Yan, an ETS-domain transcription factor, negatively modulates the Wingless pathway in the Drosophila eye

Abstract

We report the identification of yan, an ETS-domain transcription factor belonging to the Drosophila epidermal growth factor receptor (DER) pathway, as an antagonist of the Wingless signalling pathway. We demonstrate that cells lacking yan function in the Drosophila eye show increased Wingless pathway activity, and inhibition of Wingless signalling in yan(-/-) cells rescues the yan mutant phenotype. Biochemical analysis shows that Yan physically associates with Armadillo, a crucial effector of the Wingless pathway, thereby suggesting a direct regulatory mechanism. We conclude that yan represents a new and unsuspected molecular link between the Wingless and DER pathways.

Figure 1

Yan misexpression reduces Wingless pathway activity. (A) Double-stranded-RNA-mediated knockdown of yan in S2R+ cells activates the Wingless-responsive dTF12 luciferase reporter in the presence, but not in the absence, of Wingless induction, as compared with gfp knockdown (control). (B) Increasing amounts of yan^ACT cDNA results in a dose-dependent reduction of dTF12 on co-transfection with wingless cDNA. Error bars in panels A and B represent average variation in normalized luciferase reporter activity within four replica data points for each described condition. (C–E) The C96–GAL4 driver is specific to the wing margin (demonstrated by UAS–GFP) and alone does not affect Senseless expression (C–C′). Misexpression of UAS–Yan^ACT by C96–GAL4 (C96>Yan^ACT; E) results in reduction or complete loss of Senseless expression, similar to what is observed on C96–GAL4 misexpression of UAS–Axin (C96>Axin; D), a known negative regulator of the Wingless pathway. (F–J) In the embryo at stages 10 and 11, paired (prd)–GAL4 leads to expression of UAS–Axin–GFP in alternating segments (GFP expression; G–G′), resulting in a downregulation of Wingless-dependent Engrailed expression (G–G′, white arrowheads) as compared with wild-type (F). Prd>Yan^ACT results in downregulation of Engrailed (H, white arrowheads), and coexpression of UAS–Arm^* with UAS–Yan^ACT (prd>Arm^*; Yan^ACT; I) restored Engrailed expression. Prd–GAL4 expression of dominant-negative DER (DER-DN; J) has no effect on En expression. (C–J) scale bar, 50 μM. Arm, Armadillo; cDNA, complementary DNA; DER, Drosophila epidermal growth factor receptor; En, Engrailed; GFP, green fluorescent protein; RLU, relative luciferase units; Sens, senseless; UAS, upstream activating sequence; Wg, Wingless; WT, wild type.

Figure 2

Wingless signalling is activated in yan^−/− clones. (A–C) Generation of yan^−/−-null clones results in patches of necrotic tissue, naked-cuticle-lacking photoreceptors (white and black arrowheads, C) and tubular outgrowths (yellow arrowheads, D–D′) in adult Drosophila eyes, as compared with wild type (A). Similar adult phenotypes were observed in axin^−/− clones (B). (E–H″′) Within yan^−/− clones, the early eye-determining gene Dacshund is reduced (F–G′), the R8 photoreceptor marker Sens is lost and Armadillo immunofluorescence is increased (H–H″″) posterior to the morphogenetic furrow (white arrowheads throughout). (I–J″) Fz3–RFP is an in vivo reporter and overlaps with endogenous Wingless expression (as monitored using wg-lacZ, I) at the lateral margins of the eye disc. Fz3–RFP is ectopically expanded in yan^−/− clones near the lateral margins (yellow arrowhead, J′–J″). Anterior is to the left. Arm, Armadillo; Dac, Dacshund; GFP, green fluorescent protein; PR, photoreceptor; RFP, red fluorescent protein; Sens, Senseless; Wg, Wingless; WT, wild type.

Figure 3

Cells are apically constricted in yan^−/− clones and axin^−/− clones in the eye. (A–A″) Wild-type control eye imaginal disc shows increased Armadillo immunofluorescence within the morphogenetic furrow (white arrowheads); higher magnification (A′–A″) shows that cells in the morphogenetic furrow are apically constricted and Armadillo localizes to the cell membrane. Posterior to the morphogenetic furrow, cells relax and Armadillo localizes to ‘rosettes’ incorporated into the adherents junctions between differentiating photoreceptors (yellow arrowheads, A″). (B–B″) Cells within yan^−/− clones remain apically constricted several rows posterior to the morphogenetic furrow (white arrowheads throughout), similar to what we observe in axin^−/− clones (C–C″). Activation of the DER pathway on generation of FO clones expressing an activated form of DER (FO>DER^λtop, D–D″) results in ectopic Armadillo-positive rosettes, but does not result in apical constriction. Anterior is to the left. Scale bar, 25 μM. Arm, Armadillo; DER, Drosophila epidermal growth factor receptor; FO, Flip-Out; GFP, green fluorescent protein; WT, wild type.

Figure 4

Expression of negative regulators of the Wingless pathway suppresses the yan mutant phenotype in the Drosophila eye. (A–A′) Cells with control yan^−/− MARCM clones expressing UAS–GFP are apically constricted posterior to the morphogenetic furrow, whereas yan^−/− MARCM clones expressing UAS–Axin (B–B′) or UAS–dnTCF (C–C′) show suppression of the apical constriction phenotype. A–C′ represent Z-projection images of the eye imaginal disc; anterior is to the left; white arrowheads in A–C′ denote morphogenetic furrow; yellow asterisks in A–A′ and C–C′ denote areas with GFP marked tones in the peripodial membrane of the eye disc, hence absence of apical constrictions; scale bars, 25 μM. (D–H) Approximately 17–20% of control MARCM adults with genotype yan^−/−; TM6B (D) or yan^−/−; and UAS–GFP (E) show the yan^−/− phenotype, which includes necrotic patches and naked cuticle lacking photoreceptors (white and black arrowheads E–D; severe defects and death in H). yan^−/− MARCM clones expressing UAS–Axin (F) or UAS–dnTCF (G) show significant suppression of the yan^−/− phenotype, as only approximately 6–7% of adult flies showed mild defects in ommatidial morphology, as compared with sibling controls (H). Arm, Armadillo; dnTCF, dominant-negative TCF allele; GFP, green fluorescent protein; MARCM, mosaic analysis with a repressible cell marker; TCF, T-cell factor; UAS, upstream activating sequence; Wg, Wingless; WT, wild type.

Figure 5

Yan and Armadillo proteins physically interact. (A) Western blot analysis showing mobility shift in Yan protein (red arrows) in S2R+ cells in the presence of increased levels of Armadillo on transient transfection of armadillo cDNA or increasing amounts of wingless cDNA. (B,C) Endogenous Yan protein coimmunoprecipitates with Armadillo in both S2R+ cells transiently transfected with arm cDNA (B) or in embryos with Da–GAL4 expression of UAS–Arm^* (C). Notably, we observe that both Yan moieties coimmunoprecipitate with Armadillo (red and black asterisks, see text). D, a suggested model for the dual role of Yan during retinal development in Drosophila. Arm, Armadillo; cDNA, complementary DNA; da, daughterless; IB, immunoblot; insol., insoluble fraction; IP, immunoprecipitation; UAS, upstream activating sequence.