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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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Apoptotic consequences of Notch depletion in wing imaginal discs.N-RNAi was induced in the posterior compartment of the wing disc, N protein is shown in blue (A, B, D and A'', B'', D''), the posterior compartment is marked in green with GFP (A-F and C'', E'', F''). (A-B'') Caspase activity, visualized with anti-Dcp-1act (red in A, A' and B, B') is detectable in early (96 h AEL) and late third instar wing discs (120 h AEL) autonomously (arrows in A, A' and B, B') and non-autonomously (open arrows in A, A' and B, B'). The effect of AiA is enhanced in later phases of development (B, B'). (Genotype: UAS-N-RNAi; en-Gal4 UAS-GFP/+). (C-C'') JNK-signaling readout visualized with puc-lacZ expression is seen in both compartments (red in C, C' arrows; UAS-N-RNAi; en-Gal4 UAS-GFP/+; puc-lacZ/+). The wild type expression of puc-lacZ at the stalk region is marked with an asterisk (C, C'). (D-D'') Cells within S-phase are labeled with EdU (red); a minor autonomous reduction and weak increase in most central cells abutting the A/P compartment boundary is observed (arrow in D, D'; genotype as in A). (E-E'') The expression of Wg is lost at the dorso-ventral boundary in the posterior compartment (red in E, E', repressive arrow; genotype as in A). (F-F'') dpp expression is not affected by downregulation of N in the posterior compartment (red in F, F'; UAS-N-RNAi; en-Gal4 UAS-GFP/+; dpp-lacZ/+). The A/P compartment boundary is marked with a dotted line. Scale bars: 100 μm.
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pone.0124652.g006: Apoptotic consequences of Notch depletion in wing imaginal discs.N-RNAi was induced in the posterior compartment of the wing disc, N protein is shown in blue (A, B, D and A'', B'', D''), the posterior compartment is marked in green with GFP (A-F and C'', E'', F''). (A-B'') Caspase activity, visualized with anti-Dcp-1act (red in A, A' and B, B') is detectable in early (96 h AEL) and late third instar wing discs (120 h AEL) autonomously (arrows in A, A' and B, B') and non-autonomously (open arrows in A, A' and B, B'). The effect of AiA is enhanced in later phases of development (B, B'). (Genotype: UAS-N-RNAi; en-Gal4 UAS-GFP/+). (C-C'') JNK-signaling readout visualized with puc-lacZ expression is seen in both compartments (red in C, C' arrows; UAS-N-RNAi; en-Gal4 UAS-GFP/+; puc-lacZ/+). The wild type expression of puc-lacZ at the stalk region is marked with an asterisk (C, C'). (D-D'') Cells within S-phase are labeled with EdU (red); a minor autonomous reduction and weak increase in most central cells abutting the A/P compartment boundary is observed (arrow in D, D'; genotype as in A). (E-E'') The expression of Wg is lost at the dorso-ventral boundary in the posterior compartment (red in E, E', repressive arrow; genotype as in A). (F-F'') dpp expression is not affected by downregulation of N in the posterior compartment (red in F, F'; UAS-N-RNAi; en-Gal4 UAS-GFP/+; dpp-lacZ/+). The A/P compartment boundary is marked with a dotted line. Scale bars: 100 μm.

Mentions: As Pzg was shown to be required for efficient N signaling to occur, we asked whether the observed apoptotic outcome after pzg-RNAi induction might be the consequence of an impaired N signaling activity [40,41]. It is well established that a reduction in N signaling activity during imaginal development is correlated with tissue loss and apoptosis [51,72]. Therefore, we induced N-RNAi in the posterior compartment of the wing disc to compare the effects with those obtained after pzg depletion. To this end, we used the same N-RNAi line that has been shown by others to provide RNAi-mediated knock-down of N activity in wing imaginal discs [38,73]. We observed an accumulation of activated Dcp-1 caspase autonomously in N mutant cells and also non-autonomously in the anterior compartment, indicating that a downregulation of N signaling contributes to AiA (Fig 6A-B''). Moreover, a robust induction of puc-lacZ was detected in both compartments, indicating that JNK-mediated activity was induced as well (Fig 6C-C''). In contrast, cell proliferation, visualized with EdU labeling, was different from pzg-RNAi depleted cells: Cells within the N-RNAi depleted compartment were still able to cycle through the cell cycle concluded from EdU incorporation (Fig 6D-D''). Based on the modest reduction of EdU signals, less cells however, appeared to enter the S-phase. Moreover, AiP was only weakly observed as EdU labeled cells abutting the N-deficient area appeared only more densely spaced (Fig 6D-D''), and no ectopic induction of the mitogens Wg (Fig 6E-E'') or dpp-lacZ (Fig 6F-F'') was detected.


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

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

Apoptotic consequences of Notch depletion in wing imaginal discs.N-RNAi was induced in the posterior compartment of the wing disc, N protein is shown in blue (A, B, D and A'', B'', D''), the posterior compartment is marked in green with GFP (A-F and C'', E'', F''). (A-B'') Caspase activity, visualized with anti-Dcp-1act (red in A, A' and B, B') is detectable in early (96 h AEL) and late third instar wing discs (120 h AEL) autonomously (arrows in A, A' and B, B') and non-autonomously (open arrows in A, A' and B, B'). The effect of AiA is enhanced in later phases of development (B, B'). (Genotype: UAS-N-RNAi; en-Gal4 UAS-GFP/+). (C-C'') JNK-signaling readout visualized with puc-lacZ expression is seen in both compartments (red in C, C' arrows; UAS-N-RNAi; en-Gal4 UAS-GFP/+; puc-lacZ/+). The wild type expression of puc-lacZ at the stalk region is marked with an asterisk (C, C'). (D-D'') Cells within S-phase are labeled with EdU (red); a minor autonomous reduction and weak increase in most central cells abutting the A/P compartment boundary is observed (arrow in D, D'; genotype as in A). (E-E'') The expression of Wg is lost at the dorso-ventral boundary in the posterior compartment (red in E, E', repressive arrow; genotype as in A). (F-F'') dpp expression is not affected by downregulation of N in the posterior compartment (red in F, F'; UAS-N-RNAi; en-Gal4 UAS-GFP/+; dpp-lacZ/+). The A/P compartment boundary is marked with a dotted line. Scale bars: 100 μm.
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Related In: Results  -  Collection

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pone.0124652.g006: Apoptotic consequences of Notch depletion in wing imaginal discs.N-RNAi was induced in the posterior compartment of the wing disc, N protein is shown in blue (A, B, D and A'', B'', D''), the posterior compartment is marked in green with GFP (A-F and C'', E'', F''). (A-B'') Caspase activity, visualized with anti-Dcp-1act (red in A, A' and B, B') is detectable in early (96 h AEL) and late third instar wing discs (120 h AEL) autonomously (arrows in A, A' and B, B') and non-autonomously (open arrows in A, A' and B, B'). The effect of AiA is enhanced in later phases of development (B, B'). (Genotype: UAS-N-RNAi; en-Gal4 UAS-GFP/+). (C-C'') JNK-signaling readout visualized with puc-lacZ expression is seen in both compartments (red in C, C' arrows; UAS-N-RNAi; en-Gal4 UAS-GFP/+; puc-lacZ/+). The wild type expression of puc-lacZ at the stalk region is marked with an asterisk (C, C'). (D-D'') Cells within S-phase are labeled with EdU (red); a minor autonomous reduction and weak increase in most central cells abutting the A/P compartment boundary is observed (arrow in D, D'; genotype as in A). (E-E'') The expression of Wg is lost at the dorso-ventral boundary in the posterior compartment (red in E, E', repressive arrow; genotype as in A). (F-F'') dpp expression is not affected by downregulation of N in the posterior compartment (red in F, F'; UAS-N-RNAi; en-Gal4 UAS-GFP/+; dpp-lacZ/+). The A/P compartment boundary is marked with a dotted line. Scale bars: 100 μm.
Mentions: As Pzg was shown to be required for efficient N signaling to occur, we asked whether the observed apoptotic outcome after pzg-RNAi induction might be the consequence of an impaired N signaling activity [40,41]. It is well established that a reduction in N signaling activity during imaginal development is correlated with tissue loss and apoptosis [51,72]. Therefore, we induced N-RNAi in the posterior compartment of the wing disc to compare the effects with those obtained after pzg depletion. To this end, we used the same N-RNAi line that has been shown by others to provide RNAi-mediated knock-down of N activity in wing imaginal discs [38,73]. We observed an accumulation of activated Dcp-1 caspase autonomously in N mutant cells and also non-autonomously in the anterior compartment, indicating that a downregulation of N signaling contributes to AiA (Fig 6A-B''). Moreover, a robust induction of puc-lacZ was detected in both compartments, indicating that JNK-mediated activity was induced as well (Fig 6C-C''). In contrast, cell proliferation, visualized with EdU labeling, was different from pzg-RNAi depleted cells: Cells within the N-RNAi depleted compartment were still able to cycle through the cell cycle concluded from EdU incorporation (Fig 6D-D''). Based on the modest reduction of EdU signals, less cells however, appeared to enter the S-phase. Moreover, AiP was only weakly observed as EdU labeled cells abutting the N-deficient area appeared only more densely spaced (Fig 6D-D''), and no ectopic induction of the mitogens Wg (Fig 6E-E'') or dpp-lacZ (Fig 6F-F'') was detected.

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