protein itself, yield similar Wg-retention phenotypes, and because porc is required for the post-translational processing of Wg, a role for Srt in the post-translational processing of Wg is one possible function of Srt. In this role, it would be predicted that Srt might act as an enzyme that either participates in known post-translational changes to the Wg protein, such as glycosylation or palmitoylation, or identifies a new post-translational alteration in Wg that is required for its maturation. In addition to catalyzing the palmitoylation of Wg, the action of Porc is required to target Wg to lipid rafts in the plasma membrane (Zhai, 2004). This observation suggests that membrane targeting might occur by an active process mediated by specific protein(s). Another possible function of Srt could be as a Wg-specific chaperone protein that promotes proper folding and shuttles Wg through the secretory pathway to the plasma membrane once posttranslational processing is complete. Indeed, there is precedent for the need of protein-specific chaperones in the Wg pathway. In order for functional Arrow -- the Wg low-density lipoprotein co-receptor -- to reach the plasma membrane, it requires the activity of a specific chaperone protein, Boca. Recent studies suggest that at least some Wg protein is loaded into lipoprotein particles during larval development, which may be required for the movement of lipid-modified Wg in the extracellular space to establish its morphogenetic gradient in the wing. These lipoprotein particles are exogenously synthesized in the fat body and must be loaded with their lipid-modified cargo in the cells that express the ligand. Hence, there must be protein(s) present at the plasma membrane that catalyzes this process. Sprinter may be localized within membrane rafts, at the ready to load palmitoylated Wg into arriving lipoprotein particles for dissemination to the Wg target cells. Another potential role for Sprinter could be to act indirectly on Wg by supporting the posttranslational maturation or subcellular targeting of the proteins that directly regulate these processes, although a physical interaction between Wg and Srt (also known as Wls or Evi) has been reported (Bänziger, 2006; Goodman, 2007).

Localization of Srt within the secretory pathway could be predictive of its function. Srt localization in lipid rafts at the plasma membrane would suggest involvement in generating Wg-loaded lipoprotein particles. However, ER or Golgi localization could indicate a role in Wg maturation as it moves through the secretory pathway. Although determination of the subcellular localization of Srt awaits specific antibodies, it was observed that, in srt7E4-mutant tissues, there is shift in the cellular distribution of Wg toward the basolateral surface of wing-disc cells -- the surface of Wg extracellular gradient formation in target cells -- without disruption of Golgi localization. This would suggest that the srt block to Wg maturation occurs within the ER or a post-Golgi compartment of the secretory pathway (Goodman, 2007).

The retromer complex influences Wnt secretion by recycling Wntless from endosomes to the trans-Golgi network

Secreted Wnt proteins play essential roles in many biological processes during development and diseases. However, little is known about the mechanism(s) controlling Wnt secretion. Recent studies have identified Wntless (Wls) and the retromer complex as essential components involved in Wnt signaling. While Wls has been shown to be essential for Wnt secretion, the function(s) of the retromer complex in Wnt signaling is unknown. This study examined a role of Vps35, an essential retromer subunit, in Wnt signaling in Drosophila and mammalian cells. Compelling evidence is provided that the retromer complex is required for Wnt secretion. Importantly, Vps35 colocalizes in endosomes and interacts with Wls. Wls becomes unstable in the absence of retromer activity. These findings link Wls and retromer functions in the same conserved Wnt secretion pathway. It is proposed that retromer influences Wnt secretion by recycling Wntless from endosomes to the trans-Golgi network (TGN)(Belenkaya, 2008).

This study provided convincing evidence for mechanistic roles of retromer and Wls in Wnt secretion. The retromer complex is involved in Wnt secretion in both Drosophila and mammalian cells. Wls stability is regulated by Dynamin-mediated endocytosis, and retromer plays an essential role in maintaining Wls protein levels, possibly via the retrieval of Wls from endosomes to the Golgi. Together, these findings have linked retromer and Wls into the same conserved Wnt secretion pathway. It is proposed that retromer influences Wnt secretion by recycling Wntless from endosomes to the TGN (Belenkaya, 2008).

One main finding of this work is the demonstration of a retromer requirement for Wnt secretion. First, it was shown that Wg protein accumulated inside the Wg-producing cells, whereas Wg levels in the receiving cells were reduced in the absence of the retromer activity. Second, secretion of Wg, as well as of Wnt3a and Wnt5a proteins, is inhibited in cultured cells, in which retromer activity is depleted by RNAi. Finally, a role of retromer in controlling the levels of Wls protein, which is essential for Wnt secretion, is demonstrated (Belenkaya, 2008).

Previously, Coudreuse (2006) found that vps-35 mutant in C. elegans did not affect the level of the Wnt, EGL-20, within producing cells when examined using an EGL-20-GFP fusion protein. Coudreuse also failed to detect Wnt3a secretion defects in Vps35-depleted mammalian cells. These differences could be due to variations in experimental procedures. In the current experiment, mosaic clones were used to compare Wg accumulation in Vps35 mutant cells with surrounding wild-type cells, whereas Coudreuse used homozygous Vps35 mutant embryos to compare with wild-type embryos. Furthermore, Coudreuse used a relatively short time period (maximum 5 hr) to collect Wnt3a protein in Vps35-depleted cells, which perhaps was not long enough to detect a decrease in Wnt3a secretion. Finally, EGL-20-GFP and Wnt3a-proteinA chimeras used previously might be less dependent on retromer activity for secretion compared with the native forms of these proteins. In the current experiments, nontagged Wg, Wnt3a, and Wnt5a proteins were used to avoid this potential complication (Belenkaya, 2008).

Consistent with a role of retromer in Wg secretion, reduced Wg short-range and long-range signaling activities were observed in the absence of retromer activity in the wing discs. The data argue strongly that diminished levels of Wg protein contribute to the reduced short- and long-range signaling activities of Wg. In this regard, it is important to mention that the short-range signaling activity of EGL-20 was only mildly affected, while the long-range signaling of EGL-20 was fully impaired in C. elegans (Coudreuse, 2006). The distinct responses of target genes are attributed to differences in their sensitivity to extracellular Wnt protein levels. Indeed, it was noticed that while Sens is strikingly reduced, only relatively weak reduction of Dll expression is observed in the absence of retromer activity (Belenkaya, 2008).

Several models have been proposed for the function of retromer in Wnt-gradient formation. These models include roles of retromer in (1) Wnt maturation by directly interacting with other enzymes such as Porcupine and (2) facilitating the interaction of Wnt protein with lipoprotein particles (Coudreuse, 2007; Coudreuse, 2006). Although these mechanisms of retromer's role in Wnt activity cannot be ruled out completely, in light of the data, it is argued strongly that the main function of retromer in Wnt signaling is to maintain Wls protein levels. It was shown that Wls is substantially reduced in the absence of retromer activity and it interacts with retromer in cells. In further support of this view, it was shown that overexpression of Wls can restore Wg secretion defects in Dvps35 depleted wing discs. Since Wls is likely to be required for the secretion of all of Wnt members (Banziger, 2006; Hausmann, 2007), it is suggested that retromer may be essential for the secretion of all Wnt proteins (Belenkaya, 2008).

One main issue related to Wls function is its subcellular distribution. While Bartscherer (2006). identified Wls at the plasma membrane of Drosophila and HEK293T cells, Banziger (2006) showed localization of Wls in the Golgi and in vesicles between the Golgi and the surface of mammalian cells. To clarify this issue, Wls distribution was carefully examined in wing discs and in HeLa cells. The results showed that Wls protein localizes on plasma membrane, TGN, and vesicles. Data from the biotinylation experiment, the extracellular staining of Wls, and colocalization with plasma membrane markers provide compelling evidence that some of the Wls protein is present on the plasma membrane. Importantly, the data revealed colocalization of Wls protein with early endosome markers including Rab5, EEA1, and Hrs. Thus, it is likely that Wls is trafficking from the TGN onto cell membrane and is then subsequently endocytosed from the cell surface. A previous study showed that Wls is able to bind Wnt (Banziger, 2006). Therefore, it is likely that Wls acts as a Wnt cargo receptor for the delivery of Wnt ligands from the Golgi onto the cell surface for secretion. Consistent with this view, accumulation of Wg in the Golgi was observed in the absence of Vps35 activity. Perhaps, the absence of retromer activity causes depletion of Wls in the Golgi, thereby resulting in Wg accumulation in the Golgi (Belenkaya, 2008).

These experiments further demonstrate that Wls is actively internalized and degraded through Dynamin-mediated endocytosis. Since Wls is enhanced in endosomes in the absence of Hrs activity, the data also suggest that Wls is likely to undergo the lysosomal degradation. Endocytosis-mediated protein degradation has been shown to be a mechanism for regulation of a number of signaling receptors such as Patched, Thickveins, and EGF receptor. Thus, this work builds on the known principle that lysosomal targeting of receptors regulates signaling in the responding cell, by showing that lysosomal targeting of a putative cargo receptor can also attenuate the production and presentation of ligand in the first place (Belenkaya, 2008).

Extensive colocalization of Wls with Vps35 protein was found in endosomes in HeLa cells, arguing that endosomes are the main sites of retromer activity. Studies in both yeast and mammalian cells suggest an essential role of retromer in retrieving membrane proteins from endosomes back to TGN (Seaman, 2005). In light of the data, it is suggested that the retromer complex is involved in recycling Wls from endosomes to TGN for its further function in Wnt secretion. This is likely mediated by the interaction of Wls with the retromer complex. Consistent with this view, it was found that cell surface Wls can be internalized and returned to the Golgi. In the absence of retromer activity, internalized Wls is likely to be sorted into lysosomes for degradation. In support of this view, it was demonstrated that overexpression of Vps35 can significantly enhance levels of Wls in mammalian cells even in the absence of Wnt ligand (Belenkaya, 2008).

On the basis of these findings, the following model is proposed. At the ER, Wnt protein is dually lipid-modified by the Porc. The modified Wnt exits from the ER and enters the Golgi, where it binds Wls. Wls carries and sends Wnt proteins from TGN onto the cell surface for secretion. Wnt is delivered to the cell surface, the unloaded Wls protein on the plasma membrane will subsequently be internalized through a Dynamin-mediated endocytosis process. In the endosomes, the internalized Wls can have two fates. (1) The retromer complex interacts with Wls and retrieves Wls from endosomes back to TGN, thereby maintaining the normal levels of Wls protein. (2) In the absence of retromer activity, Wls protein is subsequently delivered into lysosomes for degradation. In this model, Wls acts as a Wnt cargo receptor. More experiments will be needed to define the mechanism by which Wls protein transports Wnt from TGN onto the cell surface for secretion. In addition, it also remains to be determined whether Wls is required for Wnt modification or its interaction with other proteins such as lipoprotein particles before its secretion (Belenkaya, 2008).

Wingless secretion requires endosome-to-Golgi retrieval of Wntless/Evi/Sprinter by the retromer complex

The glycolipoproteins of the Wnt family raise interesting trafficking issues, especially with respect to spreading within tissues. Recently, the retromer complex has been suggested to participate in packaging Wnts into long-range transport vehicles. Analysis of a Drosophila mutant in Vps35 show that, instead, the retromer complex is required for efficient progression of Wingless (a Drosophila Wnt) through the secretory pathway. Indeed expression of senseless, a short-range target gene, is lost in Vps35-deficient imaginal discs. In contrast, Vps35 is not required for Hedgehog secretion, suggesting specificity. Overexpression of Wntless, a transmembrane protein known to be specifically required for Wingless secretion overcomes the secretion block of Vps35-mutant cells. Furthermore, biochemical evidence confirms that Wntless engages with the retromer complex. It is proposed that Wntless accompanies Wingless to the plasma membrane where the two proteins dissociate. Following dissociation from Wingless, Wntless is internalized and returns to the Golgi apparatus in a retromer-dependent manner. Without the retromer-dependent recycling route, Wingless secretion is impaired and, as electron microscopy suggests, Wntless is diverted to a degradative compartment (Franch-Marro, 2008).

A SNX3-dependent retromer pathway mediates retrograde transport of the Wnt sorting receptor Wntless and is required for Wnt secretion

Wnt proteins are lipid-modified glycoproteins that play a central role in development, adult tissue homeostasis and disease. Secretion of Wnt proteins is mediated by the Wnt-binding protein Wntless (Wls), which transports Wnt from the Golgi network to the cell surface for release. It has recently been shown that recycling of Wls through a retromer-dependent endosome-to-Golgi trafficking pathway is required for efficient Wnt secretion, but the mechanism of this retrograde transport pathway is poorly understood. This study reports that Wls recycling is mediated through a retromer pathway that is independent of the retromer sorting nexins SNX1-SNX2 and SNX5-SNX6. The unrelated sorting nexin, SNX3, has an evolutionarily conserved function in Wls recycling and Wnt secretion, and SNX3 interacts directly with the cargo-selective subcomplex of the retromer to sort Wls into a morphologically distinct retrieval pathway. These results demonstrate that SNX3 is part of an alternative retromer pathway that functionally separates the retrograde transport of Wls from other retromer cargo (Harterink, 2011).

The identification of the Wnt sorting receptor Wls has shown that the secretion of Wnt proteins is mediated by a specialized trafficking pathway that provides an important layer of regulation to Wnt signalling. A key step in this pathway is the retromer-dependent transport of Wls from endosomes to the TGN. This study reports that Wls retrieval is mediated by a retromer pathway that functions independently of the SNX-BAR retromer coat components (Harterink, 2011).

The retromer consists of a cargo-selective subcomplex that interacts with sorting nexins of the SNX-BAR family to segregate cargo into a tubular endosomal sorting pathway. The results show that the cargo-selective subcomplex also interacts with SNX3 as part of an alternative retromer pathway that mediates the recycling of Wls. Three lines of evidence suggest that these are functionally distinct retromer pathways. First, genetic analysis in C. elegans showed that snx-3 and the SNX-BAR sorting nexins function in parallel pathways. Thus, retrieval of the phagocytic receptor CED-1 is dependent on the SNX-BAR sorting nexins but independent of SNX3, whereas Wls recycling requires SNX3 but not the SNX-BAR sorting nexins. Second, co-immunoprecipitation experiments showed that the interaction of the cargo-selective subcomplex of the retromer with the SNX-BAR sorting nexins and SNX3 is mutually exclusive. Finally, live-cell imaging revealed that the SNX3 retromer pathway sorts Wls into a vesicular retrieval pathway that is morphologically distinct from the SNX-BAR-dependent tubular endosomal sorting pathway. On the basis of these results it is concluded that the SNX-BAR and SNX3 pathways are independent and mechanistically distinct retromer pathways (Harterink, 2011).

Studies in yeast have shown that the SNX3 orthologue Grd19p also functions in retromer-dependent endosome-to-Golgi retrieval, but, in contrast to SNX3, Grd19p functions together with the SNX-BAR sorting nexins Vps5p and Vps17p in the retrieval of cargo proteins such as the iron transporter Fet3p-Ftr1p. Grd19p physically interacts with a sorting sequence in the cytoplasmic tail of Ftr1p and with the SNX-BAR retromer complex, which led to the hypothesis that Grd19p acts as a cargo-specific adaptor that links Ftr1p to the SNX-BAR retromer complex. No interaction was observed between SNX3 and Wls in co-immunoprecipitation experiments and also co-precipitation of SNX3 with the SNX-BAR sorting nexins was not found. Furthermore, it was found that mutation of the SNX-BAR sorting nexins did not affect the SNX3-dependent retrieval of Wls, indicating that the function of SNX3 is fundamentally different from that of Grd19p in yeast (Harterink, 2011).

How do the two distinct SNX3- and SNX-BAR-retromer complexes regulate sorting of different endosomal cargo? One simple model to answer this question relies on the spatial segregation of SNX3 and the SNX-BAR sorting nexins along the endosomal maturation pathway. Although there is significant co-localization between these sorting nexins, SNX3 is predominantly localized to early endosomes by its high-affinity interaction with PtdIns(3)P, whereas the SNX-BAR retromer sorting nexins reside at the interface between early and late endosomes. Endocytosed Wls therefore initially enters SNX3-labelled early endosomes, where it engages the VPS26-VPS29-VPS35 trimeric complex, recruited to this compartment by the interaction with SNX3. Through a vesicular pathway, possibly dependent on indirect binding to clathrin as well as further membrane-remodelling proteins, the SNX3 retromer complex sorts Wls for retrieval to the TGN. In the absence of SNX3, Wls can be missorted into intraluminal vesicles and hence lysosomal degradation, or can be recycled through SNX-BAR retromer to the TGN. The relative flux through these two distinct pathways therefore determines the steady-state level of Wls. As the level of Wls is severely reduced on loss of SNX3, the flux into the lysosomal degradative pathway seems to be dominant. Thus, although a proportion of Wls may undergo SNX-BAR retromer-mediated recycling in the absence of SNX3, this is insufficient to maintain the required level of Wls for Wnt gradient formation during iterative rounds of Wnt secretion and Wls retrieval from the cell surface (Harterink, 2011).

Interestingly, the steady-state trafficking of the classical SNX-BAR retromer cargo CI-MPR is primarily defined by intracellular cycling between the TGN and late endosomes with retrieval to the TGN by way of the SNX-BAR retromer. The spatial-segregation model therefore suggests that the lack of effect of SNX3 suppression on steady-state CI-MPR distribution arises from CI-MPR entering the endosomal network at a point downstream of SNX3. That said, the complexity of CI-MPR trafficking -- a proportion of this receptor traffics to the plasma membrane before undergoing endocytosis and retrograde transport to the TGN -- that such a simple spatial-segregation model may be an oversimplification. It is therefore speculated that, alongside spatial segregation, cargo binding to the VPS26-VPS29-VPS35 complex may be an important element in selecting the sorting nexin coat that specifies the subsequent retrograde trafficking route. Thus, binding of VPS26-VPS29-VPS35 to Wls may favour association with SNX3, whereas engagement with CI-MPR favours binding to the SNX-BAR coat complex (Harterink, 2011).

WLS retrograde transport to the endoplasmic reticulum during Wnt secretion

Wnts are transported to the cell surface by the integral membrane protein WLS (also known as Wntless, Evi, and GPR177). Previous studies of WLS trafficking have emphasized WLS movement from the Golgi to the plasma membrane (PM) and then back to the Golgi via retromer-mediated endocytic recycling. This study found that endogenous WLS binds Wnts in the endoplasmic reticulum (ER), cycles to the PM, and then returns to the ER through the Golgi. An ER-targeting sequence was identified at the carboxyl terminus of native WLS that is critical for ER retrograde recycling and contributes to Wnt secretory function. Golgi-to-ER recycling of WLS requires the COPI regulator ARF as well as ERGIC2, an ER-Golgi intermediate compartment protein that is also required for the retrograde trafficking of the KDEL receptor and certain toxins. ERGIC2 is required for efficient Wnt secretion. ER retrieval is an integral part of the WLS transport cycle (Yu, 2014).

Active Wnt proteins are secreted on exosome

Wnt signalling has important roles during development and in many diseases. As morphogens, hydrophobic Wnt proteins exert their function over a distance to induce patterning and cell differentiation decisions. Recent studies have identified several factors that are required for the secretion of Wnt proteins; however, how Wnts travel in the extracellular space remains a largely unresolved question. This study shows that Wnts are secreted on exosomes both during Drosophila development and in human cells. Exosomes carry Wnts on their surface to induce Wnt signalling activity in target cells. Together with the cargo receptor Evi/WIs, Wnts are transported through endosomal compartments onto exosomes, a process that requires the R-SNARE Ykt6. This study demonstrates an evolutionarily conserved functional role of extracellular vesicular transport of Wnt proteins (Gross, 2012).

The present study demonstrated by biochemical and genetic approaches that a portion of functional Wnts is secreted on exosomes. The discrepancy between the hydrophobic properties of Wnts and their trafficking over longer distances has been a puzzling observation, leading to different hypotheses on how Wnts disperse. The results demonstrate that Wnts are secreted on exosomes in vivo and ex vivo. These secreted microvesicles originate through inward budding in multivesicular bodies (MVBs) and influence intercellular trafficking. Several lines of evidence support these conclusions. First, by biochemical fractionation it was found Wnt co-segregating with exosomes derived from mammalian and Drosophila cells. Ultrastructural analysis demonstrated that Wg is found on the outer membrane of exosomes. Second, it was demonstrate that Wnts are shuttled to MVBs by their cargo receptor Evi; this step is impaired by inhibition of MVB maturation or depletion of the ESCRT-0 complex, which mediates cargo recognition of exosomal proteins. Third, it was shown that a fraction of extracellular Wg co-localizes with several exosomal markers in vivo. Fourth, Ykt6 was identified as a protein required for secretion of Wnts and exosomal proteins. The results argue for an alternative route of Wnt secretion in vivo that is independent of lipoprotein particles. The biochemical fractionation method differs from the parameters used to biochemically isolate lipoprotein particles; in addition, lipoprotein particles were not found by ultrastructural analysis in the preparations. Taken together, it is believed that this study provides solid evidence for a Wnt secretion route through exosomes (Gross, 2012).

Ykt6 belongs to the longin type of R-SNARE involved in various trafficking events. Studies in yeast have suggested that Ykt6 localizes to the Golgi and to endosomal and vacuolar membranes. This study shows that in Drosophila Ykt6 does not affect secretion of transmembrane proteins such as Ptc and Fmi, and that on loss of Ykt6 in HeLa cells Evi accumulates in early endosomes but not in the Golgi. This finding is consistent with the role of Ykt6 in early/recycling endosomes described in mammalian cells; however, further functional roles for Ykt6 might exist and might also be modulated in a cell-type-specific manner (Gross, 2012).

Interestingly, the results imply that the retromer complex acts upstream of MVB sorting of Evi-Wnt complexes, as demonstrated by the effect of ESCRT and Ykt6 RNAi on secretion of Wnt/Evi and the exosomal marker CD81. The retromer-SNX3 sorting route might decide whether unloaded Evi is recycled to the Golgi or whether Evi-Wnt complexes are packaged onto exosomes. This is supported by the finding that depletion of retromer leads to lysosomal sorting, thereby reducing the pool of Evi as well as inhibiting the secretion of Wnts on exosomes. It has been shown that SNX3 together with HGS functions in sorting and membrane invagination at the MVB level. In yeast, SNX3, similar to Ykt6, retrieves cargo receptors to Golgi before homotypic fusion of vacuoli. Thus, it is tempting to speculate that a retromer-SNX3-dependent step retrieves empty Evi to Golgi, whereas Wnts (and Evi) are packaged onto exosomes for functional release (Gross, 2012).

Different extracellular forms of Wnts might not be mutually exclusive. The results show that removal of vesicle-bound Wnt only partially reduced Wnt activity, implying that other secretion routes exist, possibly through the direct release from the plasma membrane, which might have different extracellular trafficking properties (Gross, 2012).

It is tempting to speculate that morphogens provide spatial information in other forms than concentrations of signalling molecules, involving a 'digital' rather than 'analogue' mode of encoding signals. Different packaging modes of Wnt molecules and their control in Wnt-producing cells might have a profound effect on spreading and signalling properties of morphogens (Gross, 2012).

The microRNA miR-8 is a conserved negative regulator of Wnt signaling

Wnt signaling plays many important roles in animal development. This evolutionarily conserved signaling pathway is highly regulated at all levels. To identify regulators of the Wg pathway, a genetic screen was performed in Drosophila. The microRNA miR-8 was identified as an inhibitor of Wg signaling. Expression of miR-8 potently antagonizes Wg signaling in vivo, in part by directly targeting wntless, a gene required for Wg secretion. In addition, miR-8 inhibits the pathway downstream of the Wg signal by repressing TCF protein levels. Another positive regulator of the pathway, CG32767, is also targeted by miR-8. These data suggest that miR-8 potently antagonizes the Wg pathway at multiple levels, from secretion of the ligand to transcription of target genes. In addition, mammalian homologues of miR-8 promote adipogenesis of marrow stromal cells by inhibiting Wnt signaling. These findings indicate that miR-8 family members play an evolutionarily conserved role in regulating the Wnt signaling pathway (Kennell, 2008).

Ectopic activation of Wg signaling in the developing eye using the GMR-Gal4 driver causes a dramatic reduction in eye size. To identify regulators of the Wg pathway, a genetic screen was performed to identify genes that, when misexpressed, suppress this small-eye phenotype. Wg was coexpressed with random genes that were placed under the control of bidirectional Gal4-dependent (UAS) promoters by GSV transposable element insertions. Two GSV transposon insertions (GSV1305-2 and GSV2196), known to suppress the GMR/Wg phenotype, were located upstream of the microRNA miR-8. Both GSV insertions also suppressed the phenotype resulting from ectopic expression of Arm*, a stable form of Arm, in the developing eye. To verify that the phenotype of these insertions was because of expression of miR-8 and not to expression of surrounding genes, transgenic flies were generated expressing miR-8 under the control of a Gal4-dependent promoter (UAS-miR-8). Expression of miR-8 suppressed the small-eye phenotype caused by ectopic expression of Wg or Arm*. These data suggest that miR-8 can inhibit ectopic Wg signaling in the developing fly eye (Kennell, 2008).

These studies suggest that miR-8 is a potent antagonist of Wg signaling in vivo and in cell culture. Inhibition of Wg signaling by miR-8 may be because of targeting of the pathway at multiple levels, from Wg secretion to the reception of the signal in the nucleus by TCF. Finally, these studies of miR-8 extended across species to examine the effects of miR-8 family members in a cell culture model of mouse mesenchymal stem cell differentiation. The studies suggest that miR-8 family members promote adipogenesis by inhibiting endogenous Wnt signaling. Negative regulation of Wnt signaling by these microRNAs is consistent with a study reporting that all five miR-8 family members are highly expressed in the epidermis of the skin but are excluded from the hair follicle, a site of Wnt activity. Together, these data suggest that miR-8 may play an evolutionarily conserved role in negatively regulating Wnt signaling (Kennell, 2008).

This study found that miR-8 inhibits TCF protein levels without affecting TCF mRNA, suggesting that TCF is a direct target of miR-8. However, the 3'UTRs of the reported TCF isoforms were not regulated by miR-8, suggesting that miR-8 may directly target TCF mRNA independently of its 3'UTR or through an indirect mechanism. An intriguing possibility is that miR-8 may directly target an unidentified gene that is required for TCF protein stability. Studies have shown that TCF/LEF transcription factor activity or subcellular localization is regulated by posttranslational modifications, such as sumoylation, phosphorylation, and acetylation; however, none of these studies have reported an effect of these modifications on overall TCF protein expression. Interestingly, Sox17 has been reported to negatively regulate both TCF-4 and β-catenin protein levels in human colorectal cell lines by a mechanism that appears to require the proteasome; however, a positive regulator of TCF protein stability has not yet been reported, and this study found that knockdown of the miR-8 target CG32767 did not affect TCF protein levels (Kennell, 2008).

Another microRNA, miR-315, was recently reported to be a positive regulator of the Wg pathway (Silver, 2007). miR-315 was identified in a cell culture-based screen for effects on Wg dependent reporter gene activity. In contrast to the current findings, that study did not identify miR-8 as a pathway regulator in its screen. These discrepant findings may be because of the use of different cell lines (Kc167 vs clone8) and overall approaches (in vivo vs cell culture-based screen). This study did not test the effects of miR-8 expression in clone8 cells, although robust inhibition of the Wg pathway in the wing imaginal disk, the original source of the cell line, was noted (Kennell, 2008).

miR-8 loss of function mutant flies were reported recently (Kerres, 2007). The authors described a subtle mutant phenotype with defective leg and wing extension and behavioral defects, in part, because of an increased expression of Atrophin. Although the miR-8 mutants do not demonstrate an obvious Wg-related phenotype, Wg signaling does play an important role in Drosophila leg and wing development, and increased expression of other direct or indirect targets of miR-8 (e.g., TCF, wls, CG32767) also may contribute to the mutant phenotype in the leg and elsewhere (Kennell, 2008).

The subtle phenotype of the miR-8 mutant is not surprising, given similar reports for other microRNA loss of function mutants. The differences in scale of the loss-of-function versus the gain-of-function phenotype that is reported in this study may be, in part, because of redundancy, because all three targets that were identified contain predicted binding sites for multiple microRNAs. Mutation of multiple microRNAs may be required to produce dramatic effects on some signaling and developmental pathways. In addition, negative and positive regulators of the Wg pathway, such as nkd and fz3, are dispensable in certain tissues, including the wing imaginal disk. This may be indicative of redundant negative and positive regulator activities in these tissues. Redundancy may protect the Wg pathway in certain tissues from aberrations because of alterations in regulator gene expression. Overall, the studies of miR-8 suggest that the role of miR-8 is not as a developmental switch, but instead miR-8 may act as a modulator of multiple pathways in vivo by precisely tuning target gene expression in concert with other microRNAs and genes (Kennell, 2008).

Sol narae (Sona) is a Drosophila ADAMTS involved in Wg signaling

ADAMTS (a disintegrin and metalloproteases with thrombospondin motif) family consists of secreted proteases, and is shown to cleave extracellular matrix proteins. Their malfunctions result in cancers and disorders in connective tissues. This paper reports that a Drosophila ADAMTS named Sol narae (Sona; CG9850) promotes Wnt/Wingless (Wg) signaling. sona loss-of-function mutants are lethal and rare escapers had malformed appendages, indicating that sona is essential for fly development and survival. sona exhibited positive genetic interaction with wntless (wls) that encodes a cargo protein for Wg. Loss of sona decreased the level of extracellular Wg, and also reduced the expression level of Wg effector proteins such as Senseless (Sens), Distalless (Dll) and Vestigial (Vg). Sona and Wg colocalized in Golgi and endosomal vesicles, and were in the same protein complex. Furthermore, co-expression of Wg and Sona generated ectopic wing margin bristles. This study suggests that Sona is involved in Wg signaling by regulating the level of extracellular Wg (Kim, 2016).

Proteases were originally started out as simple destructive enzymes in order to digest proteins and to provide amino acids to ancient organisms, but many proteases evolved in later times are specialized to change activity, localization, and binding properties of proteins and thereby affect many cellular functions. More than four hundred thousand proteases in all organisms can be classified into 9 categories and numerous subfamilies. Among these proteases, ADAMTS family as a subclass of ADAM (a disintegrin and metalloproteases) family constitutes a group of zinc-dependent secreted proteases widely expanded during metazoan evolution, including 6 members in flies, 5 members in nematodes, and 19 members in mammals (Kim, 2016).

These ADAMTSs are involved in many biological actions by processing mostly ECM and some non-ECM substrates. For example, ADAMTS-1 cleaves versican and aggrecan, and plays a key role in the ovulation process. ADAMTS-2, 3, and 14 cleave procollagen I , and mutations in ADAMTS-2 cause Ehlers-Danlos syndrome, a connective tissue disorder. ADAMTS-7 and 12 are significantly upregulated in arthritic patients. Besides ECM proteins, ADAMTS-13 cleaves von Willebrand factor (vWF) in blood, and mutations in ADAMTS-13 result in thrombotic thrombocytopenic purpura (TTP). In addition, ADAMTSs either enhance or inhibit cancer development. The level of ADAMTS-7 is upregulated in carcinoma and ADAMTS-1 promotes tumor development through the induction of stromal reaction. In contrast, ADAMTS-9 suppresses the formation of carcinoma by inhibiting angiogenesis, and stable expression of ADAMTS-16 decreases proliferation of cancer cells. Loss of ADAMTS-12 in mammals also increased tumor growth and progression (Kim, 2016).

ADAMTS is synthesized as a zymogen and has a relatively long prodomain. The physical interaction between the prodomain and the metalloprotease domain is essential for the latency of enzyme activity. Removal of prodomain in most ADAMTSs is mediated by furin, a proprotein convertase, in the secretory pathway. However, prodomains of ADAMTS-9, -10, -15 are processed by furin in ECM. In case of MIG-17 that is involved in male gonadal formation in C. elegans, the prodomain is cleaved autocatalytically. Thus, the activation mechanism of ADAMTS family appears to be diverse and may be tightly controlled in order to ensure the generation of active forms at the right time and place (Kim, 2016).

This study reports that an ADAMTS encoded by the CG9850 gene in Drosophila melanogaster is capable of promoting Wnt/Wg signaling. Wnt family proteins are conserved morphogens for growth, development and adult homeostasis in all metazoans. CG9850 was named sol narae (sona) meaning 'small wing' in Korean, based on the small wing phenotype of mutant escapers. Fly Wg, a homolog of mammalian Wnt-1, is a prototype of Wnt family proteins essential for the development of all fly appendages, and the wing imaginal disc has been an excellent system to study Wg signaling. Wg is known to be secreted from Wg-producing cells at the dorsal-ventral (DV) midline in the wing pouch and forms a concentration gradient in extracellular matrix (ECM). Wg binding to Frizzled receptors on the plasma membrane of Wg-responding cells activates Wg signaling cascade, and Wg effector proteins including Sens, Dll and Vg are expressed in different regions of wing pouch (Kim, 2016).

This study has focused on answering the following questions. Where and when is sona transcribed and translated? Where is the active form of Sona present? Which gene shows genetic interaction with sona? What are the in vivo roles of sona? sona exhibits a positive genetic interaction with wntless (wls) whose function is essential for secretion of Wg, and sona is positively involved in Wg signaling. Based on data provided in this report, it is proposed that Sona may modify proteins involved in Wg signaling (Kim, 2016).

ADAMTSs are secreted metalloproteases that are known to be involved in mainly ECM remodeling. Among six ADAMTSs in the fly, Papilin is essential for the formation of basement membrane and fly development, Stall functions in ovarian follicle formation and exhibits positive genetic interaction with Delta and ADAMTS-A is important for cell migration, especially in detaching cells from the apical ECM in salivary gland. This report has shown that Sona is a fly ADAMTS essential for fly development and survival. Transient coexpression of Sona and Wg increased the number of wing margin bristles, indicating that Sona is positively involved in Wg signaling. Accordingly, loss of sona decreased the level of Wg effector proteins as well as the level of extracellular Wg. Based on these results, it is proposed that Sona, as an ADAMTS, modifies yet unidentified protein(s) essential for Wg signaling (Kim, 2016).

During fly development, sona was transcribed at a high level in discrete regions in imaginal discs, which corresponded to the malformed regions in adult appendages of sona escapers. For instance, dorsal eye disc, the center of antenna disc, and outer ring of leg disc expressed the high level of sona transcripts, and sona escapers accordingly had disoriented ommatidial bristles in the dorsal eye, malformed arista, and kinked femur. Wing disc also exhibited the complicated mosaic pattern of sona transcription, and adult wings of sona escapers were small and abnormally shaped. Involvement of Sona in modulating the level of extracellular Wg may explain why these malformed adult structures are generated in sona escapers because Wg is specifically expressed in eye, wing and leg discs and determines the fate of organs (Kim, 2016).

The genetic link between Sona and Wg signaling was identified in a genetic screen in which a wls allele could rescue the lethal phenotype caused by the overexpression of Sona. Likewise, wing notching by the loss of wls was rescued by overexpression of sona. Furthermore, the loss of sona decreased the level of extracellular Wg. Taken together, these results raised a possibility that Sona may be involved in Wg signaling by affecting Wg secretion. How may Sona positively regulate Wg secretion? To act on Wg secretion, Sona has to be activated intracellularly, and function in secretory pathways. It has been shown that the prodomains of most ADAMTSs are cleaved in trans-Golgi network to become active. Thus, activated intracellular Sona may cleave unidentified proteins involved in Wg secretion and thereby promote the secretion of Wg. Indeed, intracellular Sona was enriched in the apical region while extracellular Sona are more enriched in the basolateral region. Similarly, intracellular and extracellular Wg are enriched in the apical and basolateral regions, respectively. It has been recently shown that Wg is secreted to the apical side and then reentered cells by endocytosis, and then moves to the basal side and secreted by transcytosis. It will be interesting to figure out whether Sona and Wg may be secreted together by transcytosis (Kim, 2016).

Besides the function of intracellular Sona for Wg secretion, presence of active Sona in conditioned medium of S2 cell culture suggests that extracellular active Sona may be involved in Wg signaling by modifying unknown ECM components. Immunocytochemical analysis of Sona confirmed that the active form of Sona devoid of the prodomain is present in basal ECM of wing discs. Therefore, active Sona may cleave ECM proteins that affect stability or activity of Wg. Well-studied ECM proteins essential for Wg signaling and formation of Wg gradient are Heparan sulfate proteoglycans (HSPG) such as Division abnormally delayed (Dally) and Dally-like (Dlp). These HSPGs can be modified by proteins such as Notum and Matrix metalloprotease 2 (Mmp2). Notum blocks Wg activity as α/β-hydrolase by modifying Dally and Dlp, and Mmp2 cleaves Dlp to inhibit the interaction between Dlp and Wg. Thus, Sona may act on these HSPGs or related ECM proteins to affect the stability or activity of extracellular Wg (Kim, 2016). Involvement of Sona in Wg signaling raises a possibility that some mammalian ADAMTSs may also be involved in Wnt signaling. Some mammalian ADAMTSs are known to function as positive factors for tumor invasion and progression. Overexpression of Wnts or downstream components of Wnt signaling also induces various tumors such as colon cancer, breast cancer, and leukemia. Wnt signaling is also essential for the growth and remodeling of bones and connective tissues. Overlapping functions of ADAMTSs and Wnt signaling supports the view that some mammalian ADAMTSs may be linked to Wnt signaling. Further work on identifying the intracellular or extracellular substrate(s) of Sona is required to fully understand how Sona is positively involved in Wg signaling (Kim, 2016).

Functions of Wntless orthologs in other species

C. elegans AP-2 and Retromer control Wnt signaling by regulating MIG-14/Wntless

While endocytosis can regulate morphogen distribution, its precise role in shaping these gradients is unclear. Even more enigmatic is the role of retromer, a complex that shuttles proteins between endosomes and the Golgi apparatus, in Wnt gradient formation. This study reports that DPY-23, the C. elegans mu subunit of the clathrin adaptor AP-2 that mediates the endocytosis of membrane proteins, regulates Wnt function. dpy-23 mutants display Wnt phenotypes, including defects in neuronal migration, neuronal polarity, and asymmetric cell division. DPY-23 acts in Wnt-expressing cells to promote these processes. MIG-14, the C. elegans homolog of the Wnt-secretion factor Wntless, also acts in these cells to control Wnt function. In dpy-23 mutants, MIG-14 accumulates at or near the plasma membrane. By contrast, MIG-14 accumulates in intracellular compartments in retromer mutants. Based on these observations, it is proposed that intracellular trafficking of MIG-14 by AP-2 and retromer plays an important role in Wnt secretion (Pan, 2008).

Three groups have found a new molecule known as Wntless, Evi, or Sprinter, that is necessary for Wnt function (Banziger, 2006; Bartscherer, 2006; Goodman, 2006). Wntless interacts physically with Wingless, targeting it to the cell surface for secretion (Banziger, 2006). C. elegans MIG-14, which is also known as MOM-3, is the homolog of Wntless (Banziger, 2006). The mig-14 alleles were originally identified in screens for mutants with defects in QL migration, which the Wnt EGL-20 regulates. A screen for mutants with defects in the asymmetric division of the EMS blast cell identified the original mom-3 allele. MOM-3 acts in P2, the cell that secretes the Wnt MOM-2 and signals to EMS, causing it to divide asymmetrically (Pan, 2008).

This paper establishes a connection between endocytosis, retromer function, and MIG-14. The C. elegans gene dpy-23 encodes the mu subunit of the AP-2 clathrin adaptor complex that is necessary for receptor-mediated endocytosis and functions in several Wnt-related processes. The observations indicate that efficient Wnt secretion requires endocytosis and trafficking of MIG-14 by retromer (Pan, 2008).

Wnt function requires the C. elegans AP-2 μ subunit DPY-23. Together with retromer, DPY-23 regulates the intracellular distribution of MIG-14, a Wnt-binding factor required for Wnt secretion. It is speculated that newly synthesized EGL-20/Wnt binds to MIG-14 in the Golgi, targeting the Wnt to the cell membrane for secretion. In this model, AP-2-mediated endocytosis and retromer retrieval at the sorting endosome would recycle MIG-14 to the Golgi, where it can bind to EGL-20/Wnt for next cycle of secretion (Pan, 2008).

Studies in Drosophila demonstrated a role for endocytosis in the formation of a Wingless gradient. Models based on a nonautonomous requirement for dynamin in Wnt function implicated endocytosis as part of a relay that transferred Wingless from one cell to the next. Other studies have proposed that the Wingless gradient was generated by diffusion. It was proposed that the effects of dynamin loss on Wnt function reflected a lack of Wingless secretion from cells expressing the morphogen. While the current results do not directly resolve this controversy, the requirement for DPY-23 in MIG-14 endocytosis supports the hypothesis that endocytosis is necessary for Wnt secretion and provides a mechanism for how endocytosis regulates Wnt secretion (Pan, 2008).

A previous study argued that retromer was not necessary for Wnt secretion, but instead was necessary for production of a functional Wnt (Coudreuse, 2006). In this study it was also proposed that retromer was necessary for long-range Wnt signaling, but only played a minor role in short-range signaling. It was argued that retromer mutants produced Wnt molecules that could only act on nearby cells but failed to act on more distant cells. The current findings that retromer is required for MIG-14 trafficking and the previous discovery that Wntless, the Drosophila MIG-14 homolog, is necessary for Wingless secretion are at odds with the interpretation that retromer plays a specific role in production of a Wnt that acts in long-range signaling (Banziger, 2006; Bartscherer, 2006; Pan, 2008 and references therein).

An argument for retromer playing a specific role in long-range signaling was based on the observations that retromer mutants have little effect on processes that require MOM-2 and LIN-44, Wnts that are produced near responding cells (Coudreuse, 2006). Further support for the long-range hypothesis was based on the higher frequency of V5 defects in egl-20 mutants compared to retromer mutants (Coudreuse, 2006). This difference contrasted with the high frequency of QL migration defects in both egl-20 and retromer mutants. The discrepancy between the V5 and QL defects in the two types of mutants was explained by the closer proximity of the V5 cell to the EGL-20 source. The model that retromer plays a specific role in long-range Wnt signaling has led to speculation that the trafficking events regulated by this complex might control the production of a specifically modified form of Wnt (Coudreuse, 2007; Coudreuse, 2006; Hausmann, 2007), for example, a Wnt that could associate with lipoprotein particles (Pan, 2008).

A simpler hypothesis is favored where retromer is required for MIG-14 recycling and where blocked recycling leads to defects in Wnt secretion. The observation that excess MIG-14 can ameliorate the Wnt phenotypes of dpy-23 and vps-35 mutants is consistent with the notion that low levels of functional Wnts are still secreted in these mutants. It is proposed that the phenotypic differences observed between retromer and egl-20 mutants may result from differential sensitivities of various responding cells to lowered Wnt levels, and a similar explanation could account for the phenotypic differences between dpy-23 and Wnt mutants (Pan, 2008).

While the phenotypes of mig-14 mutants have most of the defects displayed by either single Wnt mutants or Wnt mutant combinations, dpy-23 mutants do not exhibit certain Wnt mutant phenotypes. They do not have the severe ALM polarity defects that are exhibited by cwn-1; egl-20 or cwn-1; cwn-2 double mutants and completely lack the PLM polarity defects of lin-44 mutant. Yet the dpy-23 defects in HSN and QL migration are extremely severe. One explanation for these differences between dpy-23 and mig-14 mutants, as well as the differences between retromer and mig-14 mutants, is that different Wnt-producing cells vary in their dependence on AP-2 or retromer to secrete Wnts. It is speculated that endocytosis and retomer recycle MIG-14 for multiple rounds of Wnt secretion. If this hypothesis is correct, phenotypic differences could reflect the ability of some cells to synthesize sufficient MIG-14 resulting in less dependence on recycling. Alternatively, independent mechanisms for trafficking MIG-14 could operate in different Wnt-secreting cells (Pan, 2008).

Retromer dependent recycling of the Wnt secretion factor Wls is dispensable for stem cell maintenance in the mammalian intestinal epithelium

In C. elegans and Drosophila, retromer mediated retrograde transport of Wntless (Wls) from endosomes to the trans-Golgi network (TGN) is required for Wnt secretion. When this retrograde transport pathway is blocked, Wls is missorted to lysosomes and degraded, resulting in reduced Wnt secretion and various Wnt related phenotypes. In the mammalian intestine, Wnt signaling is essential to maintain stem cells. This prompted asking if retromer mediated Wls recycling is also important for Wnt signaling and stem cell maintenance in this system. To answer this question, a conditional Vps35 (fl) allele was generated. As Vps35 is an essential subunit of the retromer complex, this genetic tool allowed inducibe interference with retromer function in the intestinal epithelium. Using a pan-intestinal epithelial Cre line (Villin-CreERT2), no defects were observed in crypt or villus morphology after deletion of Vps35 from the intestinal epithelium. Wnt secreted from the mesenchyme of the intestine may compensate for a reduction in epithelial Wnt secretion. To exclude the effect of the mesenchyme, intestinal organoid cultures were generated. Loss of Vps35 in intestinal organoids did not affect the overall morphology of the organoids. It was possible to culture Vps35 Δ/Δ organoids for many passages without Wnt supplementation in the growth medium. However, Wls protein levels were reduced and a subtle growth defect was observed in the Vps35 Δ/Δ organoids. These results confirm the role of retromer in the retrograde trafficking of Wls in the intestine, but show that retromer mediated Wls recycling is not essential to maintain Wnt signaling or stem cell proliferation in the intestinal epithelium (dE Groot, 2013).

Smed-Evi/Wntless is required for β-catenin-dependent and -independent processes during planarian regeneration

Planarians can regenerate a whole animal from only a small piece of their body, and have become an important model for stem cell biology. To identify regenerative processes dependent on Wnt growth factors in the planarian Schmidtea mediterranea (Smed), RNAi phenotypes of Evi, a transmembrane protein specifically required for the secretion of Wnt ligands, were examined. During regeneration, Smed-evi loss-of-function prevents posterior identity, leading to two-headed planarians that resemble Smed-catenin1 RNAi animals. In addition, regeneration defects of the nervous system were observed that are not found after Smed-catenin1 RNAi. By systematic knockdown of all putative Smed Wnts in regenerating planarians, Smed-WntP-1 and Smed-Wnt11-2 were identifed as the putative posterior organizers; Smed-Wnt5 is a regulator of neuronal organization and growth. Thus, this study provides evidence that planarian Wnts are major regulators of regeneration, and that they signal through β-catenin-dependent and -independent pathways (Adell, 2009).

Cell shape and Wnt signaling redundantly control the division axis of C. elegans epithelial stem cells

Tissue-specific stem cells combine proliferative and asymmetric divisions to balance self-renewal with differentiation. Tight regulation of the orientation and plane of cell division is crucial in this process. This study reports an investigation of the reproducible pattern of anterior-posterior-oriented stem cell-like divisions in the C. elegans seam epithelium. In a genetic screen, an alg-1 Argonaute mutant was identified with additional and abnormally oriented seam cell divisions. ALG-1 is the main subunit of the microRNA-induced silencing complex (miRISC) and was previously shown to regulate the timing of postembryonic development. Time-lapse fluorescence microscopy of developing larvae revealed that reduced alg-1 function successively interferes with Wnt signaling, cell adhesion, cell shape and the orientation and timing of seam cell division. Wnt inactivation, through mig-14 Wntless mutation, was found to disrupt tissue polarity but not anterior-posterior division. However, combined Wnt inhibition and cell shape alteration resulted in disordered orientation of seam cell division, similar to the alg-1 mutant. These findings reveal additional alg-1-regulated processes, uncover a previously unknown function of Wnt ligands in seam tissue polarity, and show that Wnt signaling and geometric cues redundantly control the seam cell division axis (Wildwater, 2011).


Search PubMed for articles about Drosophila Wntless

Adell, T., et al. (2009). Smed-Evi/Wntless is required for β-catenin-dependent and -independent processes during planarian regeneration. Development 136: 905-910. PubMed ID: 19211673

Banziger, C., et al. (2006). Wntless, a conserved membrane protein dedicated to the secretion of Wnt proteins from signaling cells. Cell 125: 509-522. PubMed ID: 16678095

Bartscherer, et al. (2006). Secretion of Wnt ligands requires Evi, a conserved transmembrane protein. Cell 125: 523-533. PubMed ID: 16678096

Belenkaya, T. Y., et al. (2008). The retromer complex influences Wnt secretion by recycling wntless from endosomes to the trans-Golgi network. Dev. Cell 14(1): 120-31. PubMed ID: 18160348

Coudreuse, D. Y., Roel, G., Betist, M. C., Destree, O. and Korswagen, H. C. (2006). Wnt gradient formation requires retromer function in Wnt-producing cells. Science 312: 921-924. PubMed ID: 16645052

Coudreuse, D. and Korswagen, H. C. (2007). The making of Wnt: new insights into Wnt maturation, sorting and secretion. Development 134: 3-12. PubMed ID: 17138665

Culi, J. and Mann, R. S. (2003). Boca, an endoplasmic reticulum protein required for Wingless signaling and trafficking of LDL receptor family members in Drosophila. Cell 112: 343-354. PubMed ID: 12581524

de Groot, R. E., Farin, H. F., Macurkova, M., van Es, J. H., Clevers, H. C. and Korswagen, H. C. (2013). Retromer dependent recycling of the Wnt secretion factor Wls is dispensable for stem cell maintenance in the mammalian intestinal epithelium. PLoS One 8: e76971. PubMed ID: 24130821

Franch-Marro, X., et al. (2008). Wingless secretion requires endosome-to-Golgi retrieval of Wntless/Evi/Sprinter by the retromer complex. Nat. Cell Biol. 10(2): 170-7. PubMed ID: 18193037

Goodman, R. M., et al. (2007). Sprinter: a novel transmembrane protein required for Wg secretion and signaling. Development 133: 4901-4911. PubMed ID: 17108000

Gross, J. C., Chaudhary, V., Bartscherer, K. and Boutros, M. (2012). Active Wnt proteins are secreted on exosomes. Nat Cell Biol 14: 1036-1045. PubMed ID: 22983114

Harterink, M., et al. (2011). A SNX3-dependent retromer pathway mediates retrograde transport of the Wnt sorting receptor Wntless and is required for Wnt secretion. Nature Cell Biol. 13: 914-923. PubMed ID: 21725319

Hausmann, G., Banziger, C. and Basler, K. (2007). Helping Wingless take flight: how WNT proteins are secreted. Nat. Rev. Mol. Cell Biol. 8: 331-336. PubMed ID: 17342185

Hsieh, J. C., et al. (2003). Mesd encodes an LRP5/6 chaperone essential for specification of mouse embryonic polarity. Cell 112(3): 355-67. PubMed ID: 12581525

Kennell, J. A., Gerin, I., MacDougald, O. A. and Cadigan, K. M. (2008). The microRNA miR-8 is a conserved negative regulator of Wnt signaling. Proc. Natl. Acad. Sci. 105(40): 15417-22. PubMed ID: 18824696

Kim, G. W., Won, J. H., Lee, O. K., Lee, S. S., Han, J. H., Tsogtbaatar, O., Nam, S., Kim, Y. and Cho, K. O. (2016). Sol narae (Sona) is a Drosophila ADAMTS involved in Wg signaling. Sci Rep 6: 31863. PubMed ID: 27535473

Pan, C.-L., et al. (2008). C. elegans AP-2 and Retromer control Wnt signaling by regulating MIG-14/Wntless. Dev. Cell 14: 132-139. PubMed ID: 18160346

Pfeiffer, S., et al. (2002). Producing cells retain and recycle Wingless in Drosophila embryos. Curr. Biol. 12: 957-962. PubMed ID: 12062063

Seaman, M. N. (2005). Recycle your receptors with retromer. Trends Cell Biol. 15: 68-75. PubMed ID: 15695093

Wildwater, M., et al. (2011). Cell shape and Wnt signaling redundantly control the division axis of C. elegans epithelial stem cells. Development 138(20): 4375-85. PubMed ID: 21937595

Yu, J., Chia, J., Canning, C. A., Jones, C. M., Bard, F. A. and Virshup, D. M. (2014). WLS retrograde transport to the endoplasmic reticulum during Wnt secretion. Dev Cell 29: 277-291. PubMed ID: 24768165

Zhai, L., Chaturvedi, D. and Cumberledge, S. (2004). Drosophila wnt-1 undergoes a hydrophobic modification and is targeted to lipid rafts, a process that requires porcupine. J. Biol. Chem. 279: 33220-33227. PubMed ID: 15166250

date revised: 22 July 2014

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