The Drosophila FoxA ortholog Fork head regulates growth and gene expression downstream of Target of rapamycin

Abstract

Forkhead transcription factors of the FoxO subfamily regulate gene expression programs downstream of the insulin signaling network. It is less clear which proteins mediate transcriptional control exerted by Target of rapamycin (TOR) signaling, but recent studies in nematodes suggest a role for FoxA transcription factors downstream of TOR. In this study we present evidence that outlines a similar connection in Drosophila, in which the FoxA protein Fork head (FKH) regulates cellular and organismal size downstream of TOR. We find that ectopic expression and targeted knockdown of FKH in larval tissues elicits different size phenotypes depending on nutrient state and TOR signaling levels. FKH overexpression has a negative effect on growth under fed conditions, and this phenotype is not further exacerbated by inhibition of TOR via rapamycin feeding. Under conditions of starvation or low TOR signaling levels, knockdown of FKH attenuates the size reduction associated with these conditions. Subcellular localization of endogenous FKH protein is shifted from predominantly cytoplasmic on a high-protein diet to a pronounced nuclear accumulation in animals with reduced levels of TOR or fed with rapamycin. Two putative FKH target genes, CG6770 and cabut, are transcriptionally induced by rapamycin or FKH expression, and silenced by FKH knockdown. Induction of both target genes in heterozygous TOR mutant animals is suppressed by mutations in fkh. Furthermore, TOR signaling levels and FKH impact on transcription of the dFOXO target gene d4E-BP, implying a point of crosstalk with the insulin pathway. In summary, our observations show that an alteration of FKH levels has an effect on cellular and organismal size, and that FKH function is required for the growth inhibition and target gene induction caused by low TOR signaling levels.

Figure 1. FKH levels influence larval body size.

Pictures show 72 h old larvae (±2h) which were treated at 24 h after egg deposition (AED) with 0 (A, C, E, G, I) or 50 µM (B, D, F, H, J) rapamycin for 48 h. A, B: OregonR wildtype control. C, D: y w UAS-LacZ-RNAi; arm-Gal4/+. Treating control larvae with 50 µM rapamycin leads to severe reduction of growth. E, F: w; arm-Gal4/+; UAS-FKH-RNAi/+. Untreated larvae are slightly smaller than controls, but size reduction by rapamycin feeding is less pronounced in larvae with low FKH levels. G, H: w;; ppl-Gal4/UAS-LacZ. I, J: w;; ppl-Gal4/UAS-FKH. Overexpression of FKH leads to a severe reduction in body size similar to that resulting from rapamycin feeding. At 25°C, overexpression furthermore leads to larval lethality in the 2^nd instar. Rapamycin feeding of FKH-overexpressing larvae has almost no additive effect to the reduction in body size. (K) Quantitation of larval body size shows that rapamycin-treated larvae with low FKH levels are significantly larger than control larvae and that untreated larvae with high FKH levels are significantly smaller than control larvae. Significance was tested using an unpaired 2-tailed Student's t-test. *** = p<0.001. Error bars represent standard error of the mean (SEM).

Figure 2. FKH levels influence cell size in the larval fatbody.

Pictures show confocal sections of larval fatbodies, using the fly line y w hs-flp;; Act>CD2>Gal4 UAS-GFP to drive UAS-FKH-RNAi, overexpression and control responder lines. Cells expressing the transgene are marked by the co-expression of GFP, whereas the non-fluorescent serve as wild-type controls within the same tissue sample. Larvae were reared on yeast for 64 h AED, or starved on PBS for 24 h after growing on yeast for 64 h, or treated with rapamycin for 24 h after growing on yeast for 48 h. Tissue was stained with α-GFP (green), α-CD2 (red) and DAPI (blue). (A–C, G–I) Controls show no obvious phenotype in the GFP-positive cells. (D–F) Cells expressing FKH RNAi have no growth phenotype under fed conditions, but are larger than the surrounding tissue in starved and rapamycin-treated larvae. (J–L) Cells overexpressing FKH are smaller than the surrounding tissue under fed conditions.

Figure 3. Quantitation of the cell size phenotypes shown in figure 2.

Cell size was analyzed by measuring the cell perimeter with the software ImageJ. Blue bars represent the GFP-negative wild-type cells, red bars represent the cells expressing the transgene. Larvae were fed with yeast paste, starved on PBS or treated with 50 µM rapamycin. (A) Effect of FKH knockdown on cell size. Larvae were of the genotype y w hs-flp;; Act>CD2>Gal4 UAS-GFP/UAS-FKH-RNAi (bars labeled “FKH-RNAi”) or y w hs-flp;; Act>CD2>Gal4 UAS-GFP/UAS-LacZ-RNAi (bars labeled “LacZ-RNAi”) as an unspecific control. Cells expressing FKH dsRNA are significantly larger than the surrounding tissue in larvae which were starved on PBS or treated with rapamycin. (B) Effect of FKH overexpression on cell size. Larvae were of the genotype y w hs-flp;; Act>CD2>Gal4 UAS-GFP/UAS-FKH (bars labeled “UAS-FKH”) or y w hs-flp;; Act>CD2>Gal4 UAS-GFP/UAS-LacZ (bars labeled “UAS-LacZ”) as an unspecific control. Cells which overexpress FKH are significantly smaller than the surrounding tissue in fed larvae, but not in larvae starved on PBS. They are also significantly smaller than wild-type cells in larvae treated with rapamycin. Significance was tested using an unpaired 2-tailed Student's t-test. * = p<0.05; ** = p<0.01; *** = p<0.001. Error bars represent SEM. Because the larvae are not age-matched across the different conditions (see Materials and Methods), we base our statements on the comparison of transgene-expressing cells to the wild-type control cells within the same sample, and not on the comparison of absolute cell sizes across different conditions.

Figure 4. Subcellular localization of FKH is modulated by nutrient availability and TOR signaling.

Confocal sections of larval fatbody are shown. For each section, one panel shows only the signal derived from the FKH or dFOXO antibody in green, and a second panel an additional nuclear counterstain with DAPI pseudo-colored in red. (A, B) FKH is excluded from the nucleus in fatbodies of wildtype 2^nd instar larvae fed on yeast. (C, D) FKH accumulates in the nucleus in fatbodies of wildtype larvae treated with 50 µM rapamycin. (E, F) Closeup of A, B. (G, H) FKH is nuclear in heterozygous TOR mutants (y w; dTOR^ΔP/+). (I, J) Nuclear exclusion of FKH is not observed in larvae subjected to starvation on PBS. (K, L) In contrast to FKH, dFOXO shows a clear nuclear localization in wildtype larvae starved on PBS. Scale bar is 20 µm.

Figure 5. Transcription of CG6770 and cabut is regulated by FKH and rapamycin.

Realtime qPCR was performed to quantify mRNAs in larval extracts. (A) Compared to wildtype animals, the putative FKH targets CG6770 and cabut are downregulated in yeast-fed larvae with low FKH levels (w; arm-Gal4/+; UAS-FKH-RNAi/+) and upregulated in larvae overexpressing FKH (w;; ppl-Gal4/UAS-FKH). (B) CG6770 and cabut transcription is induced in wildtype larvae fed with 50 µM rapamycin. (C) CG6770 and cabut mRNA levels are significantly elevated upon rapamycin treatment and in larvae with high FKH levels in comparison to the wildtype and unspecific controls (w;; ppl-Gal4/UAS-LacZ). Rapamycin treatment of larvae with high FKH levels has no significant additive effect on target gene expression. Significance was tested using an unpaired 2-tailed Student's t-test. * = p<0.05; ** = p<0.01; *** = p<0.001. Error bars represent SEM.

Figure 6. FKH is required for the response to rapamycin and lowered TOR levels.

Realtime qPCR was performed to quantify mRNAs in larval extracts. (A) Transcription of CG6770 and cabut is induced upon rapamycin treatment (Rapa) and downregulated in larvae with low FKH levels (w; arm-Gal4/+; UAS-FKH-RNAi/+). Compared to rapamycin-fed wildtype and unspecific control animals (w; arm-Gal4/+; UAS-LacZ-RNAi/+), expression of CG6770 and cabut is significantly lower in rapamycin-fed FKH-RNAi larvae. (B) Consistent with the high expression of CG6770 and cabut in rapamycin-treated larvae, these genes are transcriptionally upregulated in dTOR mutants (y w; dTOR^ΔP/+ and y w; dTOR^ΔP/dTOR^ΔP). The elevated expression is suppressed in larvae transheterozygous for dTOR and FKH (y w; dTOR^ΔP/+; fkh¹/+ and y w; dTOR^ΔP/+; fkh⁶/+), indicating that FKH function is required for target gene induction by low TOR signaling. (C) The body weight of adult flies heterozygous for dTOR^ΔP is increased by the presence of one copy of the fkh¹ allele. Both male and female transheterozygous flies are slightly but significantly heavier than dTOR^ΔP/+ flies. Significance was tested using an unpaired 2-tailed Student's t-test. * = p<0.05; ** = p<0.01; *** = p<0.001. Error bars represent SEM.

Figure 7. FKH and TOR impact on the expression of the dFOXO target d4E-BP.

Realtime qPCR was applied to quantify mRNAs in larval extracts. (A) Compared to wildtype and unspecific control animals, transcript levels of d4E-BP are low in larvae expressing FKH dsRNA (w; arm-Gal4/+; UAS-FKH-RNAi/+) and high in larvae overexpressing FKH (w;; ppl-Gal4/UAS-FKH). d4E-BP is upregulated in larvae treated with rapamycin, and the elevated d4E-BP transcription resulting from rapamycin feeding is completely suppressed by FKH RNAi. (B) d4E-BP is upregulated in dTOR mutants (y w; dTOR^ΔP/+ and y w; dTOR^ΔP/dTOR^ΔP) and the expression is suppressed in larvae transheterozygous for dTOR and FKH mutations (y w; dTOR^ΔP/+; fkh¹/+ and y w; dTOR^ΔP/+; fkh⁶/+). This suggests that d4E-BP is a transcriptional target not only of the insulin pathway and dFOXO, but also of the TOR pathway and FKH. Significance was tested using an unpaired 2-tailed Student's t-test. * = p<0,05 ** = p<0,01 *** = p<0,001. Error bars represent SEM.

Figure 8. A simplified working model of nutrient-dependent gene expression by TOR and FKH.

(A) Under conditions of dietary protein abundance, the TOR signaling module is active and exerts a negative regulation on FKH, which is consequently sequestered in the cytoplasm and unable to modulate gene transcription. (B) When TOR complex 1 activity is inhibited by rapamycin or protein deprivation, the repression of FKH activity is diminished. A significant fraction of the cellular FKH pool accumulates in the nucleus and activates expression of the growth-inhibiting genes CG6770, cabut and d4E-BP.