Onents of the Wingless pathway, wingless and armadillo, for interactions with lqfR/Tel2. Wingless ligand binds its receptor Frizzled which results in accumulation in the nucleus of the transcriptional regulator Armadillo [36]. We found strong Dimethylenastron web genetic interactions between lqfR/tel2 and each of the two Wingless pathway genes. Heterozygotes for a hypomorphic allele and a null allele of lqfR (lqfRP/ lqfRD117) are semi-viable and the adult escapers may have normal, slightly roughened, or kidney-shaped eyes [32] (Fig. 4). Flies that were lqfRP/lqfRD117 and also heterozygous for a loss-of-function allele of armadillo (arm3 or arm8) died in their pupal cases. (arm-/ arm+ flies appear wild-type.) The pupae had small or absent eyes, small head capsules, and also had 58-49-1 site morphological defects in the head cuticle, wings, and legs (Fig. 4). wingless loss-of-functionFigure 3. Subcellular localization of Myc-tagged LqfR proteins. Confocal microscope images of third instar larval eye disc tissue from two different discs (each row is a single disc) are shown. The portion of the eye disc shown is the peripodial epithelium, a layer of cells that lies atop the cell layer that forms the retina. The peripodial cells are large and flat the nuclei and cytoplasm are distinguished more easily than in the retinal cells. The discs were immunostained with antibodies to the Myc epitope (green) and the DNA stain TOPRO3 (purple). The Myctagged proteins indicated were expressed by UAS transgenes using an Actin5C-gal4 driver. scale bar: ,10 mm. doi:10.1371/journal.pone.0046357.gOnly Tel2 Portion of Fly EpsinR/Tel2 Is Essentialmutations (wgI-17 or wgI-8) had similar but weaker effects when heterozygous in a lqfRP/lqfRD117 background; there were viable adult escapers 
with severely defective eyes varying from kidneyshaped to nearly absent, and also with defects in the head cuticle (Fig. 4). (wg2/wg+ animals appear wild-type.) These strong genetic interactions suggest that lqfR/tel2 may function in the Wingless signaling pathway.Wingless target gene expression depends to some extent on lqfR/tel2 functionThe dominant enhancement of lqfR/tel2 mutant phenotypes by loss of function mutations in Wingless pathway genes suggests that lqfR/tel2 facilitates Wingless pathway activation. To test this idea, we generated lqfR/tel2 null clones in eye discs and monitored expression of the Wingless target genes dachsous (ds) [36,37] and optomotor blind (omb) [38]. Weak effects on target gene expression were apparent in both cases. As the effects on ds expression was stronger, this data is shown below. The dachsous gene encodes an atypical cadherin adhesion protein involved in cell polarity and cell growth and is a transcriptional target of Arm [37,38]. ds-lacZ enhancer trap lines express b-galactosidase in response to Wg pathway activation [38]. Wingless ligand is expressed in the dorsal- and ventral-most margins of the eye disc and the protein forms a gradient with its lowest point at the dorsal/ventral axis (the equator) [40]. bgalactosidase expression by ds-lacZ reflects the Wg gradient [39]. We found that ds-lacZ expression was reduced in lqfR/tel2 null clones (Fig. 5). Moreover, we found that a dachsous loss-of-function mutation, ds38K, is as strong a dominant enhancer of the lqfRP/ lqfRD117 mutant phenotype as are armadillo mutations (Fig. 5). These results suggest that in the absence of LqfR/Tel2, Wg signaling is less efficient than it is normally. The effects of lqfR/tel2 loss of funct.Onents of the Wingless pathway, wingless and armadillo, for interactions with lqfR/Tel2. Wingless ligand binds its receptor Frizzled which results in accumulation in the nucleus of the transcriptional regulator Armadillo [36]. We found strong genetic interactions between lqfR/tel2 and each of the two Wingless pathway genes. Heterozygotes for a hypomorphic allele and a null allele of lqfR (lqfRP/ lqfRD117) are semi-viable and the adult escapers may have normal, slightly roughened, or kidney-shaped eyes [32] (Fig. 4). Flies that were lqfRP/lqfRD117 and also heterozygous for a loss-of-function allele of armadillo (arm3 or arm8) died in their pupal cases. (arm-/ arm+ flies appear wild-type.) The pupae had small or absent eyes, small head capsules, and also had morphological defects in the head cuticle, wings, and legs (Fig. 4). wingless loss-of-functionFigure 3. Subcellular localization of Myc-tagged LqfR proteins. Confocal microscope images of third instar larval eye disc tissue from two different discs (each row is a single disc) are shown. The portion of the eye disc shown is the peripodial epithelium, a layer of cells that lies atop the cell layer that forms the retina. The peripodial cells are large and flat the nuclei and cytoplasm are distinguished more easily than in the retinal cells. The discs were immunostained with antibodies to the 
Myc epitope (green) and the DNA stain TOPRO3 (purple). The Myctagged proteins indicated were expressed by UAS transgenes using an Actin5C-gal4 driver. scale bar: ,10 mm. doi:10.1371/journal.pone.0046357.gOnly Tel2 Portion of Fly EpsinR/Tel2 Is Essentialmutations (wgI-17 or wgI-8) had similar but weaker effects when heterozygous in a lqfRP/lqfRD117 background; there were viable adult escapers with severely defective eyes varying from kidneyshaped to nearly absent, and also with defects in the head cuticle (Fig. 4). (wg2/wg+ animals appear wild-type.) These strong genetic interactions suggest that lqfR/tel2 may function in the Wingless signaling pathway.Wingless target gene expression depends to some extent on lqfR/tel2 functionThe dominant enhancement of lqfR/tel2 mutant phenotypes by loss of function mutations in Wingless pathway genes suggests that lqfR/tel2 facilitates Wingless pathway activation. To test this idea, we generated lqfR/tel2 null clones in eye discs and monitored expression of the Wingless target genes dachsous (ds) [36,37] and optomotor blind (omb) [38]. Weak effects on target gene expression were apparent in both cases. As the effects on ds expression was stronger, this data is shown below. The dachsous gene encodes an atypical cadherin adhesion protein involved in cell polarity and cell growth and is a transcriptional target of Arm [37,38]. ds-lacZ enhancer trap lines express b-galactosidase in response to Wg pathway activation [38]. Wingless ligand is expressed in the dorsal- and ventral-most margins of the eye disc and the protein forms a gradient with its lowest point at the dorsal/ventral axis (the equator) [40]. bgalactosidase expression by ds-lacZ reflects the Wg gradient [39]. We found that ds-lacZ expression was reduced in lqfR/tel2 null clones (Fig. 5). Moreover, we found that a dachsous loss-of-function mutation, ds38K, is as strong a dominant enhancer of the lqfRP/ lqfRD117 mutant phenotype as are armadillo mutations (Fig. 5). These results suggest that in the absence of LqfR/Tel2, Wg signaling is less efficient than it is normally. The effects of lqfR/tel2 loss of funct.