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Expression of Drosophila FOXO regulates growth and can phenocopy starvation.

Kramer JM, Davidge JT, Lockyer JM, Staveley BE - BMC Dev. Biol. (2003)

Bottom Line: Analysis of the wings and eyes of these small flies indicates that the reduction in size is due to decreases in cell size and cell number.Overexpression of dFOXO in the developing eye leads to a characteristic phenotype with reductions in cell size and cell number.This phenotype can be rescued by co-expression of upstream insulin signaling components, dPI3K and dAkt, however, this rescue is not seen when FOXO is mutated to a constitutively active form. dFOXO is conserved in both sequence and regulatory mechanisms when compared with other FOXO homologues.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, Memorial University of Newfoundland, St, John's, Newfoundland, (A1B 3X9), Canada. x04jmk@mun.ca

ABSTRACT

Background: Components of the insulin signaling pathway are important regulators of growth. The FOXO (forkhead box, sub-group "O") transcription factors regulate cellular processes under conditions of low levels of insulin signaling. Studies in mammalian cell culture show that activation of FOXO transcription factors causes cell death or cell cycle arrest. The Caenorhabditis elegans homologue of FOXO, Daf-16, is required for the formation of dauer larvae in response to nutritional stress. In addition, FOXO factors have been implicated in stress resistance and longevity.

Results: We have identified the Drosophila melanogaster homologue of FOXO (dFOXO), which is conserved in amino acid sequence compared with the mammalian FOXO homologues and Daf-16. Expression of dFOXO during early larval development causes inhibition of larval growth and alterations in feeding behavior. Inhibition of larval growth is reversible upon discontinuation of dFOXO expression. Expression of dFOXO during the third larval instar or at low levels during development leads to the generation of adults that are reduced in size. Analysis of the wings and eyes of these small flies indicates that the reduction in size is due to decreases in cell size and cell number. Overexpression of dFOXO in the developing eye leads to a characteristic phenotype with reductions in cell size and cell number. This phenotype can be rescued by co-expression of upstream insulin signaling components, dPI3K and dAkt, however, this rescue is not seen when FOXO is mutated to a constitutively active form.

Conclusions: dFOXO is conserved in both sequence and regulatory mechanisms when compared with other FOXO homologues. The establishment of Drosophila as a model for the study of FOXO transcription factors should prove beneficial to determining the biological role of these signaling molecules. The alterations in larval development seen upon overexpression of dFOXO closely mimic the phenotypic effects of starvation, suggesting a role for dFOXO in the response to nutritional adversity. This work has implications in the understanding of cancer and insulin related disorders, such as diabetes and obesity.

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Expression of dFOXO in first instar larvae phenocopiesstarvation and effects feeding behavior. Expression of dFOXOand mFOXO1-AA early in larval development using the (A) ActGal4 and(C) hsGal4 driver lines leads to developmental arrest similarto that seen in starved larvae. Developmentally arrested larvaeare capable of surviving for up to seven days after egg laying (AEL).(B) Expression of dFOXO (red bars) and mFOXO1-AA (green bars) leadsto alterations in feeding behavior when compared to controls (greybars). The percentage of wandering larvae is significantly greaterin larvae expressing dFOXO and mFOXO1-AA at 48 hours and 72 hoursAEL (p = 0.05). Expression of dPI3K-DN (blue bars) did not increaselarval wandering. (D) Developmental arrest is reversible upon removalof dFOXO expression (red bars), but not upon removal of mFOXO1-AAexpression (green bars). Grey bars represent the controls. Eachbar reflects the average of three separate trials, with 50 larvaeper trial. Genotypes are; (A-top, B-grey bars) w; ActGal4/+,(A-middle, B-red bars) w; ActGal4/+; UAS-dFOXO/+, (A-bottom,B-green bars) w, UAS-mFoxo1-AA/w; ActGal4/+, (C-top, D-greybars) w; hsGal4/+, (C-middle, D-red bars), w; hsGal4/UAS-dFOXO,(C-bottom, D-green bars) w, UAS-mFoxo1-AA/w; hsGal4/+,(B-blue bars) w; ActGal4/UAS-dPI3K-DN.
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Figure 2: Expression of dFOXO in first instar larvae phenocopiesstarvation and effects feeding behavior. Expression of dFOXOand mFOXO1-AA early in larval development using the (A) ActGal4 and(C) hsGal4 driver lines leads to developmental arrest similarto that seen in starved larvae. Developmentally arrested larvaeare capable of surviving for up to seven days after egg laying (AEL).(B) Expression of dFOXO (red bars) and mFOXO1-AA (green bars) leadsto alterations in feeding behavior when compared to controls (greybars). The percentage of wandering larvae is significantly greaterin larvae expressing dFOXO and mFOXO1-AA at 48 hours and 72 hoursAEL (p = 0.05). Expression of dPI3K-DN (blue bars) did not increaselarval wandering. (D) Developmental arrest is reversible upon removalof dFOXO expression (red bars), but not upon removal of mFOXO1-AAexpression (green bars). Grey bars represent the controls. Eachbar reflects the average of three separate trials, with 50 larvaeper trial. Genotypes are; (A-top, B-grey bars) w; ActGal4/+,(A-middle, B-red bars) w; ActGal4/+; UAS-dFOXO/+, (A-bottom,B-green bars) w, UAS-mFoxo1-AA/w; ActGal4/+, (C-top, D-greybars) w; hsGal4/+, (C-middle, D-red bars), w; hsGal4/UAS-dFOXO,(C-bottom, D-green bars) w, UAS-mFoxo1-AA/w; hsGal4/+,(B-blue bars) w; ActGal4/UAS-dPI3K-DN.

Mentions: Drosophila larvae feed continuously for about 5 days after egglaying (AEL). During this time the appetite and growth rate of thelarvae is enormous. If young larvae are deprived of food, they donot grow and tend to disperse randomly[16,17,42]. When the foodsupply is replenished, the larvae immediately move towards it andcontinue eating until they are close to pupation. If the food supplyis depleted, the larvae will disperse again[42].We utilized the UAS/Gal4 ectopic expression system [43] to overexpress dFOXO in the developinglarvae under the control of the ActGal4 driver[44]. This resulted in complete developmentalarrest of the larvae, which remained as first instar for up to 7days (Figure 2A),similar to the life expectancy of starved larvae [16-18].This trend was also seen using a constitutively active version of Murine Foxo1(mFoxo1) containing an alanine substitution at the T1 (T24A), andS1 (S253A) Akt phosphorylation sites (mFoxo1-AA) [45] (Figure 2A).In addition, larvae expressing dFOXO and mFoxo1-AA were often foundto be wandering far from their food supply. We monitored feedingbehavior by assessing the number of larvae away from their foodat 48 and 72 hours after egg laying (AEL). Larvae expressing dFOXOand mFoxo1-AA showed a 3–4 fold increase in wandering over larvaeexpressing Gal4 alone (Figure 2B).Thus, dFOXO expression drastically alters feeding behavior and isable to induce a starvation type response in larvae which have anadequate food supply.


Expression of Drosophila FOXO regulates growth and can phenocopy starvation.

Kramer JM, Davidge JT, Lockyer JM, Staveley BE - BMC Dev. Biol. (2003)

Expression of dFOXO in first instar larvae phenocopiesstarvation and effects feeding behavior. Expression of dFOXOand mFOXO1-AA early in larval development using the (A) ActGal4 and(C) hsGal4 driver lines leads to developmental arrest similarto that seen in starved larvae. Developmentally arrested larvaeare capable of surviving for up to seven days after egg laying (AEL).(B) Expression of dFOXO (red bars) and mFOXO1-AA (green bars) leadsto alterations in feeding behavior when compared to controls (greybars). The percentage of wandering larvae is significantly greaterin larvae expressing dFOXO and mFOXO1-AA at 48 hours and 72 hoursAEL (p = 0.05). Expression of dPI3K-DN (blue bars) did not increaselarval wandering. (D) Developmental arrest is reversible upon removalof dFOXO expression (red bars), but not upon removal of mFOXO1-AAexpression (green bars). Grey bars represent the controls. Eachbar reflects the average of three separate trials, with 50 larvaeper trial. Genotypes are; (A-top, B-grey bars) w; ActGal4/+,(A-middle, B-red bars) w; ActGal4/+; UAS-dFOXO/+, (A-bottom,B-green bars) w, UAS-mFoxo1-AA/w; ActGal4/+, (C-top, D-greybars) w; hsGal4/+, (C-middle, D-red bars), w; hsGal4/UAS-dFOXO,(C-bottom, D-green bars) w, UAS-mFoxo1-AA/w; hsGal4/+,(B-blue bars) w; ActGal4/UAS-dPI3K-DN.
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Related In: Results  -  Collection

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Figure 2: Expression of dFOXO in first instar larvae phenocopiesstarvation and effects feeding behavior. Expression of dFOXOand mFOXO1-AA early in larval development using the (A) ActGal4 and(C) hsGal4 driver lines leads to developmental arrest similarto that seen in starved larvae. Developmentally arrested larvaeare capable of surviving for up to seven days after egg laying (AEL).(B) Expression of dFOXO (red bars) and mFOXO1-AA (green bars) leadsto alterations in feeding behavior when compared to controls (greybars). The percentage of wandering larvae is significantly greaterin larvae expressing dFOXO and mFOXO1-AA at 48 hours and 72 hoursAEL (p = 0.05). Expression of dPI3K-DN (blue bars) did not increaselarval wandering. (D) Developmental arrest is reversible upon removalof dFOXO expression (red bars), but not upon removal of mFOXO1-AAexpression (green bars). Grey bars represent the controls. Eachbar reflects the average of three separate trials, with 50 larvaeper trial. Genotypes are; (A-top, B-grey bars) w; ActGal4/+,(A-middle, B-red bars) w; ActGal4/+; UAS-dFOXO/+, (A-bottom,B-green bars) w, UAS-mFoxo1-AA/w; ActGal4/+, (C-top, D-greybars) w; hsGal4/+, (C-middle, D-red bars), w; hsGal4/UAS-dFOXO,(C-bottom, D-green bars) w, UAS-mFoxo1-AA/w; hsGal4/+,(B-blue bars) w; ActGal4/UAS-dPI3K-DN.
Mentions: Drosophila larvae feed continuously for about 5 days after egglaying (AEL). During this time the appetite and growth rate of thelarvae is enormous. If young larvae are deprived of food, they donot grow and tend to disperse randomly[16,17,42]. When the foodsupply is replenished, the larvae immediately move towards it andcontinue eating until they are close to pupation. If the food supplyis depleted, the larvae will disperse again[42].We utilized the UAS/Gal4 ectopic expression system [43] to overexpress dFOXO in the developinglarvae under the control of the ActGal4 driver[44]. This resulted in complete developmentalarrest of the larvae, which remained as first instar for up to 7days (Figure 2A),similar to the life expectancy of starved larvae [16-18].This trend was also seen using a constitutively active version of Murine Foxo1(mFoxo1) containing an alanine substitution at the T1 (T24A), andS1 (S253A) Akt phosphorylation sites (mFoxo1-AA) [45] (Figure 2A).In addition, larvae expressing dFOXO and mFoxo1-AA were often foundto be wandering far from their food supply. We monitored feedingbehavior by assessing the number of larvae away from their foodat 48 and 72 hours after egg laying (AEL). Larvae expressing dFOXOand mFoxo1-AA showed a 3–4 fold increase in wandering over larvaeexpressing Gal4 alone (Figure 2B).Thus, dFOXO expression drastically alters feeding behavior and isable to induce a starvation type response in larvae which have anadequate food supply.

Bottom Line: Analysis of the wings and eyes of these small flies indicates that the reduction in size is due to decreases in cell size and cell number.Overexpression of dFOXO in the developing eye leads to a characteristic phenotype with reductions in cell size and cell number.This phenotype can be rescued by co-expression of upstream insulin signaling components, dPI3K and dAkt, however, this rescue is not seen when FOXO is mutated to a constitutively active form. dFOXO is conserved in both sequence and regulatory mechanisms when compared with other FOXO homologues.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, Memorial University of Newfoundland, St, John's, Newfoundland, (A1B 3X9), Canada. x04jmk@mun.ca

ABSTRACT

Background: Components of the insulin signaling pathway are important regulators of growth. The FOXO (forkhead box, sub-group "O") transcription factors regulate cellular processes under conditions of low levels of insulin signaling. Studies in mammalian cell culture show that activation of FOXO transcription factors causes cell death or cell cycle arrest. The Caenorhabditis elegans homologue of FOXO, Daf-16, is required for the formation of dauer larvae in response to nutritional stress. In addition, FOXO factors have been implicated in stress resistance and longevity.

Results: We have identified the Drosophila melanogaster homologue of FOXO (dFOXO), which is conserved in amino acid sequence compared with the mammalian FOXO homologues and Daf-16. Expression of dFOXO during early larval development causes inhibition of larval growth and alterations in feeding behavior. Inhibition of larval growth is reversible upon discontinuation of dFOXO expression. Expression of dFOXO during the third larval instar or at low levels during development leads to the generation of adults that are reduced in size. Analysis of the wings and eyes of these small flies indicates that the reduction in size is due to decreases in cell size and cell number. Overexpression of dFOXO in the developing eye leads to a characteristic phenotype with reductions in cell size and cell number. This phenotype can be rescued by co-expression of upstream insulin signaling components, dPI3K and dAkt, however, this rescue is not seen when FOXO is mutated to a constitutively active form.

Conclusions: dFOXO is conserved in both sequence and regulatory mechanisms when compared with other FOXO homologues. The establishment of Drosophila as a model for the study of FOXO transcription factors should prove beneficial to determining the biological role of these signaling molecules. The alterations in larval development seen upon overexpression of dFOXO closely mimic the phenotypic effects of starvation, suggesting a role for dFOXO in the response to nutritional adversity. This work has implications in the understanding of cancer and insulin related disorders, such as diabetes and obesity.

Show MeSH
Related in: MedlinePlus