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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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dFOXO inactivation is essential for dAkt, but not dPI3K,mediated increases in cell size. Ommatidia area was measuredas a means to determine the effect of FOXO overexpression on cellsize. Expression of dFOXO (bar 2), mFoxo1 (bar 3), and mFoxo1-AA(bar 4) under the control of GMR-Gal4 causes a significantdecrease in ommatidia area when compared to the expression of Gal4alone (bar 1). In addition, GMR-Gal4 was used to drivethe expression of dPI3K (bars 5–8), and UAS-dAkt (bars9–12), either alone (grey bars), or in the presence of UAS-dFOXO (redbars), UAS-mFoxo1 (light green bars), or UAS-mFoxo1-AA (darkgreen bars). Two sided t-tests were preformed to determine statisticalsignificance (p = 0.001). Genotypes are: (1) w; GMR-Gal4/+,(2) w; GMR-Gal4/+; UAS-dFOXO/+, (3) w; GMR-Gal4, UAS-mFoxo1/+,(4) w, UAS-mFoxo1-AA/w; GMR-Gal4/+, (5) w; UAS-dPI3K/GMR-Gal4,(6) w; UAS-dPI3K/ GMR-Gal4; UAS-dFOXO/+, (7) w; GMR-Gal4,UAS-mFoxo1/UAS-dPI3K, (8) w, UAS-mFoxo1-AA/w; GMR-Gal4/UAS-dPI3K,(9) w; UAS-dAkt/GMR-Gal4, (10) w; UAS-dAkt/GMR-Gal4;UAS-dFOXO/+ (11) w; GMR-Gal4, UAS-mFoxo1/UAS-dAkt (12) w,UAS-mFoxo1-AA/w; GMR-Gal4/UAS-dAkt.
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Figure 5: dFOXO inactivation is essential for dAkt, but not dPI3K,mediated increases in cell size. Ommatidia area was measuredas a means to determine the effect of FOXO overexpression on cellsize. Expression of dFOXO (bar 2), mFoxo1 (bar 3), and mFoxo1-AA(bar 4) under the control of GMR-Gal4 causes a significantdecrease in ommatidia area when compared to the expression of Gal4alone (bar 1). In addition, GMR-Gal4 was used to drivethe expression of dPI3K (bars 5–8), and UAS-dAkt (bars9–12), either alone (grey bars), or in the presence of UAS-dFOXO (redbars), UAS-mFoxo1 (light green bars), or UAS-mFoxo1-AA (darkgreen bars). Two sided t-tests were preformed to determine statisticalsignificance (p = 0.001). Genotypes are: (1) w; GMR-Gal4/+,(2) w; GMR-Gal4/+; UAS-dFOXO/+, (3) w; GMR-Gal4, UAS-mFoxo1/+,(4) w, UAS-mFoxo1-AA/w; GMR-Gal4/+, (5) w; UAS-dPI3K/GMR-Gal4,(6) w; UAS-dPI3K/ GMR-Gal4; UAS-dFOXO/+, (7) w; GMR-Gal4,UAS-mFoxo1/UAS-dPI3K, (8) w, UAS-mFoxo1-AA/w; GMR-Gal4/UAS-dPI3K,(9) w; UAS-dAkt/GMR-Gal4, (10) w; UAS-dAkt/GMR-Gal4;UAS-dFOXO/+ (11) w; GMR-Gal4, UAS-mFoxo1/UAS-dAkt (12) w,UAS-mFoxo1-AA/w; GMR-Gal4/UAS-dAkt.

Mentions: The constitutively active mFoxo1-AA construct [45] was also expressed in the developingeye. Expression of this construct causes a phenotype similar tothat of dFOXO and mFoxo1, with characteristic lack of ommatidiaand mechanosensory bristles (Figure 4M).When mFoxo1-AA is co-expressed with dPI3K-DN the eye is nearly obliterated(Figure 4N),as seen with dFOXO and mFoxo1 (Figures 4F and 4J).Co-expression of mFoxo1-AA with dPI3K leads to a partial rescueof the phenotype, with still an obvious lack of ommatidia and mechanosensorybristles (Figure 4O).In contrast, co-expression of mFoxo1-AA with dAkt does not causerescue of the ommatidia or mechanosensory bristles (Figure 4P), indicating thatthis construct is not responsive to dAkt signaling. The partial rescueof the dFOXO phenotype by dPI3K appears to be mediated through alterationsin cell size (Figure 5) rather thancell number, as there is still an obvious lack of ommatidia andmechanosensory bristles (Figure 4O). Thisdata indicates that inactivation of dFOXO is required for the fulleffects of growth mediated by dPI3K and dAkt.


Expression of Drosophila FOXO regulates growth and can phenocopy starvation.

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

dFOXO inactivation is essential for dAkt, but not dPI3K,mediated increases in cell size. Ommatidia area was measuredas a means to determine the effect of FOXO overexpression on cellsize. Expression of dFOXO (bar 2), mFoxo1 (bar 3), and mFoxo1-AA(bar 4) under the control of GMR-Gal4 causes a significantdecrease in ommatidia area when compared to the expression of Gal4alone (bar 1). In addition, GMR-Gal4 was used to drivethe expression of dPI3K (bars 5–8), and UAS-dAkt (bars9–12), either alone (grey bars), or in the presence of UAS-dFOXO (redbars), UAS-mFoxo1 (light green bars), or UAS-mFoxo1-AA (darkgreen bars). Two sided t-tests were preformed to determine statisticalsignificance (p = 0.001). Genotypes are: (1) w; GMR-Gal4/+,(2) w; GMR-Gal4/+; UAS-dFOXO/+, (3) w; GMR-Gal4, UAS-mFoxo1/+,(4) w, UAS-mFoxo1-AA/w; GMR-Gal4/+, (5) w; UAS-dPI3K/GMR-Gal4,(6) w; UAS-dPI3K/ GMR-Gal4; UAS-dFOXO/+, (7) w; GMR-Gal4,UAS-mFoxo1/UAS-dPI3K, (8) w, UAS-mFoxo1-AA/w; GMR-Gal4/UAS-dPI3K,(9) w; UAS-dAkt/GMR-Gal4, (10) w; UAS-dAkt/GMR-Gal4;UAS-dFOXO/+ (11) w; GMR-Gal4, UAS-mFoxo1/UAS-dAkt (12) w,UAS-mFoxo1-AA/w; GMR-Gal4/UAS-dAkt.
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Figure 5: dFOXO inactivation is essential for dAkt, but not dPI3K,mediated increases in cell size. Ommatidia area was measuredas a means to determine the effect of FOXO overexpression on cellsize. Expression of dFOXO (bar 2), mFoxo1 (bar 3), and mFoxo1-AA(bar 4) under the control of GMR-Gal4 causes a significantdecrease in ommatidia area when compared to the expression of Gal4alone (bar 1). In addition, GMR-Gal4 was used to drivethe expression of dPI3K (bars 5–8), and UAS-dAkt (bars9–12), either alone (grey bars), or in the presence of UAS-dFOXO (redbars), UAS-mFoxo1 (light green bars), or UAS-mFoxo1-AA (darkgreen bars). Two sided t-tests were preformed to determine statisticalsignificance (p = 0.001). Genotypes are: (1) w; GMR-Gal4/+,(2) w; GMR-Gal4/+; UAS-dFOXO/+, (3) w; GMR-Gal4, UAS-mFoxo1/+,(4) w, UAS-mFoxo1-AA/w; GMR-Gal4/+, (5) w; UAS-dPI3K/GMR-Gal4,(6) w; UAS-dPI3K/ GMR-Gal4; UAS-dFOXO/+, (7) w; GMR-Gal4,UAS-mFoxo1/UAS-dPI3K, (8) w, UAS-mFoxo1-AA/w; GMR-Gal4/UAS-dPI3K,(9) w; UAS-dAkt/GMR-Gal4, (10) w; UAS-dAkt/GMR-Gal4;UAS-dFOXO/+ (11) w; GMR-Gal4, UAS-mFoxo1/UAS-dAkt (12) w,UAS-mFoxo1-AA/w; GMR-Gal4/UAS-dAkt.
Mentions: The constitutively active mFoxo1-AA construct [45] was also expressed in the developingeye. Expression of this construct causes a phenotype similar tothat of dFOXO and mFoxo1, with characteristic lack of ommatidiaand mechanosensory bristles (Figure 4M).When mFoxo1-AA is co-expressed with dPI3K-DN the eye is nearly obliterated(Figure 4N),as seen with dFOXO and mFoxo1 (Figures 4F and 4J).Co-expression of mFoxo1-AA with dPI3K leads to a partial rescueof the phenotype, with still an obvious lack of ommatidia and mechanosensorybristles (Figure 4O).In contrast, co-expression of mFoxo1-AA with dAkt does not causerescue of the ommatidia or mechanosensory bristles (Figure 4P), indicating thatthis construct is not responsive to dAkt signaling. The partial rescueof the dFOXO phenotype by dPI3K appears to be mediated through alterationsin cell size (Figure 5) rather thancell number, as there is still an obvious lack of ommatidia andmechanosensory bristles (Figure 4O). Thisdata indicates that inactivation of dFOXO is required for the fulleffects of growth mediated by dPI3K and dAkt.

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