Wnt proteins form a group of secreted ligands that regulate a number of events in development and disease. In Drosophila, many of these processes are mediated through a canonical pathway, which is initiated by Wingless (Wg), the prototypic Wnt signal. Recent genetic and molecular studies have uncovered new aspects on how Wg is produced, received, transduced and converted into target gene control, and on how the Wg canonical pathway gains specificity to elicit distinct cellular responses. Another Wnt-activated pathway, initiated by DWnt-4, facilitates cell movement during ovarian morphogenesis. The cell motility pathway is distinct from both the canonical Wnt pathway and from the Planar Cell Polarity (PCP) pathway.

Introduction

Wnt genes encode evolutionary conserved secreted glycoproteins that act as signaling molecules essential for a number of fundamental processes in development and disease. The wg gene, the Drosophila counterpart of vertebrate Wnt-1, was first identified as a hypomorphic mutation leading to wing deficient flies. Further characterization demonstrated that wg is involved in many developmental processes such as embryonic segment polarity and patterning, midgut morphogenesis, malpighian tubule organogenesis or limb development. The segment polarity phenotype was later instrumental for the identification of genes modifying the cellular or developmental response to Wg, thus of molecules required for transducing the signal, which resulted in the definition of a pathway specifically activated by Wg/Wnt-1 molecules, the socalled “canonical Wnt pathway” (see Chapter 1). The pathway includes reception by Frizzled transmembrane proteins, transduction through a cascade of cytoplasmic components, nuclear translocation of Armadillo (Arm), and control of Wg-responsive genes by a transcriptional effector Arm-dTCF complex (for a review see ref.1).

Unlike wg, the six additional Wnt genes existing in Drosophila were identified from approaches other than genetics (see Table 1). DWnt-2 and DWnt-3/5, sequences homologous to vertebrate Wnt-7 and to Wnt-5, were isolated through a PCR-based molecular screen. DWnt-4, which is very divergent from all other Wnt sequences, was discovered as a target gene of Hox proteins in the developing embryo. Finally, the last three Wnt genes, respectively related to vertebrates Wnt-6, Wnt-8 and Wnt-10, were identified by genome wide sequencing. Out of the six genes, mutations are available for DWnt-2 and DWnt-4 only and genetics has provided evidence for a function of DWnt-2 in the determination of male-specific pigment cells in the developing gonad and for a function of DWnt-4 in the control of cell movement during oogenesis.

Table 1

Drosophila Wnt genes have structural homologs in vertebrates.

Most of the review focuses on Wg signaling. We shall first briefly present the molecules that constitute the core of the canonical Wg pathway and emphasize recent findings that highlight the mechanisms operating at some steps. We will next discuss how the canonical pathway gains specificity to elicit distinct cellular responses and then will revisit the question of Wg functioning as an instructive morphogen. Finally, we will comment on the signaling cascade activated by DWnt-4, as it constitutes at present the only pathway currently proposed in Drosophila which is distinct from both the canonical Wnt pathway and from the PCP pathway.

The Wg Pathway

The prototypic Wnt gene wg is required in a variety of developmental events. Most of the genes coding for intermediates in the induced signaling pathway were then screened on the criterion that their loss of function phenotypes shared traits with the wg phenotype. Combination of genetic and molecular studies attributed these molecules with particular functions in the production, reception, transduction or transcriptional translation of the Wg signal. Here we shall focus on newly discovered components or mechanisms operating in the cascade and shall discuss the implications for the current view of the canonical Wg pathway.

Correct localization of extracellular signaling molecules is often decisive to bring about normal development of animals. Several levels of intracellular and extracellular control accomplish the targeted production and release of Wg. Some of the molecular players are mentioned here and more mechanistic aspects, especially how to generate a Wg gradient, are discussed in a separate section.

It is well established that wg transcripts are asymmetrically localized at the apical side of embryonic cells.² Only recently, however, it was shown that this localization is important for the function. Minimal sequences that direct this localization could be identified in the wg 3'UTR and their manipulation in living embryos demonstrated that apical localization of wg mRNA is essential for signaling. Thus, the segment polarity phenotype of wg mutant embryos, loss of naked cuticle in each segment, can be rescued with apically but not basally localizing wg transcripts.³ This localization requires an active transport of wg transcripts along cytoskeleton elements towards the apical end of cells. Intracytoplasmic transport proceeds along a microtubule network, using the motor protein ATPase dynein as a carrier, instead of myosin motors and actin filaments.⁴ These localization events therefore result in the synthesis of Wg protein in close proximity to where it will be secreted.

Processing and export of Wg from the producing cells is a rather complex process, not fully understood yet. Known pieces in this puzzle are that Wg processing engages Porcupine, a multipass transmembrane protein of the endoplasmic reticulum,⁵ and that domains in Wg critical for secretion are identified.⁶ Once secreted, Wg establishes strong molecular interactions with heparan sulphate proteoglycans (HSPGs) at the cell surface and extracellular matrix. This is important for both signaling and diffusion of Wg away from secreting cells. Interest- ingly, some of the HSPG mutations, like sugarless and sulfateless affect both Wg and FGF signaling, while others, like tout-velu affect Hedgehog (Hh) signaling but not Wg and FGF, and dally affects both Wg and Decapentaplegic (Dpp) signaling but not in all tissues. This indicates that HSPGs bind extracellular ligands of different pathways in a developmentally regulated fashion (for a review see ref. 7). New studies of membrane dynamics in Drosophila imaginal discs have resolved the apparent paradox that molecules such as Wg and Hh bind tightly to membranes and travel long distances. Greco et a^l8 described a cell biology mechanism that disperses membranes vesicles, which they named argosomes, over a large distance through the disc epithelium and showed that Wg colocalizes with argosomes derived from Wg-producing cells. This led the authors to suggest that a primary function of the interaction with HSPGs is to allow the incorporation of Wg into argosomes, which would then serve as vehicles for the spread of Wg. Another important advance regards the routing modulation of Wg signaling. Dubois et al.⁹ showed that directional clues for Wg to signal originate from a lysosomal protein degradation system operating posterior to Wg-producing cells in each parasegment of the embryo and from spatially regulated receptor binding and endocytosis on the other side.

The first Wg receptors identified were dFz-2 and Fz, two members of the Frizzled family, seven pass transmembrane molecules with an extracellular cysteine-rich domain shown to bind Wnts. Mutant phenotypes indicate that Fz acts in PCP (see chapter 6), through the Jun kinase (JNK) pathway and as a receptor for Wg using the canonical pathway, whereas dFz-2 is restricted to Wg signaling.¹⁰ While redundant roles for the two receptors were first discussed upon genetic experiments, dFz-2 was finally proposed to be a higher affinity receptor for Wg than Fz.^11,12 The respective roles of Fz and dFz-2 in Wg reception still is, however, a matter of debate, especially whether they could participate in different receptor complexes to mediate the distinct cellular responses to Wg. Particularly interesting in this context is the discovery of Arrow, a co-receptor used by dFz-2 to activate the pathway.¹³ arrow mutants develop a phenotype identical to wg loss of function but do not show any PCP phenotype, which clearly links its function to the canonical signaling. Arrow is a single pass transmembrane protein which harbors in its extracellular domain four EGF-like repeats and three LDL-like receptor repeats, features shared with its mammalian LRP homologs. Consistent with a function of Arrow in Wg reception, epistasis experiments showed that it acts downstream of Wg and upstream of Dishevelled (Dsh), the first intracellular component of the transduction pathway. Direct evidence for a LRP/Fz co-receptor complex, comes from vertebrates where the extracellular domains of LRP6 and Fz interact in a Wnt-dependent fashion (for a review see ref. 14).

Dsh is the most immediate intracellular mediator of the canonical pathway. Its role is to antagonize a multiprotein complex that leads to Arm degradation in the absence of signaling. Although it is well known that Dsh transduces the Wg signal to Axin and the serine threonine kinase Shaggy/Zeste white3 (Zw3), there are still molecular and mechanistic gaps to be filled. Especially the signal relay to Dsh is enigmatic, since Dsh does not directly bind Frizzleds.

Dsh was first identified and named for its loss of function phenotype that revealed a role in PCP. A wealth of information is currently being generated about this aspect of Dsh function (see Chapter 6), and also for this pathway Dsh is most proximal to the receptor so far. It is moreover required for signaling by DWnt-4 (see below). Dsh thus appears to function as a dispatching molecule that directs Fz-mediated signals towards distinct transduction routes. Three evolutionary conserved domains are characteristic of Dsh, the DIX, PDZ and DEP domains. Most interestingly, a differential domain usage guides the function in Wg signaling versus the PCP pathway, whereby DIX signals to the canonical pathway, DEP to PCP, and PDZ possibly to both.¹⁵¹⁸ More molecules are to be discovered around Dsh to understand in mechanistic terms how the selection of the appropriate pathway is achieved.

Two recently identified genes, arrow and PAR-1, likely play a role in this process. The presence of Arrow in the receptor complex (see above) commits Dsh to specify the Wg pathway, although whether Arrow directly contacts Dsh or acts through the recruitment of unknown cytoplasmic proteins remains to be established. PAR-1 is a Dsh-associated kinase that in cell culture assays potentiates signaling by Wg and blocks the JNK pathway.¹⁹ Gain of function and gene inactivation by RNA interference in fly consistently involved PAR-1 in the naked/denticled cell fate choice mediated by Wg in the embryonic ectoderm. These results suggest that Dsh phosphorylation by PAR-1 should be the molecular switch committing the Wg pathway. Further studies are needed, however, to fix the point since Dsh phosphorylation also correlates with the Dsh PCP function in vertebrates^17,20 and Drosophila.¹⁸ PAR-1 mutant isolation and epistasis analysis of functional interactions with mutations in dsh and arrow would thus unravel how a Fz activated receptor communicates with Dsh to ultimately activate either the Wg or the PCP pathway.

Naked (Nkd) is another recently identified factor interacting with Dsh through the PDZ domain.²¹ Nkd acts as a regulatory component in a feedback loop that restrains signaling in cells distant from the Wg source by affecting the transduction step between Dsh and Zw3 in a cell-autonomous manner. Although both Zw3 and Nkd kinases antagonize the pathway, their roles are clearly distinct. Zw3, which phosphorylates and causes degradation of Arm in the absence of Wg, becomes inactivated upon Wg reception. Nkd is induced in cells receiving Wg signal and functions directly through Dsh to limit its activity by setting the levels of Zw3 activity and therefore of Arm stabilization and downstream gene activation. Whether Nkd could play in PCP also was suggested, but not proved, by overexpression studies in fly and in vertebrate cell lines where the JNK pathway seems to be activated.²² Nkd might thus serve as a molecular switch converse to PAR-1, to limit Wg signaling and possibly activate PCP.

Armadillo, β-catenin in vertebrates, is a central player of the canonical Wnt pathway. This multifunctional protein has a pivotal role since, apart from its role as Wg transcriptional effector, it is involved in cadherin-mediated cell adhesion as a component of adherens junctions. In the absence of Wg signaling, the cytoplasmic pool of Arm is maintained at a low level following a process that depends on the formation of a multimeric complex composed of Zw3 and of the scaffolding proteins Axin and adenomatous polyposis coli (APC). This so-called “destruction complex” acts in two steps. Zw3 first phosphorylates critical sites in the N-terminal part of Arm which is then targeted for degradation by the ubiquitin-dependent proteasome. Wnt-induced inactivation of Zw3 prevents phosphorylation and subsequent proteosomal degradation, which stabilizes Arm in the cytoplasm in a cadherin free form. As a consequence of increased accumulation in the cytoplasm, Arm becomes translocated into the nucleus where it associates with the sequence-specific DNA binding protein dTCF/Pangolin to regulate Wg-responsive gene activity.²³ The sequence of cytoplasmic events in the canonical Wg/Wnt-1 pathway is solidly pictured and mechanistically well understood. Nuclear events, especially how nuclear import of Arm is controlled and how it operates in transcriptional regulation remain, however, far less well elucidated.

The ability of Arm to move back and forth between the nucleus and the cytoplasm is known for a long time.²⁴ Arm/β-catenin does not contain a nuclear localization sequence but shares homology with the importin/karyopherin family of transport receptors and was shown to bind directly to the nuclear pore machinery, consistent with an intrinsic importin-like activity required for nuclear import. Moreover, in vitro assays using permeabilized cells showed that the cytosol contains inhibitory activities anchoring β-catenin outside the nucleus, which suggests that its import may be regulated by upstream events in the signaling pathway.^25,26 This proposal has received strong support from recent findings in Drosophila: Axin titration to the cell membrane, or removal of axin in germ line clones, forces Arm into the nucleus, clearly demonstrating that Axin acts as a cytoplasmic anchor for Arm; dTCF was proposed to fulfill the converse function of anchoring Arm in the nucleus, based on the observation that the overexpression of a dominant negative form dTCF appears to block the nuclear accumulation of Arm which is observed when cytoplasmic Axin is depleted.²⁷ Although this observation clearly indicates that a nuclear anchoring system does exist to prevent export, it remains debatable whether dTCF may serve itself as an anchor, since Arm variants defective in dTCF binding still accumulate in the nucleus.²⁸ Arm nuclear retention could rather depend on interaction with other factor(s) involved in the formation of chromatin-bound Arm-dTCF transregulatory complexes (see below). As Arm/β-catenin can dynamically cross nuclear membrane pores in either directions,²⁹ a third piece in the mechanism was missing concerning the export process. Two recent reports showed that the APC nucleo-cytoplasmic protein contains a nuclear export signal, which is required for efficient export from the nucleus.^30,31 The emerging view therefore is that Arm/β-catenin subcellular localization is controlled by a combination of several mechanisms, Zw3-induced proteosomal degradation, cytoplasmic and nuclear anchoring as well as APC-mediated active export.

Another interesting emerging theme concerns the role of chromatin structural changes in the modulation of Wg/Wnt-1 responsive gene activation. In the absence of Wg signaling, dTCF recruits the corepressor Groucho (Gro) to maintain an inactive state of target genes.³² Gro has been shown to repress transcription by directly interacting with histones and the histone deacetylase (HDAC) Rpd3.³³ As reversible acetylation of histone tails plays a major role in chromatin remodeling, with the general thought that HDACs act as transcriptional corepressors and histone acetyltransferases (HAT) as coactivators,³⁴ this connection indicates that the regulation of chromatin structure is important for dTCF target gene control. A recent study moreover demonstrated a role for the Osa/Brahma chromatin remodeling complex (the SWI/SNF complex) in this mechanism.³⁵ Loss of function of osa, as well as that of brahma and moira, induces ectopic expression of Wg targets; conversely, osa overexpression represses endogenous transcription of the same genes. Osa, Brahma and Moira belong to the Trithorax group of proteins that act together with Polycomb group proteins in epigenetic mechanisms of transcriptional control of a variety of genes during development.³⁶ The same study also revealed that osa genetically interacts with gro and rpd3, leading to the proposal that the Osa/Brahma complex functions in a larger complex containing dTCF, Gro and Rpd3 to silence Wg target genes. In this silencing process, an intriguing role has been attributed to the CBP HAT that would maintain dTCF in an acetylated state, preventing interaction with Arm.³⁷

How does the nuclear accumulation of Arm, following cell stimulation by Wg, then relieve silencing? Arm/β-catenin was recently found to interact physically with both the SWI/ SNF component Brg1 and the CBP HAT and evidence that these interactions are functional in vivo was provided from cell culture assays³⁸ and genetic interaction studies in fly.³⁹ Arm would thus displace Gro and Rpd3 from the silencing complex, redirect SWI/SNF and dTCF towards the formation of an activating complex able, by recruiting the HAT activity of CBP, to remodel the chromatin structure of target gene promoters into an open conformation accessible to the basal transcription machinery. Other chromatin proteins likely participate in this dynamic,⁴⁰ among which the RuvB-like DNA helicases Pontin52 and Reptin52 have been shown to exert an additional control level in the canonical pathway.⁴¹ These two proteins, found in several chromatin modifying and remodeling complexes from yeast to mammals,^42,43 physically interact with Arm/β-catenin and with the TATA box binding protein TBP. Genetic experiments revealed opposite functions for the encoding genes, pontin producing a coactivator and reptin a corepressor of Arm. It has thus been proposed that the relative levels between the two proteins give rise to complexes that either promote or repress Wg target gene transcription. ⁴¹ The current view emerging from these studies therefore is that nuclear Arm recruits in multisubunit complex(es) a number of chromatin proteins that are required for the various aspects of chromatin dynamics related to transcriptional regulation, including histone tail modification, local relaxation, DNA unwinding, connection to transcriptional machinery and transcription initiation.

Specificity in Wg Signaling

Wingless is involved in a number of different developmental processes. It is obvious that a number of interfering regulatory inputs cooperate with Wg signaling in the control of these processes. Relatively few mechanisms have been described, however, as to how stage or tissue specificity in the cellular response to Wg is achieved. We have selected below a few examples illustrating that distinct mechanisms can confer specificity.

Does Wg Always Act as a Morphogen?

The concept of morphogen has long held attraction for developmental biologists because a single event, emission of a diffusible molecule from a source, provides parsimony for complex pattern formation. As a minimal definition, an extracellular morphogen is a molecule that can spread out from a localized source and directly instruct cell identities in a concentration-dependent manner. For secreted signaling proteins to fulfill these criteria implies the formation of a concentration gradient, a direct action at distance instead of an action by relay of a secondary signal and the commitment of responsive cells into distinct developmental fates.

It is now well established that Wg acts as a morphogen in the wing imaginal disc.^51,52 It is secreted from a narrow stripe of cells at the dorsoventral compartment boundary, forms a basally located gradient and acts in a long range manner to induce a graded expression of target genes such as achaete, Distalless and vestigial. Clonal analyses of loss and gain of function for Wg signaling moreover showed that target gene activation proceeds in direct response to Wg. New results emerge on the formation and maintenance of the gradient. Two mechanisms have been recently proposed. First, once secreted Wg spreads by extracellular diffusion along cell membranes through stabilizing interactions with dFz-2, which seems to adopt a graded distribution opposite to the Wg gradient and with components of the extracellular matrix such as the HSPG Dally-like.^53,54 The function of HSPGs, and possibly of dFz-2, would thus be to shape the gradient by limiting the diffusion of extracellular Wg. The second mechanism proposes a vesicle-mediated transport based on repeated cycles of endocytosis and secretion. This is supported by the recent demonstration that Wg is distributed in a gradient both intracellularly and extracellularly and is associated to endosomes/argosomes in cells distant from the Wg producing source.⁸ One function for HSPGs would thus be to allow Wg incorporation into argosomes and release at the cell membrane. The two mechanisms are not mutually exclusive, however, and the role of HSPGs might be twofold, first in shaping the gradient, allowing a transcytosis-like transport throughout the disc epithelium and second in a progressively decreasing release of extracellular Wg and stabilization at the membrane of receiving cells.

Whether Wg acts as a morphogen in other developmental contexts is far from clear. In the embryonic ectoderm, where its function has been extensively studied, Wg is produced from posterior most cells in each parasegment, moves on both sides into adjacent cells and then adopts a graded distribution in anterior cells only. Significant progress was made to understand how the gradient becomes asymmetrical by mid-embryogenesis through a combination of differential degradation and regulated endoctosis,⁹ which correlates with the asymmetrical instructive role played by Wg signaling in cell fate determination. Although it fulfils the criteria of a localized source, gradient formation and action at a distance, Wg however disobeys other major rules expected of a morphogen. First, there is no evidence for concentration-dependent signaling activity. Instead, a flat field of Wg protein provided by a thermo-inducible HS-wg transgene can considerably rescue the epidermal phenotype of wg deficient embryos.⁵⁵ Second, experiments using a membrane-tethered form of Wg showed that ligand transport mediated by cell movement is sufficient to account for the normal range of Wg, indicating that restricted diffusion is dispensable for embryonic ectoderm patterning.⁵⁶

DWnt-4 Controls Cell Movement Through a Unique Signaling Pathway

Seven Wnt genes exist in Drosophila. Mouse structural orthologs have been identified for all of them, but for DWnt-4 (Table 1). Functional data are available for only three, wg, DWnt-2 and DWnt-4. Nothing but some expression data⁵⁹ is currently known about the three Wnt genes identified from Drosophila genome sequencing, DWnt-6, DWnt-8 and DWnt-10. DWnt-3 is a secreted protein that accumulates in the embryonic nerve cord and whose ectopic expression leads to specific disruption of the commissural axon tracts of the central nervous system.⁶⁰ DWnt-2 encodes a signal required for the determination of male-specific pigment cells in the developing gonad and should be responsible for this sexually dimorphic trait.⁶¹ The signal transduction mechanism used by DWnt-2 has not been characterized.

DWnt-4 has received more attention. DWnt-4 and wg map to the same locus and show closely related developmental expression patterns, suggesting, despite of strong divergence in terms of coding sequence and of intron/exon structure, they have been maintained in a close physical linkage during evolution because of shared cis-regulatory elements.⁶¹ When provided ectopically, DWnt-4 can have antagonistic, similar or distinct functions than Wg, depending on the developmental context: it antagonizes late embryonic Wg signaling in the Drosophila epidermis and blocks Wg-induced body axis duplication in Xenopus;⁶² wg and DWnt-4 induce different phenotypes in the dorsal embryonic epidermis;⁶³ the two genes elicit similar cellular responses during imaginal development.⁶⁴ These apparently conflicting results could be explained by invoking functional redundancy or competition of the Wnt ligands, or interfering interaction between overproduced DWnt-4 and the Wg reception machinery. It has been recently demonstrated that DWnt-4 indeed signals through the Fz-2 receptor.⁶⁵

The message of the Cohen study⁶⁵ is actually of far broader interest in the Wnt field than only providing an explanation to the various phenotypes induced by ectopic DWnt-4. The message is that DWnt-4 facilitates cell movement through a unique signaling mechanism that is distinct from the canonical or planar polarity pathways. During vertebrate gastrulation Wnt signaling regulates lamellipodia behavior through Dsh²⁰ and controls convergent extension and cell movements.^66,67 The underlying cellular and molecular mechanisms are poorly defined, however. Cohen and colleagues have generated mutations in DWnt-4 and shown that it facilitates cell movement during ovarian morphogenesis through a signaling mechanism that results in Focal Adhesion Kinase accumulation. This kinase is required for the formation and disassembly of focal adhesion complexes, which is critical in the control of cell movement.⁶⁸ Fz-2 is the primary receptor for DWnt-4-mediated cell movement. However, the downstream effector of canonical signaling, dTCF, is not required. A mutation in Dsh that specifically disrupts planar polarity but transduces canonical signals⁶⁹ does disrupt cell movement, but the planar polarity mutants frizzled, prickled, inturned, and fuzzy do not.⁷⁰ The signaling pathway used by DWnt-4 therefore requires Fz-2 and Dsh but is neither the canonical Wnt pathway nor the planar polarity pathway.

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