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Loss of putzig Activity Results in Apoptosis during Wing Imaginal Development in Drosophila.

Zimmermann M, Kugler SJ, Schulz A, Nagel AC - PLoS ONE (2015)

Bottom Line: We present evidence that downregulation of Notch activates Dcp1 caspase and JNK signaling, however, neither induces ectopic wg nor dpp expression.In contrast, the consequences of Dref-RNAi were largely indistinguishable from pzg-RNAi with regard to apoptosis induction.Moreover, overexpression of Dref ameliorated the downregulation of pzg compatible with the notion that the two are required together to maintain cell and tissue homeostasis in Drosophila.

View Article: PubMed Central - PubMed

Affiliation: Institute of Genetics, University of Hohenheim, 70599 Stuttgart, Germany.

ABSTRACT
The Drosophila gene putzig (pzg) encodes a nuclear protein that is an integral component of the Trf2/Dref complex involved in the transcription of proliferation-related genes. Moreover, Pzg is found in a complex together with the nucleosome remodeling factor NURF, where it promotes Notch target gene activation. Here we show that downregulation of pzg activity in the developing wing imaginal discs induces an apoptotic response, accompanied by the induction of the pro-apoptotic gene reaper, repression of Drosophila inhibitor of apoptosis protein accumulation and the activation of the caspases Drice, Caspase3 and Dcp1. As a further consequence 'Apoptosis induced Proliferation' (AiP) and 'Apoptosis induced Apoptosis' (AiA) are triggered. As expected, the activity of the stress kinase Jun N-terminal kinase (JNK), proposed to mediate both processes, is ectopically induced in response to pzg loss. In addition, the expression of the mitogen wingless (wg) but not of decapentaplegic (dpp) is observed. We present evidence that downregulation of Notch activates Dcp1 caspase and JNK signaling, however, neither induces ectopic wg nor dpp expression. In contrast, the consequences of Dref-RNAi were largely indistinguishable from pzg-RNAi with regard to apoptosis induction. Moreover, overexpression of Dref ameliorated the downregulation of pzg compatible with the notion that the two are required together to maintain cell and tissue homeostasis in Drosophila.

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pzg depletion results in an inappropriate JNK-signaling activation.pzg was downregulated in the posterior part of the wing disc (Pzg protein is shown in blue). (A-B'') puc-lacZ activity is induced in pzg-RNAi mutant cells reflecting JNK-signaling activity (red in A-A', B-B', arrows) (en-Gal4 UAS-GFP UAS-pzg-RNAi/+; puc-lacZ/+). In addition, especially in late third instar larval discs, a non-autonomous activity can be detected in the anterior compartment (open arrows in A', B'). (C-F'') Further consequences of JNK-mediated developmental apoptosis induction can be observed: (C-D'') Dilp8 protein is secreted in the pzg depleted cells, however not before late third larval instar (red in D, D', arrow). (E-E'') In pzg-RNAi mutant cells, Arm protein accumulation is disturbed (red, repressive arrow). (F-F'') pzg mutant cells penetrate into the anterior compartment while retaining their posterior identity (green, arrows in F, F'). (C-F'') en-Gal4 UAS-GFP UAS-pzg-RNAi/+. The A/P compartment boundary is marked with a dotted line. Scale bars: 100 μm.
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pone.0124652.g003: pzg depletion results in an inappropriate JNK-signaling activation.pzg was downregulated in the posterior part of the wing disc (Pzg protein is shown in blue). (A-B'') puc-lacZ activity is induced in pzg-RNAi mutant cells reflecting JNK-signaling activity (red in A-A', B-B', arrows) (en-Gal4 UAS-GFP UAS-pzg-RNAi/+; puc-lacZ/+). In addition, especially in late third instar larval discs, a non-autonomous activity can be detected in the anterior compartment (open arrows in A', B'). (C-F'') Further consequences of JNK-mediated developmental apoptosis induction can be observed: (C-D'') Dilp8 protein is secreted in the pzg depleted cells, however not before late third larval instar (red in D, D', arrow). (E-E'') In pzg-RNAi mutant cells, Arm protein accumulation is disturbed (red, repressive arrow). (F-F'') pzg mutant cells penetrate into the anterior compartment while retaining their posterior identity (green, arrows in F, F'). (C-F'') en-Gal4 UAS-GFP UAS-pzg-RNAi/+. The A/P compartment boundary is marked with a dotted line. Scale bars: 100 μm.

Mentions: A key factor known to convey apoptosis in Drosophila and mammals alike is the c-Jun N-terminal kinase (JNK) pathway (reviewed in [60,61]). To measure JNK signaling activity in pzg mutant cells, we made use of the JNK downstream effector puckered (puc) and monitored the expression of a puc-lacZ enhancer trap line [62]. In wild type third instar wing discs, puc-lacZ expression is detected in the small stalk region attaching the disc to the larval epidermis [62]. Knock down of pzg robustly activated the puc-lacZ reporter gene within the depleted area and weakly in adjacent regions notably in older wing discs (Fig 3A-B'' and S2F-F'' Fig), consistent with the induction of JNK-mediated cell death. Great experimental insights were gained in the recent years demonstrating that JNK-mediated cell death in Drosophila is crucial for eliminating aberrant cells, thereby ensuring further development and morphogenesis (reviewed in [16,29,61]). This includes also the induction of a developmental delay and inhibition of metamorphosis, allowing the larva to compensate growth deficits and repair injured tissue. The gene encoding Dilp8, a member of the insulin-relaxin peptide family, was found to be upregulated in response to JNK signaling and is thought to delay metamorphosis by inhibiting ecdysone biosynthesis [53,63]. Tracing Dilp8 in wing discs, where pzg-RNAi was induced in the posterior half, revealed no obvious accumulation in younger discs (approximately 96 h AEL; Fig 3C-C''), whereas Dilp8 was highly enriched in later stages (approximately 120 h AEL; Fig 3D-D'').


Loss of putzig Activity Results in Apoptosis during Wing Imaginal Development in Drosophila.

Zimmermann M, Kugler SJ, Schulz A, Nagel AC - PLoS ONE (2015)

pzg depletion results in an inappropriate JNK-signaling activation.pzg was downregulated in the posterior part of the wing disc (Pzg protein is shown in blue). (A-B'') puc-lacZ activity is induced in pzg-RNAi mutant cells reflecting JNK-signaling activity (red in A-A', B-B', arrows) (en-Gal4 UAS-GFP UAS-pzg-RNAi/+; puc-lacZ/+). In addition, especially in late third instar larval discs, a non-autonomous activity can be detected in the anterior compartment (open arrows in A', B'). (C-F'') Further consequences of JNK-mediated developmental apoptosis induction can be observed: (C-D'') Dilp8 protein is secreted in the pzg depleted cells, however not before late third larval instar (red in D, D', arrow). (E-E'') In pzg-RNAi mutant cells, Arm protein accumulation is disturbed (red, repressive arrow). (F-F'') pzg mutant cells penetrate into the anterior compartment while retaining their posterior identity (green, arrows in F, F'). (C-F'') en-Gal4 UAS-GFP UAS-pzg-RNAi/+. The A/P compartment boundary is marked with a dotted line. Scale bars: 100 μm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4403878&req=5

pone.0124652.g003: pzg depletion results in an inappropriate JNK-signaling activation.pzg was downregulated in the posterior part of the wing disc (Pzg protein is shown in blue). (A-B'') puc-lacZ activity is induced in pzg-RNAi mutant cells reflecting JNK-signaling activity (red in A-A', B-B', arrows) (en-Gal4 UAS-GFP UAS-pzg-RNAi/+; puc-lacZ/+). In addition, especially in late third instar larval discs, a non-autonomous activity can be detected in the anterior compartment (open arrows in A', B'). (C-F'') Further consequences of JNK-mediated developmental apoptosis induction can be observed: (C-D'') Dilp8 protein is secreted in the pzg depleted cells, however not before late third larval instar (red in D, D', arrow). (E-E'') In pzg-RNAi mutant cells, Arm protein accumulation is disturbed (red, repressive arrow). (F-F'') pzg mutant cells penetrate into the anterior compartment while retaining their posterior identity (green, arrows in F, F'). (C-F'') en-Gal4 UAS-GFP UAS-pzg-RNAi/+. The A/P compartment boundary is marked with a dotted line. Scale bars: 100 μm.
Mentions: A key factor known to convey apoptosis in Drosophila and mammals alike is the c-Jun N-terminal kinase (JNK) pathway (reviewed in [60,61]). To measure JNK signaling activity in pzg mutant cells, we made use of the JNK downstream effector puckered (puc) and monitored the expression of a puc-lacZ enhancer trap line [62]. In wild type third instar wing discs, puc-lacZ expression is detected in the small stalk region attaching the disc to the larval epidermis [62]. Knock down of pzg robustly activated the puc-lacZ reporter gene within the depleted area and weakly in adjacent regions notably in older wing discs (Fig 3A-B'' and S2F-F'' Fig), consistent with the induction of JNK-mediated cell death. Great experimental insights were gained in the recent years demonstrating that JNK-mediated cell death in Drosophila is crucial for eliminating aberrant cells, thereby ensuring further development and morphogenesis (reviewed in [16,29,61]). This includes also the induction of a developmental delay and inhibition of metamorphosis, allowing the larva to compensate growth deficits and repair injured tissue. The gene encoding Dilp8, a member of the insulin-relaxin peptide family, was found to be upregulated in response to JNK signaling and is thought to delay metamorphosis by inhibiting ecdysone biosynthesis [53,63]. Tracing Dilp8 in wing discs, where pzg-RNAi was induced in the posterior half, revealed no obvious accumulation in younger discs (approximately 96 h AEL; Fig 3C-C''), whereas Dilp8 was highly enriched in later stages (approximately 120 h AEL; Fig 3D-D'').

Bottom Line: We present evidence that downregulation of Notch activates Dcp1 caspase and JNK signaling, however, neither induces ectopic wg nor dpp expression.In contrast, the consequences of Dref-RNAi were largely indistinguishable from pzg-RNAi with regard to apoptosis induction.Moreover, overexpression of Dref ameliorated the downregulation of pzg compatible with the notion that the two are required together to maintain cell and tissue homeostasis in Drosophila.

View Article: PubMed Central - PubMed

Affiliation: Institute of Genetics, University of Hohenheim, 70599 Stuttgart, Germany.

ABSTRACT
The Drosophila gene putzig (pzg) encodes a nuclear protein that is an integral component of the Trf2/Dref complex involved in the transcription of proliferation-related genes. Moreover, Pzg is found in a complex together with the nucleosome remodeling factor NURF, where it promotes Notch target gene activation. Here we show that downregulation of pzg activity in the developing wing imaginal discs induces an apoptotic response, accompanied by the induction of the pro-apoptotic gene reaper, repression of Drosophila inhibitor of apoptosis protein accumulation and the activation of the caspases Drice, Caspase3 and Dcp1. As a further consequence 'Apoptosis induced Proliferation' (AiP) and 'Apoptosis induced Apoptosis' (AiA) are triggered. As expected, the activity of the stress kinase Jun N-terminal kinase (JNK), proposed to mediate both processes, is ectopically induced in response to pzg loss. In addition, the expression of the mitogen wingless (wg) but not of decapentaplegic (dpp) is observed. We present evidence that downregulation of Notch activates Dcp1 caspase and JNK signaling, however, neither induces ectopic wg nor dpp expression. In contrast, the consequences of Dref-RNAi were largely indistinguishable from pzg-RNAi with regard to apoptosis induction. Moreover, overexpression of Dref ameliorated the downregulation of pzg compatible with the notion that the two are required together to maintain cell and tissue homeostasis in Drosophila.

Show MeSH
Related in: MedlinePlus