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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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Dref overexpression ameliorates pzg-RNAi apoptosis defects.Comparison of the apoptotic consequences of pzg depletion in the presence (A-E’) or absence (B-F’) of ectopic Dref expression. (A-D') Compared to the control (B,B',D,D'), overexpression of Dref in pzg-RNAi depleted cells ameliorates the cell cycle arrest defect (A,A',C,C'), visualized either with EdU (S-phase, red in A-B') or anti-PH3 (M-phase, red in C-D'). Repression of cell division by pzg depletion (repressive arrow in B', D'), is absent (C') or less pronounced in the Dref overexpression background (open repressive arrow in A'). Activation of Dcp-1 (anti-Dcp-1act, red in E-F') upon pzg downregulation (arrow in F'), is much weaker when Dref is overexpressed (open arrow in E'). Genotypes: (A, A'; C, C'; E, E') en-Gal4 UAS-GFP UAS-pzg-RNAi/+; UAS-Dref/+. (B, B'; D, D'; F, F') en-Gal4 UAS-GFP UAS-pzg-RNAi / UAS-lacZ. Posterior is right and dorsal up. The A/P compartment boundary is marked with a dashed line. Scale bars: 100 μm.
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pone.0124652.g008: Dref overexpression ameliorates pzg-RNAi apoptosis defects.Comparison of the apoptotic consequences of pzg depletion in the presence (A-E’) or absence (B-F’) of ectopic Dref expression. (A-D') Compared to the control (B,B',D,D'), overexpression of Dref in pzg-RNAi depleted cells ameliorates the cell cycle arrest defect (A,A',C,C'), visualized either with EdU (S-phase, red in A-B') or anti-PH3 (M-phase, red in C-D'). Repression of cell division by pzg depletion (repressive arrow in B', D'), is absent (C') or less pronounced in the Dref overexpression background (open repressive arrow in A'). Activation of Dcp-1 (anti-Dcp-1act, red in E-F') upon pzg downregulation (arrow in F'), is much weaker when Dref is overexpressed (open arrow in E'). Genotypes: (A, A'; C, C'; E, E') en-Gal4 UAS-GFP UAS-pzg-RNAi/+; UAS-Dref/+. (B, B'; D, D'; F, F') en-Gal4 UAS-GFP UAS-pzg-RNAi / UAS-lacZ. Posterior is right and dorsal up. The A/P compartment boundary is marked with a dashed line. Scale bars: 100 μm.

Mentions: Apart from its role in N target gene activation, Pzg is important for cell proliferation as a member of the Trf2/Dref complex in Drosophila [40,74]. The transcription factor Dref regulates the expression of many proliferation-related genes, and has been described as a master key factor for cell proliferation (reviewed in [37]). We hence examined the consequences of Dref-RNAi depletion on apoptosis, AiP and AiA. To this end the UAS-Dref-RNAi line was used that has been shown before to specifically target Dref activity [75]. Inducing Dref-RNAi in the posterior half of the wing disc indeed triggered a strong apoptotic response, including robust Dcp1 caspase activation in the anterior half of late third instar wing discs (appr. 120 h AEL), indicative of AiA (Fig 7A-B''). Moreover, JNK signaling, i.e. puc-lacZ, was induced autonomously in the affected compartment, and to a lesser degree also in the anterior compartment of the wing disc (Fig 7C-C''). As expected, reduction of Dref activity was correlated with impaired cell proliferation as demonstrated by reduced EdU labeling in these areas (Fig 7D-D''). Proliferation rates were, however, significantly increased in the tissue abutting the Dref mutant cells (Fig 7D-D''). Moreover, similar to the effects of pzg-RNAi depletion, ectopic Wg induction was detected in the Dref-RNAi mutant tissue (Fig 7E-E''), whereas dpp activity appeared unaffected (Fig 7F-F''). As expected from our earlier work, Wg expression along the dorso-ventral boundary was not affected by the depletion of Dref, in support of the notion that the effects of Pzg on N regulation are Dref independent [40, 41]. Overall, induction of apoptosis with all its consequences is very similar upon the depletion of either Dref or Pzg during larval wing development. We therefore asked whether an increase of Dref activity might ameliorate the apoptotic consequences of pzg depletion. To this end we overexpressed UAS-pzg-RNAi together with UAS-Dref in the posterior wing disc compartment. To account for possible titration effects of Gal4 by the extra UAS-dose, we compared the effects of Dref overexpression with those obtained in a pzg-RNAi plus UAS-lacZ background. We observed a weaker apoptotic response in the UAS-lacZ background, (Fig 8). However, a concomitant increase of Dref activity ameliorated the pzg-RNAi proliferation defects much more robustly and strongly attenuated the induction of activated Dcp-1 caspase (Fig 8). Therefore, although Pzg and Dref are part of a large multi-subunit complex, increasing the amount of Dref protein helps to counterbalance the apoptotic effects resulting from pzg loss.


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

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

Dref overexpression ameliorates pzg-RNAi apoptosis defects.Comparison of the apoptotic consequences of pzg depletion in the presence (A-E’) or absence (B-F’) of ectopic Dref expression. (A-D') Compared to the control (B,B',D,D'), overexpression of Dref in pzg-RNAi depleted cells ameliorates the cell cycle arrest defect (A,A',C,C'), visualized either with EdU (S-phase, red in A-B') or anti-PH3 (M-phase, red in C-D'). Repression of cell division by pzg depletion (repressive arrow in B', D'), is absent (C') or less pronounced in the Dref overexpression background (open repressive arrow in A'). Activation of Dcp-1 (anti-Dcp-1act, red in E-F') upon pzg downregulation (arrow in F'), is much weaker when Dref is overexpressed (open arrow in E'). Genotypes: (A, A'; C, C'; E, E') en-Gal4 UAS-GFP UAS-pzg-RNAi/+; UAS-Dref/+. (B, B'; D, D'; F, F') en-Gal4 UAS-GFP UAS-pzg-RNAi / UAS-lacZ. Posterior is right and dorsal up. The A/P compartment boundary is marked with a dashed line. Scale bars: 100 μm.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4403878&req=5

pone.0124652.g008: Dref overexpression ameliorates pzg-RNAi apoptosis defects.Comparison of the apoptotic consequences of pzg depletion in the presence (A-E’) or absence (B-F’) of ectopic Dref expression. (A-D') Compared to the control (B,B',D,D'), overexpression of Dref in pzg-RNAi depleted cells ameliorates the cell cycle arrest defect (A,A',C,C'), visualized either with EdU (S-phase, red in A-B') or anti-PH3 (M-phase, red in C-D'). Repression of cell division by pzg depletion (repressive arrow in B', D'), is absent (C') or less pronounced in the Dref overexpression background (open repressive arrow in A'). Activation of Dcp-1 (anti-Dcp-1act, red in E-F') upon pzg downregulation (arrow in F'), is much weaker when Dref is overexpressed (open arrow in E'). Genotypes: (A, A'; C, C'; E, E') en-Gal4 UAS-GFP UAS-pzg-RNAi/+; UAS-Dref/+. (B, B'; D, D'; F, F') en-Gal4 UAS-GFP UAS-pzg-RNAi / UAS-lacZ. Posterior is right and dorsal up. The A/P compartment boundary is marked with a dashed line. Scale bars: 100 μm.
Mentions: Apart from its role in N target gene activation, Pzg is important for cell proliferation as a member of the Trf2/Dref complex in Drosophila [40,74]. The transcription factor Dref regulates the expression of many proliferation-related genes, and has been described as a master key factor for cell proliferation (reviewed in [37]). We hence examined the consequences of Dref-RNAi depletion on apoptosis, AiP and AiA. To this end the UAS-Dref-RNAi line was used that has been shown before to specifically target Dref activity [75]. Inducing Dref-RNAi in the posterior half of the wing disc indeed triggered a strong apoptotic response, including robust Dcp1 caspase activation in the anterior half of late third instar wing discs (appr. 120 h AEL), indicative of AiA (Fig 7A-B''). Moreover, JNK signaling, i.e. puc-lacZ, was induced autonomously in the affected compartment, and to a lesser degree also in the anterior compartment of the wing disc (Fig 7C-C''). As expected, reduction of Dref activity was correlated with impaired cell proliferation as demonstrated by reduced EdU labeling in these areas (Fig 7D-D''). Proliferation rates were, however, significantly increased in the tissue abutting the Dref mutant cells (Fig 7D-D''). Moreover, similar to the effects of pzg-RNAi depletion, ectopic Wg induction was detected in the Dref-RNAi mutant tissue (Fig 7E-E''), whereas dpp activity appeared unaffected (Fig 7F-F''). As expected from our earlier work, Wg expression along the dorso-ventral boundary was not affected by the depletion of Dref, in support of the notion that the effects of Pzg on N regulation are Dref independent [40, 41]. Overall, induction of apoptosis with all its consequences is very similar upon the depletion of either Dref or Pzg during larval wing development. We therefore asked whether an increase of Dref activity might ameliorate the apoptotic consequences of pzg depletion. To this end we overexpressed UAS-pzg-RNAi together with UAS-Dref in the posterior wing disc compartment. To account for possible titration effects of Gal4 by the extra UAS-dose, we compared the effects of Dref overexpression with those obtained in a pzg-RNAi plus UAS-lacZ background. We observed a weaker apoptotic response in the UAS-lacZ background, (Fig 8). However, a concomitant increase of Dref activity ameliorated the pzg-RNAi proliferation defects much more robustly and strongly attenuated the induction of activated Dcp-1 caspase (Fig 8). Therefore, although Pzg and Dref are part of a large multi-subunit complex, increasing the amount of Dref protein helps to counterbalance the apoptotic effects resulting from pzg loss.

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