dFoxO promotes Wingless signaling in Drosophila

Abstract

The Wnt/β-catenin signaling is an evolutionarily conserved pathway that regulates a wide range of physiological functions, including embryogenesis, organ maintenance, cell proliferation and cell fate decision. Dysregulation of Wnt/β-catenin signaling has been implicated in various cancers, but its role in cell death has not yet been fully elucidated. Here we show that activation of Wg signaling induces cell death in Drosophila eyes and wings, which depends on dFoxO, a transcription factor known to be involved in cell death. In addition, dFoxO is required for ectopic and endogenous Wg signaling to regulate wing patterning. Moreover, dFoxO is necessary for activated Wg signaling-induced target genes expression. Furthermore, Arm is reciprocally required for dFoxO-induced cell death. Finally, dFoxO physically interacts with Arm both in vitro and in vivo. Thus, we have characterized a previously unknown role of dFoxO in promoting Wg signaling, and that a dFoxO-Arm complex is likely involved in their mutual functions, e.g. cell death.

Figure 1. Activation of Wg signaling induces cell death in Drosophila.

Light micrographs of Drosophila adult eyes and wings, and fluorescent micrographs of 3^rd instar eye and wing discs are shown. Compared with the GMR-Gal4 control (a,a’), expression of Wg, Dsh or Arm induces cell death in eye discs indicated by AO staining (b’–d’) and produces adult eyes with reduced size (b–d). Compared with the ptc-Gal4 control (e,e’), expression of Dsh, Arm or Pan induces cell death in wing discs (f’–h’) and produces a loss-of-ACV phenotype in adult wings (f–h, the lower panels are high magnification of the boxed areas in upper panels). Scale bars: 100 μm in (a–d) and (e–h, upper panels); 50 μm in (a’–d’), (e–h, lower panels) and (e’–h’).

Figure 2. dFoxO is required for Arm-induced cell death.

Light micrographs of Drosophila adult eyes and fluorescent micrographs of 3^rd instar eye discs are shown. Compared with the GMR-Gal4 control (a), expression of Arm triggers cell death and produces a small eye phenotype (b), which remains unaffected by expressing LacZ (c), but is partially suppressed by knocking-down of dfoxo (d), or in heterozygous dfoxo (e) or dfoxo (f) background, and fully suppressed in dfoxo/dfoxo mutants (g). As positive controls, knocking-down of arm (h) or pan (i) suppresses the GMR > Arm small eye phenotype. GMR > Arm-induced AO staining (j) is suppressed partially in heterozygous dfoxo mutants (k), and fully in dfoxo/dfoxo trans-heterozygous mutants (l). Sample numbers: a, 87; b, 110; c, 100; d, 75; e, 77; f, 56; g, 75; h, 97; i, 78; j, 22; k, 14; l, 17. Scale bars: 100 μm in (a–i) and 50 μm in (j–l).

Figure 3. dFoxO is required for Arm-induced hid and rpr expression.

Compared with the sd-Gal4 control (a,g), expression of Arm activates rpr–LacZ (b) and hid-LacZ (h) expression in the wing pouch, which remain unaffected by the expression of GFP (c,i), but are significantly suppressed by knocking-down of dfoxo (d,j), or heterozygous mutation of dfoxo (e,k) or dfoxo^Δ94 (f,l). Scale bars: 100 μm.

Figure 4. dFoxO is required for the wing patterning functions of Wg signaling.

Light micrographs of Drosophila adult wings are shown. Compared with the ptc-Gal4 control (a, the right panel shows high magnification of the boxed area containing ACV in the left panel), expression of Arm induces ectopic bristles (indicated by the red arrow head) and loss-of-ACV phenotypes (b), which remain unaffected by the expression of LacZ (c), but are largely suppressed by RNAi-mediated knocking-down of dfoxo (d) or heterozygous mutation of dfoxo^Δ94 (e). Compared with the sd-Gal4 control (f, the right panel shows high magnification of the boxed area containing anterior-distal wing margin in the left panel), expression of Arm induces ectopic bristles near the wing margin (g, indicated by red arrow heads), which remains unaffected by the expression of LacZ (h), but is suppressed by knocking-down of dfoxo (i) or heterozygous mutation of dfoxo^Δ94 (j). knocking-down of wg by ptc-Gal4 generates a mild wing margin notch phenotype between the L3 and L4 veins (k, indicated by the red box and amplified in the right panel), which is rescued by the expression of dFoxO (l), but is enhanced by heterozygous mutation of dfoxo (m) or dfoxo (n). Sample numbers: a, 226; b, 201; c, 108; d, 197; e, 160; f,168; g, 166; h, 146; i, 130; j, 178; k,160; l, 62; m, 174; n, 226. Scale bars: 100 μm in (a–n) and 50 μm in high magnification figures.

Figure 5. dFoxO is required for Wg target gene activation.

Light micrographs of Drosophila 3^rd instar wing discs (a–f) and salivary glands (g–l) with X-Gal staining are shown. Compared with the ptc-Gal4 control (a,g), ectopic Arm-induced wf-LacZ expression in the wing disc (b, indicated by black arrow heads) or salivary gland (h) remains unaffected by the expression of GFP (c and i), but is significantly suppressed by knocking-down of dfoxo (d,j), or heterozygous mutation of dfoxo (e,k) or dfoxo^Δ94 (f,l). Scale bars: 100 μm in (a–f) and 200 μm in (g–l).

Figure 6. Arm binds to dFoxO and is required for dFoxO-induced cell death.

(a) Co-immunoprecipitation between transfected Myc-Arm and Flag-dFoxO in Drosophila S2 cells. (b) Co-immunoprecipitation between ectopically expressed Arm and GFP-dFoxO in Drosophila eye. (c–k) Light micrographs of Drosophila adult eyes are shown. Compared with the GMR-Gal4 control (c), expression of dFoxO triggers cell death and produces a small eye phenotype (d), which remains unaffected by the expression of LacZ (e), but is considerably suppressed by the expression of two independent arm RNAi (f,g), or heterozygous mutation of arm (h), or the expression of two independent pan RNAi (i,j). The GMR > dFoxO eye phenotype is also suppressed by knocking-down of dfoxo (k), which serves as a positive control. Sample numbers from c to k: c, 123; d, 104; e, 74; f, 67; g, 73; h, 64; i, 97; j, 106; k, 53. Scale bars: 100 μm.