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Figure 1 JNK signaling regulates UV-induced apoptosis in the retina. (A–C) JNK signaling regulates the apoptotic response to UV irradiation in the developing retina. Pupal cases were removed from 24-h-old pupae to expose the developing eye. One of the eyes was subjected to UVC irradiation (5 mJ/cm2), whereas the other eye was shielded. After subsequent incubation at 25°C in the dark, morphological defects are observed in the irradiated eye (A, arrowhead). This phenotype is caused by DNA-damage-induced apoptosis (Jassim et al, 2003). (B, C) DNA damage-induced apoptosis requires JNK signaling. Loss of JNKK (Hep) function (in hemizygotes for hep1) protects eyes from UV-induced apoptosis (B), whereas increased JNK activity owing to loss of the JNK-phosphatase puc results in strongly increased defects (C). puc transcription (an indicator of JNK activation in flies) is induced in response to UV irradiation (D). Left panel: RT–PCR demonstrating rapid induction of puc transcripts in the retina within 1.5 h after UV irradiation; right panel: whole-mount X-gal staining showing activation of the puc gene in the irradiated part of the pupal head (arrowhead). The pucE69 allele contains a JNK-responsive lacZ P-element that serves as JNK reporter in vivo. (E) Quantification of tissue loss in irradiated eyes can be used to quantify the extent of apoptosis in a given genotype. The ratio between the area of left (L, irradiated) and right (R, control) eyes for each head was determined for n=10 heads of each genotype (see Materials and methods for details). Means and standard deviations are shown here. Differences between each group are statistically significant (P<0.001, Student's t-test). Quantification is shown for wild-type flies (OreR), hep1 hemizygous mutant males or homozygous mutant females, hepr75/hep1 transheterozygous females, pucE69 heterozygotes and p535A-1-4 (Rong et al, 2002) homozygotes. A requirement for p53 in protection against UV-induced apoptosis was demonstrated by Jassim et al (2003). P53 mutants are included here as control.
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 | Figure 2 JNK-mediated hid induction induces apoptosis in the retina in response to UV irradiation. (A, B) hid is induced by UV irradiation in the Drosophila retina in a JNK-dependent manner. (A) hid expression 2 h after UV irradiation was detected by monitoring lacZ expression from a hid reporter line (Russell et al, 1998; Cox et al, 2000; Cullen and McCall, 2004; Sen et al, 2004). The irradiated eye is shown on the right (UV) and the shielded eye on the left (Ctrl). (B) Similarly, hid induction in wild-type animals (w1118, left panel) could be observed using RT–PCR on retina dissected from pupae 2.5 h after irradiation (rp49 transcript levels serve as internal controls). Induction of hid was not observed in hep1 hemizygous males (right panel), indicating a requirement for JNK signaling in the transcriptional response to UV. (C, D) Overexpression of constitutively active Hep (Hepact) in developing photoreceptors and cone cells causes apoptosis. Increased TUNEL-positive cells were observed in larval eye imaginal discs expressing Hepact under the control of Sep-Gal4 (D), compared to wild-type discs (C). Sep-Gal4 is active in the developing photoreceptors and cone cells posterior to the morphogenetic furrow (arrowhead; Jasper et al, 2002). (E, F) JNK activation is sufficient to induce hid expression in the developing retina. In situ hybridization demonstrating hid induction in response to overexpression of Hepact under the control of Sep-Gal4 (F). (G–J) The resulting adult eye phenotype (G; sep-Gal4, UAS-Hepact is abbreviated as SH) is reduced in hid mutant backgrounds (in heterozygous conditions for hid (alleles W05014 and W1) as well as all three RGH genes (Df(H99)).
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Figure 3 The transcription factors Fos and Foxo are required for UV-induced apoptosis in Drosophila. (A) Constitutive activation of JNKK (Hep) in the fly retina results in a 'rough' eye phenotype (expressed in photoreceptors and cone cells under the control of sep-Gal4). This eye phenotype (referred to as SH throughout) requires the downstream kinase, JNK (not shown). (B–D) The forkhead transcription factor FOXO genetically interacts with the JNK pathway. Heterozygosity for the loss-of-function allele dfoxo21 suppresses the JNK gain-of-function phenotype (B), whereas co-overexpression of Foxo greatly enhances the eye phenotype (C). Overexpression of Foxo alone with sep-Gal4 does not impact eye morphology (D). (E–G) Similarly, decreasing Fos function by introducing the loss-of-function mutation kay2 (E), or by co-overexpressing dsRNA against Fos (F), rescues the sep>Hepact phenotype. Reducing both dfoxo and kay gene dose (G) results in an almost full recovery of normal eye morphology. (H, I, N) Homozygosity for the dfoxo loss-of-function allele dfoxo25 reduces UV-induced apoptosis. Treatment and quantification were performed as described in Figure 1. (J–N) Increased Fos expression (M, N) enhances, and loss of fos function (J, L, N) reduces UV-induced apoptosis in the eye, as compared to wild-type controls (K, N).
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 | Figure 4 Transcriptional regulation of hid by Fos and Foxo. (A, B) Foxo overexpression is sufficient to induce hid expression in the developing retina. (A) LacZ staining of flies overexpressing GFP (as control, left panel) or Foxo (right panel) posterior to the morphogenetic furrow in a W05014 (hid-lacZ) background. (B) RNA in situ hybridization detecting hid transcript in eye imaginal discs of flies overexpressing GFP (left panel) or constitutively nuclear Foxo (Foxo™; right panel) posterior to the morphogenetic furrow (arrowheads). (C) Foxo and JNK signaling induces hid expression in wing imaginal discs. hid induction was detected using RT–PCR in wing imaginal discs in which Hepact or Foxo expression was induced using the TARGET system (McGuire et al, 2003). In this system, a temperature-sensitive allele of Gal80 inhibits Gal4-mediated transcription until flies are heat-shocked. Heat shock was performed for 30 min at 37°C. At 2 h after induction of Hepact (HA) or of a constitutively nuclear form of Foxo (Foxo™), increased hid expression can be detected. Lanes are as follows: WT, wild-type wing discs (T80Gal4/CyO; Gal80ts/TM3); HA, Hepact-expressing discs (T80Gal4/UASHepact; Gal80ts/TM3); 0 h, dissected immediately after heat shock; 2 h, dissected 2 h after heat shock. (D) hid induction in wing imaginal discs in response to JNK activation is lost in a dfoxo heterozygous mutant background. Experiment was performed as in (B), with flies of the following genotypes: w; T80-Gal4/UASHepact; Gal80ts/+ (left lanes) or w; T80-Gal4/UASHepact; Gal80ts/dfoxo21. (E) Structure of the hid locus. Coordinates for chromosome 3L are listed. The first intron of hid contains multiple AP-1-binding sites (TGANTCA, blue), as well as AP-1 half-sites (TGNNTCA, brown) and Foxo response elements (TTGTTTAC, FREs, red). Arrows indicate binding sites for primers used for ChIPs in panels F and G. (F) Chromatin IP demonstrating binding of Fos to the hid locus in S2 cells. PCR on the same ChIP material against the puc locus is included as positive control and against the hsp26 locus as negative control (Lee et al, 2005). Primers used for PCR flank the 5' cluster of AP1 and Foxo-binding sites in the first hid intron and are indicated as arrows in panel E. (G) Chromatin IP demonstrating binding of Foxo to the hid locus in S2 cells. PCR on the same ChIP material against the U6 snRNA promoter is included as negative control. Wild-type Foxo (WT) or constitutively nuclear Foxo (TM) was transfected into S2 cells and immunoprecipitated using anti-Foxo antibody or pre-immune serum as control (see also Puig et al, 2003). Note that constitutively nuclear Foxo (TM) binds more efficiently to the hid locus than wild-type Foxo. Primers used for PCR flank the 5' cluster of AP1 and Foxo-binding sites in the first hid intron and are indicated as arrows in (E). (H, I) Overexpression of wild-type (H) or constitutively nuclear (I) Foxo in the developing retina leads to ommatidia loss in the adult eye. (J) The phenotype caused by Foxo overexpression in the eye can be reduced by co-overexpression of a dominant-negative form of the Drosophila Caspase DRONC (Meier et al, 2000). GMR>Foxo is abbreviated here as G>F for clarity. (K) Similarly, reduction in the gene dose of hid (W1) leads to a partial rescue of the Foxo gain-of-function phenotype. (L) Foxo-induced apoptosis requires Fos function. Reduced apoptotic defects in eyes expressing wild-type Foxo in a kay2 heterozygous background.
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Figure 5 EGFR and Insulin signaling suppress JNK-mediated apoptosis. Decreased level of EGFR activity by mutations in either EGFR (B) or ras (C) and reduced insulin signaling owing to loss of chico (D) substantially enhances the apoptotic phenotype induced by overexpression of Hepact (A). Similarly, the eye phenotype induced by Foxo overexpression (E) is enhanced by reduced EGFR (F) or ras gene dose (G). Conversely, blocking Foxo activity by co-overexpressing Akt or active PI3K blocks the Foxo gain-of-function phenotype (H, I). (J–O) UV-induced apoptosis is reduced by EGF/insulin-mediated survival signaling. Reduced activity of ras (K, O) or chico (M, O) results in strongly increased UV-induced defects in the eye. Conversely, loss of aos, a negative regulator of EGFR, or increased expression of InR, decreases UV-induced apoptotic defects (L, N, O). Genotypes are as follows: A, w; sep-Gal4, UAS-Hepact/+; B, w; sep-Gal4, UAS-Hepact/egfrf2; C, w; sep-Gal4, UAS-Hepact/+; rase1B/+; D, w; sep-Gal4, UAS-Hepact/chico1; E: w; GMR-Gal4, UAS-Foxo/+; F, w; GMR-Gal4, UAS-Foxo/egfrf2; G, w; GMR-Gal4, UAS-Foxo/+; rase1B/+; H, w; GMR-Gal4, UAS-Foxo/UAS-Akt; I, w; GMR-Gal4, UAS-Foxo/UAS-PI3Kact; J, OreR. K: rase1B/TM3; L, aos 7/TM3; M, chico1/chico1; N, w1118; sep-Gal4/UAS-InR.
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 | Figure 6 Model for the regulation of UV-induced apoptosis in the Drosophila retina. JNK activation results in Foxo- and Fos-dependent transcriptional upregulation of hid. Hid inhibits IAP and induces caspase-dependent apoptosis. Induction of hid can be blocked by InR or EGFR-initiated survival signals that inhibit Foxo activity. The relative balance between stress (JNK) and survival (RTK) signaling determines the cellular response to UV-induced DNA damage.
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