InteractiveFly: GeneBrief

Carrier of Wingless: Biological Overview | References

Gene name - Carrier of Wingless

Synonyms -

Cytological map position - 94D12-94D13

Function - secreted signaling protein

Keywords - heparin sulfate proteoglycan - secreted from the presynaptic motor neuron - acts to limit synaptic architecture and neurotransmission strength - acts cooperatively with Notum to coordinate neuromuscular synapse structural and functional differentiation via negative regulation of Wg trans-synaptic signaling within the extracellular synaptomatrix

Symbol - Cow

FlyBase ID: FBgn0039054

Genetic map position - chr3R:23,070,516-23,112,730

NCBI classification - EFh_SPARC_EC: EF-hand, extracellular calcium-binding (EC) motif, found in secreted protein acidic and rich in cysteine (SPARC)-like proteins

Cellular location - secreted

NCBI links: EntrezGene, Nucleotide, Protein

The first Wnt signaling ligand discovered, Drosophila Wingless (Wg; Wnt1 in mammals), plays critical roles in neuromuscular junction (NMJ) development, regulating synaptic architecture and function. Heparan sulfate proteoglycans (HSPGs), consisting of a core protein with heparan sulfate (HS) glycosaminoglycan (GAG) chains, bind to Wg ligands to control both extracellular distribution and intercellular signaling function. Drosophila HSPGs previously shown to regulate Wg trans-synaptic signaling at the NMJ include the glypican Dally-like Protein (Dlp) and perlecan Terribly Reduced Optic Lobes (Trol). This study investigated synaptogenic functions of the most recently described Drosophila HSPG, secreted Carrier of Wingless (Cow), which directly binds Wg in the extracellular space. At the glutamatergic NMJ, Cow secreted from the presynaptic motor neuron was found to act to limit synaptic architecture and neurotransmission strength. In cow null mutants, this study found increased synaptic bouton number and elevated excitatory current amplitudes, phenocopying presynaptic Wg overexpression. cow null mutants exhibit an increased number of glutamatergic synapses and increased synaptic vesicle (SV) fusion frequency based both on GCaMP imaging and electrophysiology recording. Membrane-tethered Wg prevents cow null defects in NMJ development, indicating that Cow mediates secreted Wg signaling. It has been shown previously that the secreted Wg deacylase Notum restricts Wg signaling at the NMJ. This study shows that Cow and Notum work through the same pathway to limit synaptic development. It is concluded Cow acts cooperatively with Notum to coordinate neuromuscular synapse structural and functional differentiation via negative regulation of Wg trans-synaptic signaling within the extracellular synaptomatrix (Kopke, 2020).

The developing nervous system requires the coordinated action of many signaling molecules to ensure proper synapse formation and function. One key class of signals is the Wnt ligands. The first discovered Wnt, Drosophila Wingless (Wg), is secreted from presynaptic neurons and glia at the developing glutamatergic neuromuscular junction (NMJ) to bind to the Frizzled-2 (Fz2) receptor in both anterograde and autocrine signaling. In the postsynaptic muscle, Wg binding to Fz2 activates the noncanonical Frizzled Nuclear Import (FNI) pathway, which leads to Fz2 endocytosis and cleavage of the Fz2 C terminus (Fz2-C; Mathew, 2005). The Fz2-C fragment is trafficked to the nucleus to control translation of synaptic mRNAs and glutamate receptors (GluRs; Speese, 2012). In presynaptic neurons, Wg binding to Fz2 activates a divergent canonical pathway inhibiting glycogen synthase kinase 3β (GSK3β) homolog Shaggy (Sgg) to control microtubule cytoskeletal dynamics via the microtubule-associated protein 1B (MAP1B) homolog Futsch, resulting in synaptic bouton growth. The Wg signaling ligand must be tightly regulated in the synaptic extracellular space (synaptomatrix) to ensure proper NMJ development (Kopke, 2020).

One critical category of proteins regulating Wg ligand in the synaptomatrix is heparan sulfate proteoglycans (HSPGs). HSPGs consist of a core protein to which heparan sulfate (HS) glycosylphosphatidylinositol (GAG) chains are covalently attached. HS GAG chains are composed of repeating disaccharide subunits expressing variable sulfation patterns (the "sulfation code"; Masu, 2016). These GAG chains bind secreted extracellular ligands to regulate intercellular signaling. There are three HSPG families: transmembrane; glycerophosphatidylinositol (GPI) anchored; and secreted. The Drosophila genome encodes only five HSPGs, with the following three known to affect NMJ development: transmembrane syndecan; GPI-anchored Dally-like protein (Dlp); and secreted perlecan. A second secreted HSPG recently characterized in Drosophila was named Carrier of Wingless (Cow; Chang, 2014). In the developing wing disk, Cow directly binds secreted Wg and promotes its extracellular transport in an HS-dependent manner. Cow shows a biphasic effect on Wg target genes. Removing Cow results in a Wg overexpression (OE) phenotype for short-range targets, and a loss-of-function phenotype for long-range targets (Chang, 2014; Kopke, 2020 and references therein).

The mammalian homolog of Cow, Testican-2, is highly expressed within the developing mouse brain, and inhibits neurite extension in cultured neurons (Schnepp, 2005), although the mechanism of action is not known. This study therefore set out to characterize Cow functions at the developing Drosophila NMJ. The larval NMJ model is used because it is large, accessible and particularly well characterized for HSPG-dependent Wg trans-synaptic signaling (Sears, 2018). Each NMJ terminal consists of a relatively stereotypical innervation pattern, with consistent axonal branching and synaptic bouton formation. Boutons are the functional unit of the NMJ, containing presynaptic components required for neurotransmission including glutamate-containing synaptic vesicle (SV) pools and specialized active zone (AZ) sites for SV fusion. AZs contain Bruchpilot (Brp) scaffolds, which both cluster Ca2+ channels and tether SVs. AZs are directly apposed to GluR clusters in the postsynaptic muscle membrane. This spatially precise juxtaposition is critical for high-speed and efficient synaptic communication between neuron and muscle (Kopke, 2020).

This study sought to test Cow functions at the NMJ, with the hypothesis that Cow should facilitate extracellular Wg transport across the synapse. Structurally, cow null mutants display overelaborated NMJs with more boutons and more synapses, phenocopying Wg overexpression. This phenotype is replicated with targeted neuronal Cow knockdown, but not muscle Cow knockdown, which is consistent with Cow secretion from the presynaptic terminal. Functionally, cow null mutants display increased synaptic transmission strength. Both electrophysiology recording and postsynaptically targeted GCaMP imaging show increased SV fusion, indicating elevated presynaptic function. Replacing native Wg with a membrane-tethered Wg blocks secretion. Tethered Wg has little effect on NMJ development, but when combined with the cow null suppresses the synaptic bouton increase, indicating that Cow mediates only secreted Wg signaling. It was recently shown that Notum, a secreted Wg deacylase, also restricts Wg signaling at the NMJ (Kopke, 2017). This study shows that combining null cow and notum heterozygous mutants causes a synergistic increase in NMJ development, indicating nonallelic noncomplementation. Moreover, combining null cow and notum homozygous mutants did not cause an increase in NMJ development compared with the single nulls, indicating an interaction within the same pathway. It is concluded that Cow functions via negative regulation of Wg trans-synaptic signaling (Kopke, 2020).

The function of signaling ligands in the extracellular space is tightly regulated to ensure coordinated intercellular development, often via glycan-dependent mechanisms. The most recently discovered Drosophila HSPG, secreted Cow, was characterized with this role (Chang, 2014). In the developing wing disk, the Wnt Wg is produced in a stripe of cells at the dorsal/ventral margin boundary, and acts as an intercellular morphogen through Fz2 receptor signaling. The glypican HSPGs Dally and Dlp, bound to outer plasma membrane leaflets via GPI anchors, bind Wg to regulate both ligand distribution and intercellular signaling. It has been proposed that Dally/Dlp HSPGs are involved in the movement of extracellular Wg to form a morphogen gradient. However, in dally dlp double mutant clones, extracellular Wg is detected far away from Wg-secreting cells, suggesting that another extracellular factor can transport Wg. Cow was shown to fill this role by binding extracellular Wg to increase stability and rate of movement from producing to receiving cells (Chang, 2014). Supporting this model, cow mutants manifest Wg ligand gain-of-function/overexpression phenotypes for short-range targets, and loss-of-function phenotypes for long-range targets (Kopke, 2020).

At the NMJ, such a long-range Wg morphogen transport function is not seemingly required, except perhaps as a clearance mechanism, but Wg extracellular regulation and short-range Wg transport to cross the synaptic cleft is critical for NMJ development. At the forming of NMJ, Wg from neurons and glia signals both presynaptically (neuronal) and postsynaptically (muscle) via Fz2 receptors. In the motor neuron, Wg signaling inhibits the GSK3β homolog Sgg to regulate the MAP1B homolog Futsch to modulate microtubule dynamics controlling NMJ bouton formation. However, Futsch distribution and microtubule dynamics do not change with elevated Wg signaling, so this pathway alone does not explain the increased bouton formation with increased Wg signaling. In the postsynaptic muscle, Wg signaling drives Fz2 endocytosis and C-terminus cleavage, with transport to the nucleus regulating mRNAs involved in synaptogenesis, including postsynaptic GluR distribution (Speese, 2012). In wg mutants, GluRs are more diffuse; with clusters irregular in size/shape, increased receptor numbers and a larger postsynaptic volume. Thus, Wg trans-synaptic signaling controls both NMJ structure and function (Kopke, 2020).

Based on the findings from Chang (2014), it was hypothesized that Cow binds Wg to facilitate the transport across the synapse to Fz2 receptors on the muscle. If this is correct, a presynaptic Wg OE phenotype would be expected in the absence of Cow (Wg buildup at the source), and a postsynaptic Wg decrease/loss phenotype (failure of Wg transport). Presynaptically, increased synaptic bouton number was found in cow null mutants phenocopying the Wg OE condition (Kopke, 2017), consistent with this hypothesis. These results indicate that Cow normally inhibits NMJ bouton formation, consistent with the effects of inhibiting presynaptic Wg signaling. Postsynaptically, an increased number of GluR clusters were found due to elevated synapse formation in cow null mutants, but no evidence of diffuse GluR clusters of irregular size/shape and larger volume, as has been reported in wg mutants. Therefore, no strong support for the second prediction of the hypothesis. GluR changes within single postsynaptic domains are challenging to see even with enhanced resolution microscopy , but future studies could focus more on GluRIIA cluster size/shape/intensity in cow mutants. If GluR defects are detected in cow nulls, it would be interesting to test the Frizzled Nuclear Import (FNI) pathway (Kopke, 2020).

Wg signaling regulates multiple steps of NMJ development including branching, satellite bouton budding, and synaptic bouton maturation. None of the cow manipulations cause changes in branching, indicating that Cow does not regulate this Wg signaling, likely working in concert with other Wg regulators. Wg loss (wgts) decreases bouton formation, while neural Wg OE increases branching, satellite, and total bouton numbers. Satellite boutons represent an immature stage of development, with small boutons connected to the mature (parent) bouton or adjacent axon. Neuronal Cow OE does not change mature bouton number, but increases satellite bouton budding. Neuronal Cow RNAi also increases satellite boutons. Thus, changing neural Cow levels in either direction elevates satellite bouton numbers, suggesting different consequences on budding versus developmental arrest. It also appears that the cellular source of secreted Cow, or the balance between sources, may be important for proper Wg regulation. Importantly, glia-secreted Wg regulates distinct aspects of synaptic development (Kerr, 2014), with loss of glial-derived Wg accounting for some, but not all, of wg mutant phenotypes. Similarly, cell-targeted cow manipulations cause different NMJ phenotypes. There is no evidence for normal Cow function in postsynaptic muscle, but it remains possible that Cow secreted from glia could regulate Wg trans-synaptic signaling (Kopke, 2020).

Increasing Wg signaling elevates evoked transmission strength and functional synapse number (Kopke, 2017), which is phenocopied in cow null mutants. Block of postsynaptic Wg signaling causes increased SV fusion frequency and amplitude of miniature excitatory junctional potentials (Speese, 2012). With neuronal cow RNAi, there is a similar increase in event frequency and amplitude. These results suggest a decrease in postsynaptic Wg signaling when cow is lost, supporting the Wg transport hypothesis. Blocking Wg secreted from neurons or glia increases muscle GluR cluster size, albeit with differential effects on neurotransmission efficacy (Kerr, 2014). Reducing neuronal Wg has no effect on mEJC frequency, but reducing glial-derived Wg increases SV fusion frequency (Kerr, 2014). Both nerve-evoked and spontaneous neurotransmission are increased in cow null mutants, together with increased Brp active zones and postsynaptic GluR clusters forming supernumerary synapses. SynapGCaMP is an exciting new tool to test function at individual synapses. With targeted neuronal cow RNAi, there is an increase in both the number of SV fusion events and the postsynaptic Ca2+ signal amplitude, which is consistent with both presynaptic and postsynaptic regulation of Wg signaling. These functional phenotypes, combined with coordinated changes in presynaptic and postsynaptic formation suggest Cow regulates trans-synaptic Wg transport (Kopke, 2020).

There were differences between spontaneous synaptic vesicle fusion findings between TEVC electrophysiological recordings and SynapGCaMP reporter (MHC-CD8-GCaMP6f-Sh) Ca2+ imaging. Motor neurons that presynaptically targeted cow RNAi showed stronger impacts on SV fusion frequency with imaging in contrast to recordings, comparable to effects in the cowGDP null mutants. Moreover, SynapGCaMP imaging revealed significantly larger SV fusion event magnitudes in contrast to the lack of change found with TEVC recording. While the basis of these differences is unknown, it is speculated that it is due to the differential nature or sensitivity of these two methods. The Ca2+ imaging is based on measuring the change in the fluorescence signal over the baseline NMJ fluorescence, and it may be that glutamate receptor Ca2+ permeability or intracellular Ca2+ signaling dynamics is changed in a way not directly related to detectable membrane current changes in the cow mutants. TEVC recordings capture whole NMJ activity, whereas with imaging type 1b bouton activity was only captured normalized to area. In future studies, SynapGCaMP imaging can be used to map spatial changes in synapse function by assaying quantal activity separately in convergent type 1s and 1b motor neuron inputs and within discrete synaptic boutons. Moreover, differences between cowGDP and cowGDP/Df conditions could be influenced by second site-enhancing mutations on the Df chromosome. Overall, it should be noted that the changes in spontaneous SV fusion frequency and amplitude in cow mutants are subtle and variable, and need to be further studied in the future (Kopke, 2020).

Wg is lipid modified via palmitoylation to become strongly membrane associated. The hydrophobic moiety is located at the interface of Wg and Fz2 binding, shielded from the aqueous environment by multiple extracellular transporters until signaling interaction with the receptor. There have been many modes of extracellular Wg transport demonstrated, primarily from work in the wing disk, including microvesicles, lipoproteins, exosomes, and cytoneme membrane extensions. These multiple mechanisms of transport are much less studied at the synapse; however, exosome-like vesicles containing the Wg-binding protein Evenness Interrupted (Evi) have been demonstrated at the Drosophila NMJ. Cow could be considered an alternative extracellular Wg transport method, acting to shield Wg while facilitating transport through the extracellular synaptomatrix. In addition, HSPGs have been shown to regulate ligands by stabilizing, degrading, or sequestering the ligand, or as bifunctional coreceptors, or as facilitators of transcytosis. Results presented in this study are consistent with the hypothesis that Cow is mediating Wg transport across the NMJ synapse, but also that Cow has an additional role in the negative regulation of Wg synaptic signaling (Kopke, 2020).

The need for secreted Wg has been recently challenged, with Wg tethering to the membrane (NRT-wg) showing Wg secretion to be largely dispensable for development. In contrast, other recent studies suggest that Wg release and spreading is necessary. This study finds that tethering Wg at the NMJ synapse increases extracellular Wg ligand levels, with no change in mature bouton numbers. This Wg accumulation shows that NRT-wg is more stable at the synaptic signaling interface, consistent with other studies. However, although Wg levels increase, Wg signaling is less effective. With NRT-wg, only the budding of new satellite bouton is increased, with no increase in mature bouton formation. Reducing Wg function causes Fz2 upregulation, so this study hypothesized that Wg signaling could be maintained by increased presynaptic Fz2 receptors. When Wg is tethered, Cow cannot mediate intercellular transport, so the hypothesis predicts a similar phenotype with Cow (NRT-wg) or without Cow (NRT-wg; cowGDP). Indeed, Cow removal in the NRT-wg condition does not impact synaptic bouton number, although it does block the increase in satellite boutons, consistent with a Cow role in greater Wg stability (Chang, 2014). These results show that Wg secretion is required for the elevated NMJ development characterizing cow mutant animals (Kopke, 2020).

To further test how Cow is working through the Wg pathway to negatively regulate NMJ development, genetic interaction tests were performed with the Wg-negative regulator Notum. At the NMJ, Wg trans-synaptic signaling is elevated in the absence of Notum, and null notum mutants display larger NMJs with more synaptic boutons, increased synapse number and elevated neurotransmission (Kopke, 2017). All these defects are phenocopied by neuronal Wg OE, showing that the positive synaptogenic phenotypes arise from lack of Wg signaling inhibition. Consistently, genetically correcting Wg levels at the synapse in notum nulls alleviates synaptogenic phenotypes (Kopke, 2017). This study shows that cow null mutants have the same phenotypes of expanded NMJs, supernumerary synaptic boutons, greater synapse number/function, and strengthened transmission, suggesting that Cow acts like Notum in regulating Wg signaling. A genetic test was performed to ask whether Cow and Notum work in this same pathway. While cow and notum null heterozygotes do not exhibit NMJ defects, cow/notum trans-heterozygotes display grossly expanded NMJs with excess boutons. This combined haplo-insufficiency (type 3 SSNC) of nonallelic noncomplementation suggests that Cow and Notum share related roles. When full double mutants were tested, there is no additive effect, showing that Cow and Notum restrict Wg signaling in the same pathway. However, this pathway convergence appears restricted only to the control of structural synaptogenesis but not of functional neurotransmission, although the control neurotransmission amplitude was elevated in these studies (Kopke, 2020).

Cow now joins the list of synaptic HSPGs with key roles in NMJ development. HSPGs have been implicated in vertebrate NMJ synapse formation for over 3 decades. The Agrin HSPG is secreted from presynaptic terminals to maintain postsynaptic acetylcholine receptor clustering. Another secreted HSPG, perlecan, regulates acetylcholinesterase localization. Drosophila NMJ analyses have begun to more systematically elucidate HSPG roles in NMJ formation and function. In particular, the glypican HSPG Dlp regulates Wg signaling to modulate both NMJ structure and function, including the regulation of active zone formation and SV release. Wg binds the core Dlp, with HS chains enhancing this binding, to retain Wg on the cell surface, where it can both compete with Fz2 receptors and facilitate Wg-Fz2 binding. This biphasic activity depends on the ratio of Wg, Fz2, and Dlp HSPG as expounded in the 'exchange factor model'. Cow may impact this exchange factor mechanism as a fourth player, acting with Dlp to modulate Wg transport and Wg-Fz2 binding at the synaptic interface. It will be important to test Dlp levels and distribution in cow nulls to see how Cow fits into this model (Kopke, 2020).

In addition to Cow, perlecan (Trol) is another secreted HSPG reported to regulate bidirectional Wg signaling at the Drosophila NMJ. Trol has been localized near the muscle membrane, where it promotes postsynaptic Wg accumulation. In the absence of Trol, Wg builds up presynaptically, causing excess satellite bouton formation. It is interesting to note that cow mutants enhance Wg signaling without increasing satellite boutons. In trol mutants, ghost boutons increase due to decreased postsynaptic Wg signaling. Note that cow mutants do not exhibit ghost boutons, which fails to support decreased postsynaptic Wg signaling. Other postsynaptic defects in trol mutants (e.g., reduced SSR, increased postsynaptic pockets) are NMJ ultrastructural features that could be a future focus using electron microscopy studies. Similar to cow mutants, extracellular Wg levels are decreased in the absence of Trol, speculated due to increased Wg proteolysis, since HS protects HS-binding proteins from degradation. In cow mutants, it is not yet known whether Wg is decreased due to elevated signaling (ligand/receptor endocytosis) or to increased degradation due to Cow no longer protecting/stabilizing the ligand. Given that synaptic Fz2 is internalized with Wg binding, future experiments could test internalized Fz2 levels in cow mutants as a proxy of Wg signaling (Kopke, 2020).

In summary, this study has confirmed new tools to study Cow HSPG function and has discovered that Cow from presynaptic motor neurons restricts NMJ bouton formation, glutamatergic synapse number, and NMJ functional differentiation. Cow acts within the same Wg trans-synaptic signaling pathway as Notum by regulating the Wg ligand in the extracellular synaptomatrix. Secreted Cow modulates extracellular Wg ligand levels, with additional functions controlling Wg signaling efficacy, which may be independent of or dependent on Wg transport. It will be interesting to determine whether Cow core protein and/or its HS chains are important for the synaptic structural and functional phenotypes. Wg must be secreted for Cow to act on it, as shown by the membrane-tethered interaction studies, showing that secreted Cow must work on the freely diffusible Wg ligand. Perhaps most informative for future studies will be dissection of the interactions, coordination or redundancy of the multiple synaptic HSPGs at the NMJ, to further the understanding of extracellular Wg trans-synaptic signaling regulation during synaptic development. Drosophila is a particularly well suited model to study HSPGs because of the relatively reduced complexity in this system (Kopke, 2020).

Carrier of Wingless (Cow), a secreted heparan sulfate proteoglycan, promotes extracellular transport of Wingless

Morphogens are signaling molecules that regulate growth and patterning during development by forming a gradient and activating different target genes at different concentrations. The extracellular distribution of morphogens is tightly regulated, with the Drosophila morphogen Wingless (Wg) relying on Dally-like (Dlp) and transcytosis for its distribution. However, in the absence of Dlp or endocytic activity, Wg can still move across cells along the apical (Ap) surface. This study identified a novel secreted heparan sulfate proteoglycan (HSPG) that binds to Wg and promotes its extracellular distribution by increasing Wg mobility, which was thus named Carrier of Wg (Cow). Cow promotes the Ap transport of Wg, independent of Dlp and endocytosis, and this function addresses a previous gap in the understanding of Wg movement. This is the first example of a diffusible HSPG acting as a carrier to promote the extracellular movement of a morphogen (Chang, 2014).

This study showed that Carrier of Wg (Cow) is a secreted HSPG that can physically interact with Wg. The binding to Wg is dependent on the HS modification on Cow, and it can occur after both proteins are secreted. The apparent rates of Cow and Wg movement were measured, and the results showed that (a) Cow moved faster than Wg, (b) overexpression of Cow enhanced the rate of Wg movement, and (c) knockdown of Cow reduced the rate of Wg movement. Thus, it is suggested that Cow serves as a carrier of Wg to enhance the rate of its extracellular movement. This enhancement of Wg movement by Cow is important for the establishment of the Wg gradient during development. Moreover, the role of Cow as a carrier for a morphogen is unique among HSPGs in that Cow is a secreted HSPG, whereas the previously studied syndecans and glypicans are membrane-bound (Chang, 2014).

The measurements of Wg and Cow mobility were performed using endogenous Wg and Cow. Therefore, the true mobility of Wg and Cow, without the presence of the other, has not been determined. Because the cow mutant phenotype is dominantly affected by reducing the wg dosage and because overexpression affected Wg signaling, it is expected that neither is present in large excess over the other. The ptc-Gal4-driven expression of Cow was also much higher than the endogenous level of Cow. Therefore, it is expected that EGFP-Cow would be expressed much more highly than endogenous Wg, and this measurement represents the mobility of free EGFP-Cow. The large excess of overexpressed Cow likely enabled the measurement of its lateral mobility independent of endocytosis (Chang, 2014).

Two methods were used to measure the mobility of Wg. The first was to use Gal4/Gal80ts and temperature shifting to transiently induce Wg expression and then measure Wg distribution within an 8-h period. The estimated rates were 2.22 μm/h and 1.31 μm/h at the Ap and Ba surfaces, respectively, for the D-V axis. The second is a modification of the method used in another study. The estimated rate of Wg movement was 5.04 μm/h at the Ap surface and 10.15 μm/h at the Ba surface. In contrast to the first method, which addresses newly synthesized Wg, the second method addresses the release of intracellularly accumulated Wg, which is therefore driven by a higher concentration gradient and thus shows higher mobility. However, the second method indicated much slower rates than the calculated rate of 50 μm/30 min reported by that study. One important difference is that the previous study used shits mutant discs, whereas this study expressed Shits in the wing disc using nub-Gal4. Moreover, this difference was likely caused by the incomplete blocking of Wg secretion by the dominant-negative Shits, as evidenced by the exWg distribution in en>Shits (Chang, 2014).

Several extracellular molecules have been reported to influence Wg trafficking, including secreted Wingless-interacting molecule (Swim) and Lipophorin, which are involved in Wg long-range travelling; exosomes, which do not affect Wg gradient formation; and Secreted Frizzled-Related Proteins (SFRPs), which have no homolog in Drosophila. This study shows that Cow is required for the short-range transport of Wg. Because Wg is lipid-modified, its diffusion may be hindered by hydrophobic interactions with cell membranes. Its interaction with Cow may also help to reduce its interaction with the cell membrane, thereby accelerating its movement (Chang, 2014).

It is noted that because Cow is a secreted protein and appears uniformly in the wing disc, it cannot provide directionality to Wg transport. Instead, it simply enhances the mobility of Wg and allows the Wg gradient to be established more quickly (Chang, 2014).

It is proposed that Cow plays two distinct roles in the formation of the exWg gradient. (1) Cow is responsible for the Ap transport of exWg. This process is independent of endocytosis and is especially important because it is responsible for moving Wg out from its producing cells, which express only low levels of DFz2 and Dlp. (2) Cow is responsible for the intercellular transfer of Wg at the Ba surface. Because Cow is diffusible, it is expected to be more efficient to carry Cow over the intercellular space than to exchange Wg between membrane-bound Dlp or other receptors on adjacent cells. In addition, Cow also slightly increased exWg stability, perhaps by binding to Wg, or by diverting Wg away from endocytosis and degradation (Chang, 2014).

Because Wg can be bound by its receptor DFz2, by Dlp and Dally, and by Cow, these factors may compete for binding to Wg. It has also been proposed that the relative levels of Wg, DFz2 and Dlp can affect the morphogen activity gradient. This study adds another potential binding partner to this process. The relative levels of Wg, DFz2, Dlp and Cow likely determine not only the shape of the Wg gradient but also its relative distribution on the Ap versus Ba surface (Chang, 2014).

The biphasic activity of Cow can be explained by its effect on Wg mobility. Cow knockdown reduced Wg mobility, causing Wg to spread less and accumulate near the Wg-producing region. For short-range targets, the effect was similar to Wg GOF, whereas for intermediate- and long-range targets, the effect was similar to Wg LOF. This model can also explain the apparent contrast between the wing and embryo phenotypes in Cow knockdown. In the embryo, Wg specifies the naked cuticle fate over 5 rows of cells in the anterior segments. Therefore, the naked cuticle fate can be viewed as a long-range target of Wg, in which Wg LOF loses the naked cuticle fate and produces a denticle fusion phenotype. Cow knockdown also produces a phenotype similar to Wg LOF. In the wing, the chemosensory bristles at the wing margin are controlled by the short-range target neur, and Cow knockdown caused an increase in chemosensory bristles, similar to the effect of Wg GOF (Chang, 2014).

Recently, it was shown that replacement of endogenous Wg with a membrane-tethered Wg is sufficient for wing development with normal patterning. It has also been suggested that early Wg expression is coupled to cellular memory of target gene expression and that the spreading of Wg is therefore dispensable for patterning. However, this hypothesis does not readily explain how different Wg target genes are expressed at different ranges from the Wg source, which can be explained by the Wg gradient model and is supported by previous studies. In addition, this study found that Dll expression is activated only after 84 h AEL, which is past the early Wg expression phase suggested for cellular memory. The contradiction between the two modes of Wg patterning mechanisms requires further study for clarification (Chang, 2014).

The mammalian testicans can regulate neurite outgrowth and proteases activity. However, the role of testicans in regulating signaling pathways has not been studied. This study on the fly testican Cow is the first to demonstrate a role for the testican family in morphogen signaling as a diffusible HSPG. In addition, this stuey showed that human Testican-2 could bind to Wnt5a extracellularly, suggesting that the testican family may have a general role in regulating Wnt distribution and thus Wnt signaling (Chang, 2014).

Misregulation of Wnt signaling is well known to contribute to human diseases, including cancer. Accordingly, components of the Wnt signaling pathway have been developed as therapeutic targets for cancer. The Reggie protein, which affects Wnt secretion and spreading, is also associated with various types of cancer. The original identification of Cow was the result of an overexpression screen of genes with elevated expression in human hepatocellular carcinoma; thus, Cow may be involved in oncogenesis. Moreover, the finding of the novel and conserved role of the testican protein family in binding to Wnt ligands may reveal their involvement in human diseases (Chang, 2014).

Functions of Cow orthologs in other species

Mouse testican-2. Expression, glycosylation, and effects on neurite outgrowth

Mouse testican-2 was cloned, sequenced, and shown to be a proteoglycan with a multidomain structure closely similar to that of the human ortholog, previously described as a calcium binding extracellular matrix molecule of the BM-40/SPARC/osteonectin family. Recombinant mouse testican-2 was used to prepare specific antibodies that allowed the detection of testican-2 in various brain structures but also in lung, testis, and in several endocrine glands. Although the testican-2 expressed in EBNA-293 cells carried both heparan sulfate and chondroitin/dermatan sulfate glycosaminoglycan chains, the tissue form always contained only heparan sulfate. Both tissue-derived and recombinant testican-2 carried N-linked glycans. Tissue-derived forms of testican-2 were detected as proteoglycans of varying size, whereas a portion of the molecules produced by EBNA-293 cells were core proteins, lacking glycosaminoglycans. Both the proteoglycan and core protein forms of testican-2 inhibited neurite extension from cultured primary cerebellar neurons and may play regulatory roles in the development of the central nervous system (Schnepp, 2005).

Molecular cloning of testican-2: defining a novel calcium-binding proteoglycan family expressed in brain

A human cDNA library was screened using an expressed sequence tag related to the BM-40/secreted protein, acidic and rich in cysteine (SPARC)/osteonectin family of proteins, and a novel cDNA was isolated. It encodes a protein precursor of 424 amino acids that consists of a signal peptide, a follistatin-like domain, a Ca2+-binding domain, a thyroglobulin-like domain, and a C-terminal region with two putative glycosaminoglycan attachment sites. The protein is homologous to testican-1 and was termed testican-2. Testican-1 is a proteoglycan originally isolated from human seminal plasma that is also expressed in brain. Northern blot hybridization of testican-2 showed a 6.1-kb mRNA expressed mainly in CNS but also found in lung and testis. A widespread expression in multiple neuronal cell types in olfactory bulb, cerebral cortex, thalamus, hippocampus, cerebellum, and medulla was detected by in situ hybridization. A recombinant fragment consisting of the Ca2+-binding EF-hand domain and the thyroglobulin-like domain of testican-2 showed a reversible Ca2+-dependent conformational change in circular dichroism studies. Testican-1 and -2 form a novel Ca2+-binding proteoglycan family built of modular domains with the potential to participate in diverse steps of neurogenesis (Vannahme, 1999).


Search PubMed for articles about Drosophila Carrier of Wingless

Chang, Y.-H., Sun, Y. H. (2014), Carrier of Wingless (Cow), a secreted heparan sulfate proteoglycan, promotes extracellular transport of Wingless. PLoS One 9:e111573. PubMed ID: 25360738

Kopke, D. L., Lima, S. C., Alexandre, C. and Broadie, K. (2017). Notum coordinates synapse development via extracellular regulation of Wingless trans-synaptic signaling. Development 144(19): 3499-3510. PubMed ID: 28860114

Kopke, D. L., Leahy, S. N., Vita, D. J., Lima, S. C., Newman, Z. L. and Broadie, K. (2020). Carrier of Wingless (Cow) regulation of Drosophila neuromuscular junction development. eNeuro. PubMed ID: 32024666

Mathew, D., Ataman, B., Chen, J., Zhang, Y., Cumberledge, S. and Budnik, V. (2005). Wingless signaling at synapses is through cleavage and nuclear import of receptor DFrizzled2. Science 310(5752): 1344-1347. PubMed ID: 16311339

Masu, M. (2016). Proteoglycans and axon guidance: a new relationship between old partners. J Neurochem 139 Suppl 2: 58-75. PubMed ID: 26709493

Schnepp, A., Komp Lindgren, P., Hulsmann, H., Kroger, S., Paulsson, M. and Hartmann, U. (2005). Mouse testican-2. Expression, glycosylation, and effects on neurite outgrowth. J Biol Chem 280(12): 11274-11280. PubMed ID: 15657052

Sears, J. C. and Broadie, K. (2017). Fragile X mental retardation protein regulates activity-dependent membrane trafficking and trans-synaptic signaling mediating synaptic remodeling. Front Mol Neurosci 10: 440. PubMed ID: 29375303

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Biological Overview

date revised: 23 March 2020

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