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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 reduces growth through alterations in cell sizeand cell number (A) Expression of UAS-dFOXO in the thirdlarval instar produces small flies (left) when compared to controls(right). w; hsGal4/CyO flies were crossed to w; UAS-dFOXO/UAS-dFOXO fliesand the progeny were heat shocked at 37°C for 4 hours during theearly third instar. (B) Flies of the genotype w; hsGal4/+; UAS-dFOXO/+ (left)were smaller than w; hsGal4/+ (right) flies when raisedat 29°C. (C) The wings of w; hsGal4/+; UAS-dFOXO/+ fliesraised at 29°C were smaller than control wings (scale bar = 1 mm).(D) Flies expressing dFOXO (red bars) also showed a significant reductionin body weight, wing area, cell number, and cell size when comparedto control flies (grey bars) (p = 0.005). (E) Flies expressing dFOXOhad smaller eyes than control flies (scale bar = 150 μm), and (F)their eyes were reduced in both the number of ommatidia and thearea of the ommatidia (red bars) when compared to controls (greybars). Genotypes are; (A-left, B-left, C-top, D-red bars, E-left,F-red bars) w; hsGal4/+; UAS-dFOXO/+, (A-right, B-right,C-bottom, D-grey bars, E-right, F-grey bars).  w; hs-Gal4/+.
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Figure 3: dFOXO reduces growth through alterations in cell sizeand cell number (A) Expression of UAS-dFOXO in the thirdlarval instar produces small flies (left) when compared to controls(right). w; hsGal4/CyO flies were crossed to w; UAS-dFOXO/UAS-dFOXO fliesand the progeny were heat shocked at 37°C for 4 hours during theearly third instar. (B) Flies of the genotype w; hsGal4/+; UAS-dFOXO/+ (left)were smaller than w; hsGal4/+ (right) flies when raisedat 29°C. (C) The wings of w; hsGal4/+; UAS-dFOXO/+ fliesraised at 29°C were smaller than control wings (scale bar = 1 mm).(D) Flies expressing dFOXO (red bars) also showed a significant reductionin body weight, wing area, cell number, and cell size when comparedto control flies (grey bars) (p = 0.005). (E) Flies expressing dFOXOhad smaller eyes than control flies (scale bar = 150 μm), and (F)their eyes were reduced in both the number of ommatidia and thearea of the ommatidia (red bars) when compared to controls (greybars). Genotypes are; (A-left, B-left, C-top, D-red bars, E-left,F-red bars) w; hsGal4/+; UAS-dFOXO/+, (A-right, B-right,C-bottom, D-grey bars, E-right, F-grey bars). w; hs-Gal4/+.

Mentions: Expression of dFOXO in the third instar larvae caused significantlethality, however, rare flies that did survive were much smallerthan control flies (Figure 3A),showing a phenotype similar to that caused by mutations in chico [12], dAkt [14] and dInr [6,9].Expression of dFOXO under the control of the ubiquitouslow level Gal4 drivers, armadillo-Gal4, and hsGal4 (raisedat 25°C with no heat shock) had very little effect on growth (datanot shown). In contrast, increasing expression of dFOXO usingthe hsGal4 driver in flies raised at 29°C lead to the developmentof small adults, which were approximately half the weight of controlflies (Figures 3B and 3D). Analysis of thewings of these flies showed that the wing area was reduced by nearlyone third and that this reduction was due to a decrease in bothcell size and cell number (Figures 3C and 3D). SEM analysis ofthe eyes revealed reductions in both ommatidia number and ommatidiaarea, which reflect cell number and cell size, respectively (Figures 3E and 3F). These results implicatedFOXO in the control of body size through alterations in cell sizeand cell number.


Expression of Drosophila FOXO regulates growth and can phenocopy starvation.

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

dFOXO reduces growth through alterations in cell sizeand cell number (A) Expression of UAS-dFOXO in the thirdlarval instar produces small flies (left) when compared to controls(right). w; hsGal4/CyO flies were crossed to w; UAS-dFOXO/UAS-dFOXO fliesand the progeny were heat shocked at 37°C for 4 hours during theearly third instar. (B) Flies of the genotype w; hsGal4/+; UAS-dFOXO/+ (left)were smaller than w; hsGal4/+ (right) flies when raisedat 29°C. (C) The wings of w; hsGal4/+; UAS-dFOXO/+ fliesraised at 29°C were smaller than control wings (scale bar = 1 mm).(D) Flies expressing dFOXO (red bars) also showed a significant reductionin body weight, wing area, cell number, and cell size when comparedto control flies (grey bars) (p = 0.005). (E) Flies expressing dFOXOhad smaller eyes than control flies (scale bar = 150 μm), and (F)their eyes were reduced in both the number of ommatidia and thearea of the ommatidia (red bars) when compared to controls (greybars). Genotypes are; (A-left, B-left, C-top, D-red bars, E-left,F-red bars) w; hsGal4/+; UAS-dFOXO/+, (A-right, B-right,C-bottom, D-grey bars, E-right, F-grey bars).  w; hs-Gal4/+.
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Figure 3: dFOXO reduces growth through alterations in cell sizeand cell number (A) Expression of UAS-dFOXO in the thirdlarval instar produces small flies (left) when compared to controls(right). w; hsGal4/CyO flies were crossed to w; UAS-dFOXO/UAS-dFOXO fliesand the progeny were heat shocked at 37°C for 4 hours during theearly third instar. (B) Flies of the genotype w; hsGal4/+; UAS-dFOXO/+ (left)were smaller than w; hsGal4/+ (right) flies when raisedat 29°C. (C) The wings of w; hsGal4/+; UAS-dFOXO/+ fliesraised at 29°C were smaller than control wings (scale bar = 1 mm).(D) Flies expressing dFOXO (red bars) also showed a significant reductionin body weight, wing area, cell number, and cell size when comparedto control flies (grey bars) (p = 0.005). (E) Flies expressing dFOXOhad smaller eyes than control flies (scale bar = 150 μm), and (F)their eyes were reduced in both the number of ommatidia and thearea of the ommatidia (red bars) when compared to controls (greybars). Genotypes are; (A-left, B-left, C-top, D-red bars, E-left,F-red bars) w; hsGal4/+; UAS-dFOXO/+, (A-right, B-right,C-bottom, D-grey bars, E-right, F-grey bars). w; hs-Gal4/+.
Mentions: Expression of dFOXO in the third instar larvae caused significantlethality, however, rare flies that did survive were much smallerthan control flies (Figure 3A),showing a phenotype similar to that caused by mutations in chico [12], dAkt [14] and dInr [6,9].Expression of dFOXO under the control of the ubiquitouslow level Gal4 drivers, armadillo-Gal4, and hsGal4 (raisedat 25°C with no heat shock) had very little effect on growth (datanot shown). In contrast, increasing expression of dFOXO usingthe hsGal4 driver in flies raised at 29°C lead to the developmentof small adults, which were approximately half the weight of controlflies (Figures 3B and 3D). Analysis of thewings of these flies showed that the wing area was reduced by nearlyone third and that this reduction was due to a decrease in bothcell size and cell number (Figures 3C and 3D). SEM analysis ofthe eyes revealed reductions in both ommatidia number and ommatidiaarea, which reflect cell number and cell size, respectively (Figures 3E and 3F). These results implicatedFOXO in the control of body size through alterations in cell sizeand cell number.

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