Wingless transduction by the Frizzled and Frizzled2 proteins of Drosophila.

Wingless (Wg) protein is a founding member of the Wnt family of secreted proteins which have profound organizing roles in animal development. Two members of the Frizzled (Fz) family of seven-pass transmembrane proteins, Drosophila Fz and Fz2, can bind Wg and are candidate Wg receptors. However, null mutations of the fz gene have little effect on Wg signal transduction and the lack of mutations in the fz2 gene has thus far prevented a rigorous examination of its role in vivo. Here we describe the isolation of an amber mutation of fz2 which truncates the coding sequence just after the amino-terminal extracellular domain and behaves genetically as a loss-of-function allele. Using this mutation, we show that Wg signal transduction is abolished in virtually all cells lacking both Fz and Fz2 activity in embryos as well as in the wing imaginal disc. We also show that Fz and Fz2 are functionally redundant: the presence of either protein is sufficient to confer Wg transducing activity on most or all cells throughout development. These results extend prior evidence of a ligand-receptor relationship between Wnt and Frizzled proteins and suggest that Fz and Fz2 are the primary receptors for Wg in Drosophila.     

The role of the cysteine-rich domain of Frizzled in Wingless-Armadillo signaling

The Frizzled (Fz) receptors contain seven transmembrane helices and an amino-terminal cysteine-rich domain (CRD) that is sufficient and necessary for binding of the ligands, the Wnts. Recent genetic experiments have suggested, however, that the CRD is dispensable for signaling. We engineered fz CRD mutant transgenes and tested them for Wg signaling activity. None of the mutants was functional in cell culture or could fully replace fz in vivo. We also show that replacing the CRD with a structurally distinct Wnt-binding domain, the Wnt inhibitory factor, reconstitutes a functional Wg receptor. We therefore hypothesized that the function of the CRD is to bring Wg in close proximity with the membrane portion of the receptor. We tested this model by substituting Wg itself for the CRD, a manipulation that results in a constitutively active receptor. We propose that Fz activates signaling in two steps: Fz uses its CRD to capture Wg, and once bound Wg interacts with the membrane portion of the receptor to initiate signaling.     

Molecular Analysis of Ems-Induced Frizzled Mutations in Drosophila Melanogaster

The frizzled (fz) gene of Drosophila is essential for the development of normal tissue polarity in the adult cuticle of Drosophila. In fz mutants the parallel array of hairs and bristles that decorate the cuticle is disrupted. Previous studies have shown that fz encodes a membrane protein with seven putative transmembrane domains, and that it has a complex role in the development of tissue polarity, as there exist both cell-autonomous and cell nonautonomous alleles. We have now examined a larger number of alleles and found that 15 of 19 alleles display cell nonautonomy. We have examined these and other alleles by Western blot analysis and found that most fz mutations result in altered amounts of Fz protein, and many also result in a Fz protein that migrates aberrantly in SDS-PAGE. We have sequenced a subset of these alleles. Cell nonautonomous fz alleles were found to be associated with mutations that altered amino acids in all regions of the Fz protein. Notably, the four cell-autonomous mutations were all in a proline residue located in the presumptive first cytoplasmic loop of the protein. We have also cloned and sequenced the fz gene from D. virilis. Conceptual translation of the D. virilis open reading frame indicates that the Fz protein is unusually well conserved. Indeed, in the putative cytoplasmic domains the Fz proteins of the two species are identical.     

Pathway specificity by the bifunctional receptor frizzled is determined by affinity for wingless

The Frizzled (Fz) protein in Drosophila is a bifunctional receptor that acts through a GTPase pathway in planar polarity signaling and as a receptor for Wingless (Wg) using the canonical Wnt pathway. We found that the ligand-binding domain (CRD) of Fz has an approximately 10-fold lower affinity for Wg than the CRD of DFz2, a Wg receptor without polarity activity. When the Fz CRD is replaced by the high-affinity CRD of DFz2, the resulting chimeric protein gains Wg signaling activity, yet also retains polarity signaling activity. In contrast, the reciprocal exchange of the Fz CRD onto DFz2 is not sufficient to confer polarity activity to DFz2. This suggests that Fz has an intrinsic capacity for polarity signaling and that high-affinity interaction with Wg couples it to the Wnt pathway.     

The Wingless morphogen gradient is established by the cooperative action of Frizzled and Heparan Sulfate Proteoglycan receptors

We have examined the respective contribution of Heparan Sulfate Proteoglycans (HSPGs) and Frizzled (Fz) proteins in the establishment of the Wingless (Wg) morphogen gradient. From the analysis of mutant clones of sulfateless/N-deacetylase-sulphotransferase in the wing imaginal disc, we find that lack of Heparan Sulfate (HS) causes a dramatic reduction of both extracellular and intracellular Wg in receiving cells. Our studies, together with others [Kirkpatrick, C.A., Dimitroff, B.D., Rawson, J.M., Selleck, S.B., 2004. Spatial regulation of Wingless morphogen distribution and signalling by Dally-like protein. Dev. Cell (in press)], reveals that the Glypican molecule Dally-like Protein (Dlp) is associated with both negative and positive roles in Wg short- and long-range signaling, respectively. In addition, analyses of the two Fz proteins indicate that the Fz and DFz2 receptors, in addition to transducing the signal, modulate the slope of the Wg gradient by regulating the amount of extracellular Wg. Taken together, our analysis illustrates how the coordinated activities of HSPGs and Fz/DFz2 shape the Wg morphogen gradient.     

Trimeric G protein-dependent frizzled signaling in Drosophila

Frizzled (Fz) proteins are serpentine receptors that transduce critical cellular signals during development. Serpentine receptors usually signal to downstream effectors through an associated trimeric G protein complex. However, clear evidence for the role of trimeric G protein complexes for the Fz family of receptors has hitherto been lacking. Here, we show roles for the Galpha(o) subunit (Go) in mediating the two distinct pathways transduced by Fz receptors in Drosophila: the Wnt and planar polarity pathways. Go is required for transduction of both pathways, and epistasis experiments suggest that it is an immediate transducer of Fz. While overexpression effects of the wild-type form are receptor dependent, the activated form (Go-GTP) can signal when the receptor is removed. Thus, Go is likely part of a trimeric G protein complex that directly transduces Fz signals from the membrane to downstream components.     

The Drosophila tissue polarity gene starry night encodes a member of the protocadherin family.

The tissue polarity genes control the polarity of hairs, bristles and ommatidia in the adult epidermis of Drosophila. We report here the identification of a new tissue polarity gene named starry night (stan). Mutations in this essential gene alter the polarity of cuticular structures in all regions of the adult body. The detailed polarity phenotype of stan on the wing suggested that it is most likely a component of the frizzled (fz) pathway. Consistent with this hypothesis, stan appears to be downstream of and required for fz function. We molecularly cloned stan and found that it encodes a huge protocadherin containing nine cadherin motifs, four EGF-like motifs, two laminin G motifs, and seven transmembrane domains. This suggests that Stan functions in signal reception, perhaps together with Fz.     

The Drosophila STE20-like kinase misshapen is required downstream of the Frizzled receptor in planar polarity signaling.

The Drosophila misshapen (msn) gene is a member of the STE20 kinase family. We show that msn acts in the Frizzled (Fz) mediated epithelial planar polarity (EPP) signaling pathway in eyes and wings. Both msn loss- and gain-of-function result in defective ommatidial polarity and wing hair formation. Genetic and biochemical analyses indicate that msn acts downstream of fz and dishevelled (dsh) in the planar polarity pathway, and thus implicates an STE20-like kinase in Fz/Dsh-mediated signaling. This demonstrates that seven-pass transmembrane receptors can signal via members of the STE20 kinase family in higher eukaryotes. We also show that Msn acts in EPP signaling through the JNK (Jun-N-terminal kinase) module as it does in dorsal closure. Although at the level of Fz/Dsh there is no apparent redundancy in this pathway, the downstream effector JNK/MAPK (mitogen-activated protein kinase) module is redundant in planar polarity generation. To address the nature of this redundancy, we provide evidence for an involvement of the related MAP kinases of the p38 subfamily in planar polarity signaling downstream of Msn.     

Non-equivalent roles of Drosophila Frizzled and Dfrizzled2 in embryonic wingless signal transduction

The highly conserved Wnt family of growth factors is essential for generating embryonic pattern in many animal species [1]. In the fruit fly Drosophila, most Wnt-mediated patterning is performed by a single family member, Wingless (Wg), acting through its receptors Frizzled (Fz) and DFrizzled2 (Dfz2). In the ventral embryonic epidermis, Wg signaling generates two different cell-fate decisions: the production of diverse denticle types and the specification of naked cuticle separating the denticle belts. Mutant alleles of wg disrupt these cellular decisions separately [2], suggesting that some aspect of ligand-receptor affinity influences cell-fate decisions, or that different receptor complexes mediate the distinct cellular responses. Here, we report that overexpression of Dfz2, but not Fz, rescues the mutant phenotype of wgPE2, an allele that produces denticle diversity but no naked cuticle. Fz was able to substitute for Dfz2 only under conditions where the Wg ligand was present in excess. The wgPE2 mutant phenotype was also sensitive to the dosage of glycosaminoglycans, suggesting that the mutant ligand is excluded from the receptor complex when proteoglycans are present. We conclude that wild-type Wg signaling requires efficient interaction between ligand and the Dfz2-proteoglycan receptor complex to promote the naked cuticle cell fate.     

The domineering non-autonomy of frizzled and van Gogh clones in the Drosophila wing is a consequence of a disruption in local signaling

The frizzled (fz) gene is required for the development of distally pointing hairs on the Drosophila wing. It has been suggested that fz is needed for the propagation of a signal along the proximal distal axis of the wing. The directional domineering non-autonomy of fz clones could be a consequence of a failure in the propagation of this signal. We have tested this hypothesis in two ways. In one set of experiments we used the domineering non-autonomy of fz and Vang Gogh (Vang) clones to assess the direction of planar polarity signaling in the wing. prickle (pk) mutations alter wing hair polarity in a cell autonomous way, so pk cannot be altering a global polarity signal. However, we found that pk mutations altered the direction of the domineering non-autonomy of fz and Vang clones, arguing that this domineering non-autonomy is not due to an alteration in a global signal. In a second series of experiments we ablated cells in the pupal wing. We found that a lack of cells that could be propagating a long-range signal did not alter hair polarity. We suggest that fz and Vang clones result in altered levels of a locally acting signal and the domineering non-autonomy results from wild-type cells responding to this abnormal signal.     

Two homologs of the Drosophila polarity gene frizzled (fz) are widely expressed in mammalian tissues.

The frizzled (fz) locus of Drosophila encodes a protein (Fz) with a seven-transmembrane-domain profile characteristic of G-protein-coupled receptors. In Drosophila, genetic evidence suggests that Fz functions to transmit and transduce polarity signals in epidermal cells during hair and bristle development. We have isolated from a UMR 106 rat osteosarcoma cell library a cDNA (fz-1) encoding a predicted 641-residue protein (Fz-1) with 46% homology with Drosophila Fz. We also identified a second cDNA (fz-2) encoding a protein (Fz-2) of 570 amino acids that is 80% homologous with Fz-1, with divergence most evident in the extracellular domains. Southern blots of rat genomic DNA indicated that fz-1 and fz-2 represent distinct genes. Northern analysis revealed the presence of a single fz-1 mRNA (4.7 kilobases) and two fz-2 mRNAs (2.5 and 4.5 kilobases) in rat tissues. The fz-1 and fz-2 genes are widely expressed in rat tissues with the highest steady-state levels of mRNA in kidney, liver, heart, uterus, and ovary. fz-1 and -2 mRNA levels were greater in neonatal than in corresponding adult tissues. Treatment of UMR 106 cells with bone resorbing agents including parathyroid hormone, epidermal growth factor, and 1,25-dihydroxyvitamin D3 produced increases in fz-1 and -2 mRNA levels. We suggest that hormonal induction of Fz proteins in osteoblasts serves to promote intercellular signaling required for functional responses such as increased bone resorption. Fz-1 and Fz-2 may represent products of a gene family whose members serve as transducers or intercellular transmitters of signals required for normal morphogenesis and/or differentiated function in diverse tissues.     

Wnt/Wingless signaling through β-catenin requires the function of both LRP/Arrow and frizzled classes of receptors

Background

Wnt/Wingless (Wg) signals are transduced by seven-transmembrane Frizzleds (Fzs) and the single-transmembrane LDL-receptor-related proteins 5 or 6 (LRP5/6) or Arrow. The aminotermini of LRP and Fz were reported to associate only in the presence of Wnt, implying that Wnt ligands form a trimeric complex with two different receptors. However, it was recently reported that LRPs activate the Wnt/β-catenin pathway by binding to Axin in a Dishevelled – independent manner, while Fzs transduce Wnt signals through Dishevelled to stabilize β-catenin. Thus, it is possible that Wnt proteins form separate complexes with Fzs and LRPs, transducing Wnt signals separately, but converging downstream in the Wnt/β-catenin pathway. The question then arises whether both receptors are absolutely required to transduce Wnt signals.

Results

We have established a sensitive luciferase reporter assay in Drosophila S2 cells to determine the level of Wg – stimulated signaling. We demonstrate here that Wg can synergize with DFz2 and function cooperatively with LRP to activate the β-catenin/Armadillo signaling pathway. Double-strand RNA interference that disrupts the synthesis of either receptor type dramatically impairs Wg signaling activity. Importantly, the pronounced synergistic effect of adding Wg and DFz2 is dependent on Arrow and Dishevelled. The synergy requires the cysteine-rich extracellular domain of DFz2, but not its carboxyterminus. Finally, mammalian LRP6 and its activated forms, which lack most of the extracellular domain of the protein, can activate the Wg signaling pathway and cooperate with Wg and DFz2 in S2 cells. We also show that the aminoterminus of LRP/Arr is required for the synergy between Wg and DFz2.

Conclusion

Our study indicates that Wg signal transduction in S2 cells depends on the function of both LRPs and DFz2, and the results are consistent with the proposal that Wnt/Wg signals through the aminoterminal domains of its dual receptors, activating target genes through Dishevelled.

     

Drosophila glypicans Dally and Dally-like shape the extracellular Wingless morphogen gradient in the wing disc

Drosophila Wingless (Wg) is the founding member of the Wnt family of secreted proteins. During the wing development, Wg acts as a morphogen whose concentration gradient provides positional cues for wing patterning. The molecular mechanism(s) of Wg gradient formation is not fully understood. Here, we systematically analyzed the roles of glypicans Dally and Dally-like protein (Dlp), the Wg receptors Frizzled (Fz) and Fz2, and the Wg co-receptor Arrow (Arr) in Wg gradient formation in the wing disc. We demonstrate that both Dally and Dlp are essential and have different roles in Wg gradient formation. The specificities of Dally and Dlp in Wg gradient formation are at least partially achieved by their distinct expression patterns. To our surprise, although Fz2 was suggested to play an essential role in Wg gradient formation by ectopic expression studies, removal of Fz2 activity does not alter the extracellular Wg gradient. Interestingly, removal of both Fz and Fz2, or Arr causes enhanced extracellular Wg levels, which is mainly resulted from upregulated Dlp levels. We further show that Notum, a negative regulator of Wg signaling, downregulates Wg signaling mainly by modifying Dally. Last, we demonstrate that Wg movement is impeded by cells mutant for both dally and dlp. Together, these new findings suggest that the Wg morphogen gradient in the wing disc is mainly controlled by combined actions of Dally and Dlp. We propose that Wg establishes its concentration gradient by a restricted diffusion mechanism involving Dally and Dlp in the wing disc.     

Tissue polarity points from cells that have higher Frizzled levels towards cells that have lower Frizzled levels

Background: The frizzled (fz) gene of Drosophila encodes the founding member of the large family of receptors for the Wnt family of signaling molecules. It was originally studied in the adult epidermis, where it plays a key role in the generation of tissue polarity. Mutations in components of the fz signal transduction pathway disrupt tissue polarity; on the wing, hairs normally point distally but their polarity is altered by these mutations.Results: We devised a method to induce a gradient of fz expression with the highest levels near the distal wing tip. The result was a large area of proximally pointing hairs in this region. This reversal of polarity was seen when fz expression was induced just before the start of hair morphogenesis when polarity is established, suggesting that the gradient of Fz protein acted fairly directly to reverse hair polarity. A similar induction of the dishevelled (dsh) gene, which acts cell autonomously and functions downstream of fz in the generation of tissue polarity, resulted in a distinct tissue polarity phenotype, but no reversal of polarity; this argues that fz signaling was required for polarity reversal. Furthermore, the finding that functional dsh was required for the reversal of polarity argues that the reversal requires normal fz signal transduction.Conclusions: The data suggest that cells sense the level of Fz protein on neighboring cells and use this information in order to polarize themselves. A polarizing signal is transmitted from cells with higher Fz levels to cells with lower levels. Our observations enable us to propose a general mechanism to explain how Wnts polarize target cells.     

Genetic evidence that Drosophila frizzled controls planar cell polarity and Armadillo signaling by a common mechanism

The frizzled (fz) gene in Drosophila controls two distinct signaling pathways: it directs the planar cell polarization (PCP) of epithelia and it regulates cell fate decisions through Armadillo (Arm) by acting as a receptor for the Wnt protein Wingless (Wg). With the exception of dishevelled (dsh), the genes functioning in these two pathways are distinct. We have taken a genetic approach, based on a series of new and existing fz alleles, for identifying individual amino acids required for PCP or Arm signaling. For each allele, we have attempted to quantify the strength of signaling by phenotypic measurements. For PCP signaling, the defect was measured by counting the number of cells secreting multiple hairs in the wing. We then examined each allele for its ability to participate in Arm signaling by the rescue of fz mutant embryos with maternally provided fz function. For both PCP and Arm signaling we observed a broad range of phenotypes, but for every allele there is a strong correlation between its phenotypic strength in each pathway. Therefore, even though the PCP and Arm signaling pathways are genetically distinct, the set of signaling-defective fz alleles affected both pathways to a similar extent. This suggests that fz controls these two different signaling activities by a common mechanism. In addition, this screen yielded a set of missense mutations that identify amino acids specifically required for fz signaling function.     

Signaling Activities of the Drosophila Wingless Gene Are Separately Mutable and Appear to Be Transduced at the Cell Surface

The Drosophila segment polarity gene wingless encodes an intercellular signaling molecule that transmits positional information during development of the embryonic epidermis. We have explored the mechanism of wg signal transduction by perturbing cellular processes genetically and by performing structure/function analysis of the Wg protein. We present evidence that Wingless protein may transduce signal at the cell surface and that Wg may bind to its cell surface receptor without necessarily activating it. We demonstrate that two specific signaling activities of the Wg molecule can be disrupted independently by mutation. Sequence analysis indicates that these different signaling activities are not promoted by discrete functional domains, but rather that the overall conformation of the molecule may control distinct signaling functions. We conclude that wg signaling may involve complex interactions between the Wg ligand and its cell surface receptor molecule(s) and that some of this complexity resides within the Wg ligand itself.     

Frizzled5/8 is required in secondary mesenchyme cells to initiate archenteron invagination during sea urchin development

Wnt signaling pathways play key roles in numerous developmental processes both in vertebrates and invertebrates. Their signals are transduced by Frizzled proteins, the cognate receptors of the Wnt ligands. This study focuses on the role of a member of the Frizzled family, Fz5/8, during sea urchin embryogenesis. During development, Fz5/8 displays restricted expression, beginning at the 60-cell stage in the animal domain and then from mesenchyme blastula stage, in both the animal domain and a subset of secondary mesenchyme cells (SMCs). Loss-of-function analyses in whole embryos and chimeras reveal that Fz5/8 is not involved in the specification of the main embryonic territories. Rather, it appears to be required in SMCs for primary invagination of the archenteron, maintenance of endodermal marker expression and apical localization of Notch receptors in endodermal cells. Furthermore, among the three known Wnt pathways, Fz5/8 appears to signal via the planar cell polarity pathway. Taken together, the results suggest that Fz5/8 plays a crucial role specifically in SMCs to control primary invagination during sea urchin gastrulation.     

The frizzled extracellular domain is a ligand for Van Gogh/Stbm during nonautonomous planar cell polarity signaling

The Frizzled (Fz) receptor is required cell autonomously in Wnt/beta-catenin and planar cell polarity (PCP) signaling. In addition to these requirements, Fz acts nonautonomously during PCP establishment: wild-type cells surrounding fz(-) patches reorient toward the fz(-) cells. The molecular mechanism(s) of nonautonomous Fz signaling are unknown. Our in vivo studies identify the extracellular domain (ECD) of Fz, in particular its CRD (cysteine rich domain), as critical for nonautonomous Fz-PCP activity. Importantly, we demonstrate biochemical and physical interactions between the FzECD and the transmembrane protein Van Gogh/Strabismus (Vang/Stbm). We show that this function precedes cell-autonomous interactions and visible asymmetric PCP factor localization. Our data suggest that Vang/Stbm can act as a FzECD receptor, allowing cells to sense Fz activity/levels of their neighbors. Thus, direct Fz-Vang/Stbm interactions represent an intriguing mechanism that may account for the global orientation of cells within the plane of their epithelial field.     

Frizzled-Dishevelled signaling specificity outcome can be modulated by Diego in Drosophila

Members of the Frizzled (Fz) family of seven-pass transmembrane receptors are required for the transduction of both Wnt-Fz/beta-catenin and Fz/planar cell polarity (PCP) signals. Although both pathways transduce signals via interactions between Fz and the cytoplasmic protein Dishevelled (Dsh), each pathway has specific and distinct effectors. One explanation for the pathway specificity is that signal-induced conformational changes result in unique Fz-Dsh interactions. Our mutational analyses of Fz-Dsh activities in vivo do however not support this model, since both pathways are affected by all mutations tested. Alternatively, the interaction of Fz or Dsh with other proteins could modulate the signaling outcome. We examined the role of a Dsh-binding PCP molecule, Diego (Dgo), in both Wnt-Fz/beta-catenin and Fz/PCP signaling. Both loss-of-function and gain-of-function results suggest that Dgo promotes Fz-Dsh/PCP signaling at the expense of Wnt-Fz/beta-catenin signaling. Our data suggest that Dgo sequesters Dsh to a functionally distinct Fz/PCP signaling compartment within the cell.