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A screen of apoptosis and senescence regulatory genes for life span effects when over-expressed in Drosophila.

Shen J, Curtis C, Tavaré S, Tower J - Aging (Albany NY) (2009)

Bottom Line: Both wingless and Ras activated form transgenes were lethal when expressed in larvae, and reduced life span when expressed in adults, consistent with results from other model systems indicating that the wingless and Ras pathways can promote senescence.Over-expression of the caspase inhibitor baculovirus p35 during larval development reduced the mean life span of male and female adults, and also produced a subset of females with increased life span.These experiments suggest that baculovirus p35 and the wingless and Ras pathways can have sex-specific and developmental stage-specific effects on adult Drosophila life span, and these reagents should be useful for the further analysis of the role of these conserved pathways in aging.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Computational Biology Program, Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089, USA.

ABSTRACT
Conditional expression of transgenes in Drosophila was produced using the Geneswitch system, wherein feeding the drug RU486/Mifepristone activates the artificial transcription factor Geneswitch. Geneswitch was expressed using the Actin5C promoter and this was found to yield conditional, tissue-general expression of a target transgene (UAS-GFP) in both larvae and adult flies. Nervous system-specific (Elav-GS) and fat body-specific Geneswitch drivers were also characterized using UAS-GFP. Fourteen genes implicated in growth, apoptosis and senescence regulatory pathways were over-expressed in adult flies or during larval development, and assayed for effects on adult fly life span. Over-expression of a dominant p53 allele (p53-259H) in adult flies using the ubiquitous driver produced increased life span in females but not males, consistent with previous studies. Both wingless and Ras activated form transgenes were lethal when expressed in larvae, and reduced life span when expressed in adults, consistent with results from other model systems indicating that the wingless and Ras pathways can promote senescence. Over-expression of the caspase inhibitor baculovirus p35 during larval development reduced the mean life span of male and female adults, and also produced a subset of females with increased life span. These experiments suggest that baculovirus p35 and the wingless and Ras pathways can have sex-specific and developmental stage-specific effects on adult Drosophila life span, and these reagents should be useful for the further analysis of the role of these conserved pathways in aging.

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Expression pattern produced by GeneSwitch drivers and UAS-GFP reporter in larvae.  The crosses are the same as                                            Figure 1, but larvae were cultured in the presence and absence of drug in                                            the food, from hatching to the indicated developmental stage.  A.                                            Expression patterns in 3rd instar larvae and dissected tissues.                                            For the Elav-GS driver ("Elav") a 1:10 dilution of drug was used because of                                            the toxic effects of drug observed in larvae with this driver. Pictures                                            were taken at the magnification of 25X, 100X, 20X, 50X, 100X, 80X, for                                            whole larvae, brain, gut, salivary gland, imaginal discs, and fat body,                                            respectively.  B. Expression patterns in the three larval stages. For Elav-GS                                            a 1:10 dilution of drug was used to avoid toxic effects. GFP pictures were                                            taken at the magnification of 100X, 50X, 25X, for 1st instar, 2nd                                            instar, and 3rd instar, respectively. C. Expression in 3rd                                            instar larvae using Act-GS-255B and titrations of drug. ETOH indicates the                                            ethanol solvent for the drug alone.  Pictures were taken at the                                            magnification of 25X.
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Figure 2: Expression pattern produced by GeneSwitch drivers and UAS-GFP reporter in larvae. The crosses are the same as Figure 1, but larvae were cultured in the presence and absence of drug in the food, from hatching to the indicated developmental stage. A. Expression patterns in 3rd instar larvae and dissected tissues. For the Elav-GS driver ("Elav") a 1:10 dilution of drug was used because of the toxic effects of drug observed in larvae with this driver. Pictures were taken at the magnification of 25X, 100X, 20X, 50X, 100X, 80X, for whole larvae, brain, gut, salivary gland, imaginal discs, and fat body, respectively. B. Expression patterns in the three larval stages. For Elav-GS a 1:10 dilution of drug was used to avoid toxic effects. GFP pictures were taken at the magnification of 100X, 50X, 25X, for 1st instar, 2nd instar, and 3rd instar, respectively. C. Expression in 3rd instar larvae using Act-GS-255B and titrations of drug. ETOH indicates the ethanol solvent for the drug alone. Pictures were taken at the magnification of 25X.

Mentions: The Geneswitch driver strains were also scored for expression patterns in 3rd instar larvae and dissected tissues (Figure 2). The Act-GS-255B driver was found to yield tissue-general expression, including abundant expression throughout the body of whole 3rd instar larvae, as well as in dissected brain, gut, salivary gland, imaginal discs and fat-body tissues; all tissues observed showed abundant GFP expression (Figure 2A). The inset for the Act-GS-255B 3rd instar larval brain shows detail of the GFP-only image, and indicates that expression was present throughout the brain, with higher-level expression in a subset of cells. The WB-FB-GS driver was found to drive abundant expression in salivary gland and anterior midgut, but notably no expression in any other larval tissues including larval fat-body. Finally the Elav-GS driver produced abundant expression in larval nervous system and no detectable expression in any other larval tissues. The inset for the Elav-GS 3rd instar larval brain shows detail of the GFP-only image, and shows that expression was present throughout the brain, with higher-level expression in a subset of cells. Notably this subset of cells was different from that observed above with Act-GS-255B. Each of the three drivers was found to produce similar patterns of expression in 1st and 2nd instar larvae as well (Figure 1B). When the Act-GS-255B driver was induced using dilutions of RU486 drug in the culture media, it produced a dose-response of GFP expression in 3rd instar larvae (Figure 1D), as well as in adult flies (data not shown).


A screen of apoptosis and senescence regulatory genes for life span effects when over-expressed in Drosophila.

Shen J, Curtis C, Tavaré S, Tower J - Aging (Albany NY) (2009)

Expression pattern produced by GeneSwitch drivers and UAS-GFP reporter in larvae.  The crosses are the same as                                            Figure 1, but larvae were cultured in the presence and absence of drug in                                            the food, from hatching to the indicated developmental stage.  A.                                            Expression patterns in 3rd instar larvae and dissected tissues.                                            For the Elav-GS driver ("Elav") a 1:10 dilution of drug was used because of                                            the toxic effects of drug observed in larvae with this driver. Pictures                                            were taken at the magnification of 25X, 100X, 20X, 50X, 100X, 80X, for                                            whole larvae, brain, gut, salivary gland, imaginal discs, and fat body,                                            respectively.  B. Expression patterns in the three larval stages. For Elav-GS                                            a 1:10 dilution of drug was used to avoid toxic effects. GFP pictures were                                            taken at the magnification of 100X, 50X, 25X, for 1st instar, 2nd                                            instar, and 3rd instar, respectively. C. Expression in 3rd                                            instar larvae using Act-GS-255B and titrations of drug. ETOH indicates the                                            ethanol solvent for the drug alone.  Pictures were taken at the                                            magnification of 25X.
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Figure 2: Expression pattern produced by GeneSwitch drivers and UAS-GFP reporter in larvae. The crosses are the same as Figure 1, but larvae were cultured in the presence and absence of drug in the food, from hatching to the indicated developmental stage. A. Expression patterns in 3rd instar larvae and dissected tissues. For the Elav-GS driver ("Elav") a 1:10 dilution of drug was used because of the toxic effects of drug observed in larvae with this driver. Pictures were taken at the magnification of 25X, 100X, 20X, 50X, 100X, 80X, for whole larvae, brain, gut, salivary gland, imaginal discs, and fat body, respectively. B. Expression patterns in the three larval stages. For Elav-GS a 1:10 dilution of drug was used to avoid toxic effects. GFP pictures were taken at the magnification of 100X, 50X, 25X, for 1st instar, 2nd instar, and 3rd instar, respectively. C. Expression in 3rd instar larvae using Act-GS-255B and titrations of drug. ETOH indicates the ethanol solvent for the drug alone. Pictures were taken at the magnification of 25X.
Mentions: The Geneswitch driver strains were also scored for expression patterns in 3rd instar larvae and dissected tissues (Figure 2). The Act-GS-255B driver was found to yield tissue-general expression, including abundant expression throughout the body of whole 3rd instar larvae, as well as in dissected brain, gut, salivary gland, imaginal discs and fat-body tissues; all tissues observed showed abundant GFP expression (Figure 2A). The inset for the Act-GS-255B 3rd instar larval brain shows detail of the GFP-only image, and indicates that expression was present throughout the brain, with higher-level expression in a subset of cells. The WB-FB-GS driver was found to drive abundant expression in salivary gland and anterior midgut, but notably no expression in any other larval tissues including larval fat-body. Finally the Elav-GS driver produced abundant expression in larval nervous system and no detectable expression in any other larval tissues. The inset for the Elav-GS 3rd instar larval brain shows detail of the GFP-only image, and shows that expression was present throughout the brain, with higher-level expression in a subset of cells. Notably this subset of cells was different from that observed above with Act-GS-255B. Each of the three drivers was found to produce similar patterns of expression in 1st and 2nd instar larvae as well (Figure 1B). When the Act-GS-255B driver was induced using dilutions of RU486 drug in the culture media, it produced a dose-response of GFP expression in 3rd instar larvae (Figure 1D), as well as in adult flies (data not shown).

Bottom Line: Both wingless and Ras activated form transgenes were lethal when expressed in larvae, and reduced life span when expressed in adults, consistent with results from other model systems indicating that the wingless and Ras pathways can promote senescence.Over-expression of the caspase inhibitor baculovirus p35 during larval development reduced the mean life span of male and female adults, and also produced a subset of females with increased life span.These experiments suggest that baculovirus p35 and the wingless and Ras pathways can have sex-specific and developmental stage-specific effects on adult Drosophila life span, and these reagents should be useful for the further analysis of the role of these conserved pathways in aging.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Computational Biology Program, Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089, USA.

ABSTRACT
Conditional expression of transgenes in Drosophila was produced using the Geneswitch system, wherein feeding the drug RU486/Mifepristone activates the artificial transcription factor Geneswitch. Geneswitch was expressed using the Actin5C promoter and this was found to yield conditional, tissue-general expression of a target transgene (UAS-GFP) in both larvae and adult flies. Nervous system-specific (Elav-GS) and fat body-specific Geneswitch drivers were also characterized using UAS-GFP. Fourteen genes implicated in growth, apoptosis and senescence regulatory pathways were over-expressed in adult flies or during larval development, and assayed for effects on adult fly life span. Over-expression of a dominant p53 allele (p53-259H) in adult flies using the ubiquitous driver produced increased life span in females but not males, consistent with previous studies. Both wingless and Ras activated form transgenes were lethal when expressed in larvae, and reduced life span when expressed in adults, consistent with results from other model systems indicating that the wingless and Ras pathways can promote senescence. Over-expression of the caspase inhibitor baculovirus p35 during larval development reduced the mean life span of male and female adults, and also produced a subset of females with increased life span. These experiments suggest that baculovirus p35 and the wingless and Ras pathways can have sex-specific and developmental stage-specific effects on adult Drosophila life span, and these reagents should be useful for the further analysis of the role of these conserved pathways in aging.

Show MeSH
Related in: MedlinePlus