Expression of Drosophila FOXO regulates growth and can phenocopy starvation - PubMed (original) (raw)

Comparative Study

Expression of Drosophila FOXO regulates growth and can phenocopy starvation

Jamie M Kramer et al. BMC Dev Biol. 2003.

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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Figures

Figure 1

dFOXO encodes a protein that retains important functional domains found in other FOXO homologues. (A) Schematic representation of the dFOXO cDNA clone LD05569 and its location in the genomic scaffolding, region AE003703, of the BDGP sequence. (B) ClustalW alignment of the proposed dFOXO amino acid sequence with that of mammalian homologues (FOXO1a, FOXO3a, and FOXO4) and Daf-16a1. Highlighted are: the T1, S1, and S2 Akt target sequences (yellow shading); the potential DYRK1a/mnb phosphorylation site (arrow, and grey shading); and the forkhead box DNA binding domain (black box). "*" indicates nucleotides that are identical in all sequences in the alignment, ":" indicates conserved substitutions, according to the chemical nature of the amino acids, and "." indicates semi-conserved substitutions. Colors indicate the chemical nature of the amino acid; Red = small hydrophobic (including aromatic), Blue = Acidic, Magenta = Basic, and Green = basic amino acids with hydroxyl groups and/or amine groups.

Figure 2

Expression of dFOXO in first instar larvae phenocopies starvation and effects feeding behavior. Expression of dFOXO and mFOXO1-AA early in larval development using the (A) ActGal4 and (C) hsGal4 driver lines leads to developmental arrest similar to that seen in starved larvae. Developmentally arrested larvae are capable of surviving for up to seven days after egg laying (AEL). (B) Expression of dFOXO (red bars) and mFOXO1-AA (green bars) leads to alterations in feeding behavior when compared to controls (grey bars). The percentage of wandering larvae is significantly greater in larvae expressing dFOXO and mFOXO1-AA at 48 hours and 72 hours AEL (p = 0.05). Expression of dPI3K-DN (blue bars) did not increase larval wandering. (D) Developmental arrest is reversible upon removal of dFOXO expression (red bars), but not upon removal of mFOXO1-AA expression (green bars). Grey bars represent the controls. Each bar reflects the average of three separate trials, with 50 larvae per 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-grey bars) 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.

Figure 3

dFOXO reduces growth through alterations in cell size and cell number (A) Expression of UAS-dFOXO in the third larval instar produces small flies (left) when compared to controls (right). w; hsGal4/CyO flies were crossed to w; UAS-dFOXO/UAS-dFOXO flies and the progeny were heat shocked at 37°C for 4 hours during the early third instar. (B) Flies of the genotype w; hsGal4/+; UAS-dFOXO/+ (left) were smaller than w; hsGal4/+ (right) flies when raised at 29°C. (C) The wings of w; hsGal4/+; UAS-dFOXO/+ flies raised at 29°C were smaller than control wings (scale bar = 1 mm). (D) Flies expressing dFOXO (red bars) also showed a significant reduction in body weight, wing area, cell number, and cell size when compared to control flies (grey bars) (p = 0.005). (E) Flies expressing dFOXO had smaller eyes than control flies (scale bar = 150 μm), and (F) their eyes were reduced in both the number of ommatidia and the area of the ommatidia (red bars) when compared to controls (grey bars). 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/+.

Figure 4

Regulation of dFOXO through insulin signaling is conserved between mammals and flies. The GMR-Gal4 driver was used to drive the expression of (B) dPI3K-DN, (C) wild type dPI3K, (D) dAkt, (E) dFOXO, (I) mFoxo1, and (M) mFoxo1-AA, both alone and in various combinations (F-H, J-L, N-P) as indicated through the rows 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.

Figure 5

dFOXO inactivation is essential for dAkt, but not dPI3K, mediated increases in cell size. Ommatidia area was measured as a means to determine the effect of FOXO overexpression on cell size. Expression of dFOXO (bar 2), mFoxo1 (bar 3), and mFoxo1-AA (bar 4) under the control of GMR-Gal4 causes a significant decrease in ommatidia area when compared to the expression of Gal4 alone (bar 1). In addition, GMR-Gal4 was used to drive the expression of dPI3K (bars 5–8), and UAS-dAkt (bars 9–12), either alone (grey bars), or in the presence of UAS-dFOXO (red bars), UAS-mFoxo1 (light green bars), or UAS-mFoxo1-AA (dark green bars). Two sided t-tests were preformed to determine statistical significance (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.

Figure 6

dFOXO responds to dRas2 signaling, but not to inhibitors of apoptosis. GMR-Gal4 was used to drive the expression of UAS-dFOXO (A) alone, and in the presence of (B) UAS-p35, (D) UAS-dEGFR, (F) UAS-Ras2 _V_14. UAS-Ras2 _V_14 was also expressed in combination with UAS-mFoxo1 (G) and UAS-mFoxo1-AA (H). Scale bars equal 150 μm. Genotypes are: (A) w; GMR-Gal4/+; UAS-dFOXO/+, (B) w; GMR-Gal4/UAS-p35; UAS-dFOXO/+, (C) w; GMR-Gal4/UAS-dEGFR, (D) w; GMR-Gal4/UAS-dEGFR; UAS-dFOXO/+, (E) w; GMR-Gal4/UAS-Ras V_14, (F) w; GMR-Gal4/UAS-Ras2 V_14; UAS-dFOXO/+, (G) w; GMR-Gal4, UAS-mFoxo1/UAS-Ras2 _V_14, and (H) w, UAS-mFoxo1-AA/w; GMR-Gal4/ UAS-Ras2 _V_14.

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Expression of Drosophila FOXO regulates growth and can phenocopy starvation - PubMed (original) (raw)