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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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Regulation of dFOXO through insulin signaling is conservedbetween mammals and flies. The GMR-Gal4 driver wasused to drive the expression of (B) dPI3K-DN, (C) wild type dPI3K,(D) dAkt, (E) dFOXO, (I) mFoxo1, and (M) mFoxo1-AA, both alone andin various combinations (F-H, J-L, N-P) as indicated through therows and columns in the figure (scale bar = 150 μm). Genotypes are:(A) w; GMR-Gal4/+, (B) w; UAS-dPI3K-DN/GMR-Gal4,(C) w; UAS-dPI3K/GMR-Gal4, (D) w; UAS-dAkt/GMR-Gal4,(E) w; GMR-Gal4/+; UAS-dFOXO/+, (F) w; UAS-dPI3K-DN/GMR-Gal4;UAS-dFOXO/+, (G) w; UAS-dPI3K/GMR-Gal4; UAS-dFOXO/+,(H) w; UAS-dAkt/GMR-Gal4; UAS-dFOXO/+ (I) w; GMR-Gal4,UAS-mFoxo1/+, (J) w; GMR-Gal4, UAS-mFoxo1/UAS-dPI3K-DN,(K) w; GMR-Gal4, UAS-mFoxo1/UAS-dPI3K, (L) w; GMR-Gal4,UAS-mFoxo1/UAS-dAkt, (M) w, UAS-mFoxo1-AA/w; GMR-Gal4/+,(N) w, UAS-mFoxo1-AA/w; GMR-Gal4/UAS-dPI3K-DN, (O) w,UAS-mFoxo1-AA/w; GMR-Gal4/UAS-dPI3K, (P) w, UAS-mFoxo1-AA/w;GMR-Gal4/UAS-dAkt.
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Figure 4: Regulation of dFOXO through insulin signaling is conservedbetween mammals and flies. The GMR-Gal4 driver wasused to drive the expression of (B) dPI3K-DN, (C) wild type dPI3K,(D) dAkt, (E) dFOXO, (I) mFoxo1, and (M) mFoxo1-AA, both alone andin various combinations (F-H, J-L, N-P) as indicated through therows and columns in the figure (scale bar = 150 μm). Genotypes are:(A) w; GMR-Gal4/+, (B) w; UAS-dPI3K-DN/GMR-Gal4,(C) w; UAS-dPI3K/GMR-Gal4, (D) w; UAS-dAkt/GMR-Gal4,(E) w; GMR-Gal4/+; UAS-dFOXO/+, (F) w; UAS-dPI3K-DN/GMR-Gal4;UAS-dFOXO/+, (G) w; UAS-dPI3K/GMR-Gal4; UAS-dFOXO/+,(H) w; UAS-dAkt/GMR-Gal4; UAS-dFOXO/+ (I) w; GMR-Gal4,UAS-mFoxo1/+, (J) w; GMR-Gal4, UAS-mFoxo1/UAS-dPI3K-DN,(K) w; GMR-Gal4, UAS-mFoxo1/UAS-dPI3K, (L) w; GMR-Gal4,UAS-mFoxo1/UAS-dAkt, (M) w, UAS-mFoxo1-AA/w; GMR-Gal4/+,(N) w, UAS-mFoxo1-AA/w; GMR-Gal4/UAS-dPI3K-DN, (O) w,UAS-mFoxo1-AA/w; GMR-Gal4/UAS-dPI3K, (P) w, UAS-mFoxo1-AA/w;GMR-Gal4/UAS-dAkt.

Mentions: When dFOXO is expressed in the developing eye under the controlof the GMR-Gal4 driver[50],the eye is smaller, lacking many ommatidia and nearly all of themechanosensory bristles (Figure 4E).The remaining ommatidia are arranged in the typical hexahedral arrayand cross sectional analysis revealed that all of the normal photoreceptorcells are present (Figure 4E,data not shown). Thus, it appears that dFOXO expression causesa reduction in the number of cells but does not interfere with cellulardifferentiation and the organization of the ommatidia themselves.We have used this eye phenotype to test for interactions betweendFOXO and other components of the insulin signaling pathway.


Expression of Drosophila FOXO regulates growth and can phenocopy starvation.

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

Regulation of dFOXO through insulin signaling is conservedbetween mammals and flies. The GMR-Gal4 driver wasused to drive the expression of (B) dPI3K-DN, (C) wild type dPI3K,(D) dAkt, (E) dFOXO, (I) mFoxo1, and (M) mFoxo1-AA, both alone andin various combinations (F-H, J-L, N-P) as indicated through therows and columns in the figure (scale bar = 150 μm). Genotypes are:(A) w; GMR-Gal4/+, (B) w; UAS-dPI3K-DN/GMR-Gal4,(C) w; UAS-dPI3K/GMR-Gal4, (D) w; UAS-dAkt/GMR-Gal4,(E) w; GMR-Gal4/+; UAS-dFOXO/+, (F) w; UAS-dPI3K-DN/GMR-Gal4;UAS-dFOXO/+, (G) w; UAS-dPI3K/GMR-Gal4; UAS-dFOXO/+,(H) w; UAS-dAkt/GMR-Gal4; UAS-dFOXO/+ (I) w; GMR-Gal4,UAS-mFoxo1/+, (J) w; GMR-Gal4, UAS-mFoxo1/UAS-dPI3K-DN,(K) w; GMR-Gal4, UAS-mFoxo1/UAS-dPI3K, (L) w; GMR-Gal4,UAS-mFoxo1/UAS-dAkt, (M) w, UAS-mFoxo1-AA/w; GMR-Gal4/+,(N) w, UAS-mFoxo1-AA/w; GMR-Gal4/UAS-dPI3K-DN, (O) w,UAS-mFoxo1-AA/w; GMR-Gal4/UAS-dPI3K, (P) w, UAS-mFoxo1-AA/w;GMR-Gal4/UAS-dAkt.
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Figure 4: Regulation of dFOXO through insulin signaling is conservedbetween mammals and flies. The GMR-Gal4 driver wasused to drive the expression of (B) dPI3K-DN, (C) wild type dPI3K,(D) dAkt, (E) dFOXO, (I) mFoxo1, and (M) mFoxo1-AA, both alone andin various combinations (F-H, J-L, N-P) as indicated through therows and columns in the figure (scale bar = 150 μm). Genotypes are:(A) w; GMR-Gal4/+, (B) w; UAS-dPI3K-DN/GMR-Gal4,(C) w; UAS-dPI3K/GMR-Gal4, (D) w; UAS-dAkt/GMR-Gal4,(E) w; GMR-Gal4/+; UAS-dFOXO/+, (F) w; UAS-dPI3K-DN/GMR-Gal4;UAS-dFOXO/+, (G) w; UAS-dPI3K/GMR-Gal4; UAS-dFOXO/+,(H) w; UAS-dAkt/GMR-Gal4; UAS-dFOXO/+ (I) w; GMR-Gal4,UAS-mFoxo1/+, (J) w; GMR-Gal4, UAS-mFoxo1/UAS-dPI3K-DN,(K) w; GMR-Gal4, UAS-mFoxo1/UAS-dPI3K, (L) w; GMR-Gal4,UAS-mFoxo1/UAS-dAkt, (M) w, UAS-mFoxo1-AA/w; GMR-Gal4/+,(N) w, UAS-mFoxo1-AA/w; GMR-Gal4/UAS-dPI3K-DN, (O) w,UAS-mFoxo1-AA/w; GMR-Gal4/UAS-dPI3K, (P) w, UAS-mFoxo1-AA/w;GMR-Gal4/UAS-dAkt.
Mentions: When dFOXO is expressed in the developing eye under the controlof the GMR-Gal4 driver[50],the eye is smaller, lacking many ommatidia and nearly all of themechanosensory bristles (Figure 4E).The remaining ommatidia are arranged in the typical hexahedral arrayand cross sectional analysis revealed that all of the normal photoreceptorcells are present (Figure 4E,data not shown). Thus, it appears that dFOXO expression causesa reduction in the number of cells but does not interfere with cellulardifferentiation and the organization of the ommatidia themselves.We have used this eye phenotype to test for interactions betweendFOXO and other components of the insulin signaling pathway.

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