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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 adult flies.  The indicated GeneSwitch                                            drivers Act-GS-255B ("255B"), Elav-GS ("Elav") and WB-FB-GS ("FB") were                                            crossed to the UAS-ultraGFP reporter and adult progeny containing both                                            constructs were scored for GFP expression in various tissues.  Control                                            flies were generated by crossing UAS-ultraGFP to white1118strain flies to produce progeny containing only UAS-ultraGFP.  Age-synchronized                                            flies were cultured in the presence and absence                                            of the drug RU486 for two weeks prior to assay, and GFP expression was                                            scored in whole adult flies and dissected tissues, as indicated.  Each                                            image is the overlay of the visible light and GFP images.  Insets show                                            details of the regions boxed in white, GFP image only.  M = male, F =                                            female.  Pictures were taken at the magnification of 20X, 50X, 32X, 20X,                                            50X, and 80X, for whole fly, head in half, body in half, gut, ovary, and                                            testes, respectively. The white arrow indicates a region of 255B Female                                            flight muscle that is obscured by a fragment of cuticle.
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Figure 1: Expression pattern produced by Geneswitch drivers and UAS-GFP reporter in adult flies. The indicated GeneSwitch drivers Act-GS-255B ("255B"), Elav-GS ("Elav") and WB-FB-GS ("FB") were crossed to the UAS-ultraGFP reporter and adult progeny containing both constructs were scored for GFP expression in various tissues. Control flies were generated by crossing UAS-ultraGFP to white1118strain flies to produce progeny containing only UAS-ultraGFP. Age-synchronized flies were cultured in the presence and absence of the drug RU486 for two weeks prior to assay, and GFP expression was scored in whole adult flies and dissected tissues, as indicated. Each image is the overlay of the visible light and GFP images. Insets show details of the regions boxed in white, GFP image only. M = male, F = female. Pictures were taken at the magnification of 20X, 50X, 32X, 20X, 50X, and 80X, for whole fly, head in half, body in half, gut, ovary, and testes, respectively. The white arrow indicates a region of 255B Female flight muscle that is obscured by a fragment of cuticle.

Mentions: To facilitate the screen of apoptosis and senescence-regulatory genes for life span effects, several Geneswitch system drivers were characterized for their tissue-specificity of transgene activation using a UAS-GFP reporter, both in adult flies and during larval development. The UAS-GFP reporter employed was "UAS-ultraGFP" which contains multiple copies of a UAS-eGFP construct, and yields particularly high levels of GFP expression [18]. Three Geneswitch system drivers were characterized: The Act-GS-255B driver strain contains multiple inserts of a construct in which the promoter from the cytoplasmic actin gene Actin 5C drives Geneswitch, and is expected to yield tissue-general expression [16]. The Elav-GS driver contains Geneswitch under control of the Elav gene promoter and produces nervous-system-specific expression [19]. Finally the whole-body fat-body Geneswitch driver strain ("WB-FB-GS") contains both a head fat-body driver (S1-32) and a body-fat-body driver (S1-106) [20-22], and is expected to yield expression in the fat-body tissue throughout the animal. The three driver strains were crossed to the UAS-ultraGFP reporter strain to produce adult progeny containing both the driver and reporter constructs, and the flies were cultured in the presence and absence of drug for two weeks. GFP expression was scored in live adult flies as well as in several dissected tissues (Figure 1). The Act-GS-255B driver was found to yield tissue-general expression of the UAS-ultraGFP reporter in adult flies. In whole adults, GFP expression was observed throughout the body of both males and females, with greater expression levels observed in females relative to males. Similarly with heads dissected in half and bodies dissected in half, expression was observed in all tissues, including abundant expression in nervous system, muscle (including flight muscle), and fat-body tissue. Note that flight muscle in male has lower expression than flight muscle in female, however inspection of the GFP-only image for male flight muscle (inset) reveals expression throughout this tissue. Abundant expression was also observed throughout dissected gut tissue, ovary and testes. The expression level was greater in some regions of the gut than others, however all regions of the gut exhibited staining, as revealed by inspection of the GFP-only images (inset). All tissues observed showed significant GFP expression, and therefore we conclude that Act-GS-255B yields truly tissue-general expression in adult flies. The WB-FB-GS driver produced GFP expression in the head-fat-body and body-fat-body tissues, as expected, as well as in the gut and testes, and very faint expression in ovary; there was no detectable expression in nervous, muscle, or other tissues. Notably, the expression in adult male head fat body was much reduced relative to female head fat body, consistent with recent characterization of the fat body drivers using a LacZ reporter [17]. Finally, the Elav-GS driver produced abundant expression in the brain and ventral nerve cord, as expected, and there was no detectable expression in any other tissues; for example, the muscle, gut and gonads were clearly negative. Note the GFP-only image for the gut (inset) shows a lack of expression. The Elav-GS driver was found to produce similar levels of UAS-GFP reporter expression in male versus female in our experiments.


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 adult flies.  The indicated GeneSwitch                                            drivers Act-GS-255B ("255B"), Elav-GS ("Elav") and WB-FB-GS ("FB") were                                            crossed to the UAS-ultraGFP reporter and adult progeny containing both                                            constructs were scored for GFP expression in various tissues.  Control                                            flies were generated by crossing UAS-ultraGFP to white1118strain flies to produce progeny containing only UAS-ultraGFP.  Age-synchronized                                            flies were cultured in the presence and absence                                            of the drug RU486 for two weeks prior to assay, and GFP expression was                                            scored in whole adult flies and dissected tissues, as indicated.  Each                                            image is the overlay of the visible light and GFP images.  Insets show                                            details of the regions boxed in white, GFP image only.  M = male, F =                                            female.  Pictures were taken at the magnification of 20X, 50X, 32X, 20X,                                            50X, and 80X, for whole fly, head in half, body in half, gut, ovary, and                                            testes, respectively. The white arrow indicates a region of 255B Female                                            flight muscle that is obscured by a fragment of cuticle.
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Related In: Results  -  Collection

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Figure 1: Expression pattern produced by Geneswitch drivers and UAS-GFP reporter in adult flies. The indicated GeneSwitch drivers Act-GS-255B ("255B"), Elav-GS ("Elav") and WB-FB-GS ("FB") were crossed to the UAS-ultraGFP reporter and adult progeny containing both constructs were scored for GFP expression in various tissues. Control flies were generated by crossing UAS-ultraGFP to white1118strain flies to produce progeny containing only UAS-ultraGFP. Age-synchronized flies were cultured in the presence and absence of the drug RU486 for two weeks prior to assay, and GFP expression was scored in whole adult flies and dissected tissues, as indicated. Each image is the overlay of the visible light and GFP images. Insets show details of the regions boxed in white, GFP image only. M = male, F = female. Pictures were taken at the magnification of 20X, 50X, 32X, 20X, 50X, and 80X, for whole fly, head in half, body in half, gut, ovary, and testes, respectively. The white arrow indicates a region of 255B Female flight muscle that is obscured by a fragment of cuticle.
Mentions: To facilitate the screen of apoptosis and senescence-regulatory genes for life span effects, several Geneswitch system drivers were characterized for their tissue-specificity of transgene activation using a UAS-GFP reporter, both in adult flies and during larval development. The UAS-GFP reporter employed was "UAS-ultraGFP" which contains multiple copies of a UAS-eGFP construct, and yields particularly high levels of GFP expression [18]. Three Geneswitch system drivers were characterized: The Act-GS-255B driver strain contains multiple inserts of a construct in which the promoter from the cytoplasmic actin gene Actin 5C drives Geneswitch, and is expected to yield tissue-general expression [16]. The Elav-GS driver contains Geneswitch under control of the Elav gene promoter and produces nervous-system-specific expression [19]. Finally the whole-body fat-body Geneswitch driver strain ("WB-FB-GS") contains both a head fat-body driver (S1-32) and a body-fat-body driver (S1-106) [20-22], and is expected to yield expression in the fat-body tissue throughout the animal. The three driver strains were crossed to the UAS-ultraGFP reporter strain to produce adult progeny containing both the driver and reporter constructs, and the flies were cultured in the presence and absence of drug for two weeks. GFP expression was scored in live adult flies as well as in several dissected tissues (Figure 1). The Act-GS-255B driver was found to yield tissue-general expression of the UAS-ultraGFP reporter in adult flies. In whole adults, GFP expression was observed throughout the body of both males and females, with greater expression levels observed in females relative to males. Similarly with heads dissected in half and bodies dissected in half, expression was observed in all tissues, including abundant expression in nervous system, muscle (including flight muscle), and fat-body tissue. Note that flight muscle in male has lower expression than flight muscle in female, however inspection of the GFP-only image for male flight muscle (inset) reveals expression throughout this tissue. Abundant expression was also observed throughout dissected gut tissue, ovary and testes. The expression level was greater in some regions of the gut than others, however all regions of the gut exhibited staining, as revealed by inspection of the GFP-only images (inset). All tissues observed showed significant GFP expression, and therefore we conclude that Act-GS-255B yields truly tissue-general expression in adult flies. The WB-FB-GS driver produced GFP expression in the head-fat-body and body-fat-body tissues, as expected, as well as in the gut and testes, and very faint expression in ovary; there was no detectable expression in nervous, muscle, or other tissues. Notably, the expression in adult male head fat body was much reduced relative to female head fat body, consistent with recent characterization of the fat body drivers using a LacZ reporter [17]. Finally, the Elav-GS driver produced abundant expression in the brain and ventral nerve cord, as expected, and there was no detectable expression in any other tissues; for example, the muscle, gut and gonads were clearly negative. Note the GFP-only image for the gut (inset) shows a lack of expression. The Elav-GS driver was found to produce similar levels of UAS-GFP reporter expression in male versus female in our experiments.

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