Juvenile hormone counteracts the bHLH-PAS transcription factors MET and GCE to prevent caspase-dependent programmed cell death in Drosophila

The molting hormone 20-hydroxyecdysone (20E) and juvenile hormone (JH)coordinately control insect development and metamorphosis. Although the molecular mechanism of JH action remains elusive(Riddiford; 2008), a great deal is known about 20E action (Riddiford et al., 2000; Yin and Thummel,2005; Zitnan et al.,2007). The 20E receptor complex is a heterodimer composed of two nuclear proteins, Ecdysone receptor (EcR) and Ultraspiracle (USP). In the absence of 20E, EcR-USP associates with co-repressors, binds to the 20E-response elements, and represses transcription of the 20E primary response genes. After binding 20E to form the 20E-EcR-USP complex, this ligand-receptor complex recruits co-activators and then induces the 20E-triggered transcriptional cascade, which includes the 20E primary and secondary response genes (Gilbert et al., 2000; Riddiford et al., 2003; Palli et al., 2005).

During the larval-pupal metamorphosis of holometabolous insects, larval organs undergo programmed cell death (PCD), including type I PCD apoptosis,type II PCD autophagy and eventually histolysis, whereas the adult progenitor cells undergo cell proliferation, differentiation and organogenesis to give rise to the adult organs (Edgar and Orr-Weaver, 2001; Ward et al.,2003). This process is largely controlled by 20E. The molecular mechanism of how 20E controls larval organ remodeling is relatively well understood in Drosophila (Yin and Thummel, 2005; Neufeld and Baehrecke, 2008). First, the 20E-EcR-USP complex and the 20E primary response genes [including Br-C (broad - FlyBase) E74 (Eip74EF), E75 (Eip75B) and E93 (Eip93F)] induce expression of several 20E secondary response genes that account for PCD, including the caspases Dronc(Nc) and Drice (Ice)(Cakouros et al., 2004; Kilpatrick et al., 2005) and the death activators reaper and Hid (Wrinkled)(Yin and Thummel, 2005). Second, Reaper and HID prevent Dronc and Drice from ubiquitin-regulated protein degradation, and Dronc and Drice activate each other by protein cleavage (Hay and Guo, 2006; Dorstyn and Kumar, 2008). Third, Reaper, HID, Dronc and Drice promote IAP1 (Thread) to undergo ubiquitin-regulated protein degradation, and vice versa(Hay and Guo, 2006). Fourth, E93 is a key determinant of autophagy, partially acting through Dronc (Lee et al.,2000). Last, the 20E signal blocks Phosphotidylinositol 3 kinase(PI3K) and Target of rapamycin (TOR) activity, which in turn inhibits autophagy (Rusten et al.,2004; Columbani et al., 2005). Overall, the initiator caspase Dronc and the effector caspase Drice play important roles in regulating the 20E-induced caspase-dependent PCD in Drosophila.

JH regulates many physiological and developmental events in insects(Riddiford, 1994; Wyatt and Davey, 1996). In the larvae of many insect orders, particularly in Coleoptera, Orthoptera and Lepidoptera, the larval-pupal metamorphosis results from a low titer of JH and a high titer of 20E. In these insects, application of JH, or JH agonists, can prevent normal metamorphic events, resulting in a supernumerary larval molt. For this reason, JH is referred to as the `status quo' hormone(Riddiford, 1994; Riddiford et al., 2003). It has been shown that JH plays an important role by preventing 20E-induced PCD during midgut remodeling of the mosquito Aedes aegypti(Wu et al., 2006; Parthasarathy and Palli,2007b) and the moth Heliothis virescens(Parthasarathy and Palli,2007a). At the molecular level, JH modifies or suppresses the 20E-triggered transcriptional cascade and downregulates caspase genes(Wu et al., 2006; Parthasarathy and Palli,2007a; Parthasarathy and Palli, 2008). In addition, JH can directly affect gene expression independent of 20E (Kethidi et al.,2004; Li et al.,2007). Unfortunately, the molecular mechanism by which JH regulates gene expression remains unknown(Gilbert et al., 2000; Riddiford, 2008).

Methoprene-tolerant (MET) was thought to be a possible JH receptor (JHR). Met encodes a transcription factor of the bHLH-PAS family(Ashok et al., 1988) and Drosophila Met mutants are resistant to the JH agonist methoprene(Wilson and Fabian, 1986). MET forms homodimers or heterodimerizes with its paralog Germ-cell expressed bHLH-PAS (GCE) in a JH-sensitive manner(Godlewski et al., 2006). Global overexpression of Met causes high mortality during larval life(Barry et al., 2008). Although the Met-null mutant is fully viable(Wilson and Ashok, 1998),animals receiving gce RNAi in a Met-null background die during the pupal-adult transition, and overexpressed gce can substitute for Met function (T.G.W., unpublished). It was also reported that Drosophila MET binds JH at physiological concentrations in vitro and that the transcriptional activity of MET is dependent on JH concentration (Miura et al.,2005). In terms of JH signal transduction, Met interacts with Br-C to regulate Drosophila development(Zhou and Riddiford, 2002; Wilson et al., 2006). However,it is not known how Met regulates JH-responsive genes in the control of physiological and developmental events. In the beetle Tribolium castaneum, Met plays a key role in JH action by preventing the premature development of adult structures during larval-pupal metamorphosis(Konopova and Jindra, 2007; Parthasarathy et al., 2008b),and Met acts upstream of Br-C(Konopova and Jindra, 2008; Suzuki et al., 2008; Parthasarathy et al., 2008a). There is no doubt that Met plays a crucial role in JH action and lies upstream in the JH signal transduction pathway(Riddiford, 2008), but whether MET is the bona fide JHR remains inconclusive.

In Drosophila, a high titer of JH at the wandering stage and a high titer of 20E during pupariation both cause and mediate the larval-pupal metamorphosis (Dubrovsky,2005). Application of JH or methoprene does not cause supernumerary larval molts, even when fed continuously throughout larval life(Wilson and Fabian, 1986; Riddiford and Ashburner,1991). However, JH is required for reproduction, including protein synthesis in the male accessory gland(Yamamoto et al., 1988) and endocytotic uptake of vitellogenin by oocytes(Postlethwait and Weiser,1973). In this paper, we show that the larval fat body of JH-deficient animals undergoes precocious and enhanced caspase-dependent PCD. Strikingly, JH prevents 20E-induced caspase-dependent PCD by counteracting MET and GCE and not via the suppression of the 20E-triggered transcriptional cascade. For the first time, we demonstrate that JH counteracts MET and GCE to prevent caspase-dependent PCD in Drosophila.

Met^w3::UAS-Met(Barry et al., 2008), Met^w3; UAS-gceRNAi (T.G.W., unpublished) and UAS-jhamt were generated in our laboratories. Four GAL4 lines were used: Aug21-GAL4 [Aug21>(Mirth et al., 2005)], Adh-GAL4 [Adh>(Grönke et al., 2003)], FB-GAL4 [FB>(Grönke et al., 2003)]and Act-GAL4 (Act>). The UAS-death activator line used was UAS-grim (Wing et al.,1998). UAS-Dronc was obtained from S. Kumar(Quinn et al., 2000). Flies from the Bloomington Drosophila Stock Center included: (1) w¹¹¹⁸, (2) Act>, (3) hs-EcR-RNAi, (4) UAS-mcd8GFP (UAS-GFP), (5) Adv/Cyo::arm-GFP, (6) TM6B/TM3:: arm-GFPand (7) SP/Cyo; TM3/TM6B.

Aug21>::UAS-GFP and Aug21>::hs-EcR-RNAi animals were produced by recombination of Aug21> with UAS-GFP and hs-EcR-RNAi, respectively. Homozygous Met^w3; UAS-gceRNAi females were crossed with Act>/Cyo, arm-GFPmales to produce Met^w3/Y; Act>/UAS-gceRNAi males. Homozygous Met^w3::UAS-Met females were crossed with FB> males to produce Met^w3::UAS-Met/Y; FB> males.

Juvenile hormone acid methyl transferase (JHAMT) activity in the brain-RG complex was measured as previously described(Li et al., 2003b; Sheng et al., 2008). JH synthesis by the brain-RG complex was monitored using a modification of the radiochemical assay (Richard et al.,1989) and reversed-phase HPLC separation(Li et al., 2003a). Third instar larvae were topically treated with 0.5 μl of a variety of concentrations (0-3 μg/μl) of methoprene dissolved in acetone(Wilson and Fabian, 1986). Treatment with 20E was performed on second or third instar larvae, which were fed on yeast mixture containing different concentrations (0-3 μg/μl) of 20E (McBrayer et al.,2007).

GFP- and non-GFP-containing embryos were separated under an Olympus SZX16 fluorescence stereomicroscope. Apoptosis was measured using the Caspase 3&7 Apoptosis Detection Kit (green nuclei) according to the manufacturer's instructions (Invitrogen). For determining whether the cell membrane was disrupted, apoptosis was also detected by propidium iodide staining (red nuclei) and nuclei with Hoechst 33342 (blue) (Beyotime). The staining was monitored under an Olympus Fluoview FV1000 confocal microscope or an Olympus IX71 inverted fluorescence microscope using the same conditions for the control and experimental samples.

The two-dimensional fluorescence difference gel electrophoresis/mass spectrum analysis (2D-DIGE/MS) was performed by Shanghai Applied Protein Technology (Jia et al., 2007). Using 2D-DIGE, fat body protein profiles were compared between Aug21>; UAS-grim and Aug21> at three developmental stages: early wandering (EW), white prepupa (WPP) and 6 hours after pupariation (6AP). MALDI-TOF (Applied Biosystems) and LTQ (Thermo Finnigan) MS analyses were used to identify the proteins differentially expressed between the two lines (Alban et al., 2003; Sun et al.,2007).

SDS-PAGE electrophoresis and western blot analysis for FBP1 were as previously described (Sun et al.,2007). Quantitative real-time PCR (qPCR) was performed in a Rotor-Gene 2000 thermocycler (Corbett Research) using rp49(RpL32) for normalization (Sheng et al., 2008). Details of the qPCR primers are available upon request.

Experimental data were analyzed by ANOVA and Student's t-test using an SAS program.

The cells comprising the corpus allatum (CA) are located within the ring gland (RG) and are responsible for JH biosynthesis in Drosophila(Richard et al., 1989; Dai and Gilbert, 1991). To assess the physiological roles of JH in Drosophila, the CA was genetically ablated using the UAS-GAL4 system(Brand and Perrimon, 1993). Aug21> is a GAL4 driver that specifically targets gene expression to the CA (Colombani et al.,2005; Mirth et al.,2005). Driven by Aug21>, UAS-grim(Wing et al., 1998) was expressed in the CA resulting in cell ablation. All of the Aug21>; UAS-grim animals died during early pupal life after normal pupariation. In addition, larval development of Aug21>; UAS-grim animals was delayed and the body weight reduced (see Fig. S1 in the supplementary material). UAS-GFP was then included in the background of the Aug21> flies to create Aug21>::UAS-GFP for monitoring the timing and extent of CA ablation. In comparison to Aug21>::UAS-GFP(Fig. 1A), all GFP-labeled CA cells were ablated in Aug21>::UAS-GFP; UAS-grimby early wandering (EW) (Fig. 1A′), although some cells were still present at earlier larval stages (data not shown). To determine whether JH titers were affected by CA ablation, three indirect assays were conducted. First, JHAMT activity(Shinoda and Itoyama, 2003) in the brain-RG complex was measured, as JHAMT overexpression in Drosophila results in elevated JH levels(Niwa et al., 2008) (W.G.B. and S. S. Tobe, unpublished) and JHAMT is a key regulatory enzyme for JH biosynthesis (Sheng et al.,2008). JHAMT activity measured at EW was undetectable in Aug21>; UAS-grim, whereas it was 2- to 2.5-fold higher in Aug21>; UAS-jhamt than in the two control lines Aug21> and UAS-grim(Fig. 1B). Second, the rate of in vitro JH biosynthesis (Yagi and Tobe,2001) by the brain-RG complex, a determining regulator of JH titer, was measured at EW. No in vitro JH biosynthesis was detected in Aug21>; UAS-grim, and the rate of in vitro JH biosynthesis in Aug21>; UAS-jhamt [∼900 disintegrations per minute (DPM) in 3 hours from five brain-RG] was∼2.5-fold higher than in the two control lines. Third, the JH agonist methoprene was tested for its ability to rescue developmental lethality. Methoprene treatment was able to rescue Aug21>; UAS-grimdevelopment to the adult stage, depending on the dose of methoprene used and the stage of the larvae treated (Fig. 1C). The application of low doses of methoprene (0.1, 0.3 or 1μg/μl) on day 1 or 2 of the third instar (L3D1 or L3D2) rescued 0-7% of the pupae to adults, whereas treatment with 3 μg/μl of methoprene on L3D1 was able to rescue ∼40% of the pupae to adults that were reproductively competent (Fig. 1D). However, once Aug21>; UAS-grim larvae reached the EW stage, methoprene failed to rescue the JH-deficient pupae to the adult stage, even at higher concentrations (>3 μg/μl). These results demonstrated that CA ablation results in JH deficiency leading to pupal lethality.

We then carefully observed the JH-deficient Aug21>; UAS-grim animals for developmental defects during the larval-pupal transition. Although a small proportion (∼10%) of the JH-deficient pupae underwent head eversion successfully, the adult organs of these animals initiated development but never completed it. As visualized beneath the cuticle by microscopy, internal portions of the pupae were seen to progressively retract from the cuticle (apolysis), creating an apparently empty space beginning 6 hours after pupariation (6AP)(Fig. 1E). During the larval-pupal metamorphosis of Drosophila, the fat body undergoes a remodeling process but remains in the posterior part of the pupa(Nelliot et al., 2006; Liu et al., 2009). The posterior portion of the JH-deficient pupae often appeared to be empty,suggesting that JH has an important role in the control of fat body development during the larval-pupal transition.

Similar to other larval organs, the Drosophila larval fat body undergoes massive destruction by PCD and necrosis(Hoshizaki, 2005; Liu et al., 2009). As predicted, fat body remodeling in the JH-deficient line was altered dramatically and differed significantly from w¹¹¹⁸, UAS-grim,Aug21> and the JH-overexpressing line Aug21>; UAS-jhamt. Since no significant differences in fat body remodeling were observed in the latter four lines, in the following studies only experimental data for one control line, Aug21>, are presented. Apoptosis and cell dissociation in the fat body of the JH-deficient line Aug21>; UAS-grim and the control line Aug21>were compared at several developmental stages: L3D1, L3D2, EW, white prepupa(WPP), 6AP and 10AP. At L3D1, L3D2 and EW, apoptosis of fat body cells was almost undetectable in the control (L3D2; Fig. 2A) but was pronounced in the JH-deficient animals (L3D2; Fig. 2A′) when stained using the Caspase 3&7 Apoptosis Detection Kit. From EW to WPP, apoptosis became stronger in the control but weaker in the JH-deficient animals (data not shown). At WPP and 6AP, the majority of the fat body cells in the JH-deficient animals died as a result of apoptosis, showing a disrupted cell membrane when stained with propidium iodide (6AP; Fig. 2B′),but only a small portion of cells of the control were stained by propidium iodide (6AP; Fig. 2B). At 10AP,fat body cells in the control appeared to round up and began to lose their tight associations with one another (Fig. 2C), but nearly all fat body cells in the JH-deficient animals were completely dissociated into individual cell masses(Fig. 2C′). In conclusion, the fat body in the JH-deficient pupae underwent precocious and enhanced PCD and eventually failed to complete the remodeling process,demonstrating that JH plays a crucial role in the control of fat body remodeling in Drosophila by preventing PCD.

The important role of JH in preventing PCD in the Drosophila fat body prompted us to compare the protein profiles in the fat body of the JH-deficient line Aug21>; UAS-grim and the control line Aug21> at three developmental stages (EW, WPP and 6AP) using 2D-DIGE/MS analysis. A representative 2D-DIGE image, with merged Cy2 (blue),Cy3 (green) and Cy5 (red) fluorescence is shown in Fig. 3A-A‴. Across all the loading samples, ∼1500 protein spots were reproducibly detected in each gel. The number of differentially expressed protein spots between the JH-deficient and the control animals gradually increased (>1.5-fold) from EW to WPP to 6AP. Most of the differentially expressed protein spots in the fat body of the JH-deficient animals at WPP (90%, P<0.01; 70%, P<0.05%) were downregulated, whereas more than half of them were upregulated at EW or 6AP (Fig. 3B,C). The 111 differentially expressed protein spots(P<0.05) between the two lines can be divided into five groups(Fig. 3D). Approximately 70% of the protein spots were either upregulated (34, group 1) or downregulated (40,group 2) at the three developmental stages in the JH-deficient animals(Fig. 3D).

Twenty-six of the most differentially expressed protein spots were subjected to MALDI-TOF and/or LTQ MS analysis. Twenty-one proteins were identified and five of them were detected twice, in different spots(Table 1). The mRNA levels of ten of the proteins were then analyzed by qPCR. The most significant protein in group 1 is Dronc (Fig. 4A,A′), an initiator caspase that can be upregulated by the 20E-EcR-USP complex, Br-C and E75 (Cakouros et al., 2004), which was increased at all three stages in the JH-deficient animals. The mRNA level of Drice(Fig. 4B), an effector caspase that can be upregulated by Br-C and activated by Dronc(Kilpatrick et al., 2005), was also increased at all three stages in the JH-deficient animals. Regucalcin, a molecular marker of senescence (Fujita,1999), was also identified in group 1, suggesting senescence of fat body cells (see Fig. S2A,A′ in the supplementary material). The protein FBP1 in group 2 (Fig. 4C,C′) was decreased at the three stages studied in the JH-deficient animals. Western blot analysis showed that in the JH-overexpressing line Aug21>; UAS-jhamt, the estimated,elevated levels of JH were able to convert FBP1 from an inactive form (P69) to an active form (P50) (Fig. 4C″). Changes in the levels of Lsp2 and Fbp1 mRNAs followed a similar pattern (see Fig. S2B′ in the supplementary material). Most proteins in group 2(Table 1) are involved in energy metabolism and detoxification. The mRNA levels of all the tested genes were downregulated in the JH-deficient animals at WPP and upregulated in the JH-overexpressing animals at EW (see Fig. S2C-M in the supplementary material). The one protein identified in group 4 was Krüppel (KR), which was downregulated in the JH-deficient animals at WPP(Fig. 4D,D′). The mRNA levels of two Kr paralogous genes, Kr-H1 and Kr-H2,shared a similar pattern to that of Kr (see Fig. S2N′,O′in the supplementary material). The 2D-DIGE/MS and qPCR analyses indicate that the failure of fat body remodeling in the JH-deficient animals is a result of multiple developmental defects, including precocious and enhanced caspase-dependent PCD. Importantly, the two caspase genes Dronc and Drice, which can be upregulated and activated by 20E action(Cakouros et al., 2004; Kilpatrick et al., 2005), were significantly upregulated in the fat body of the JH-deficient animals,suggesting that JH antagonizes 20E-induced caspase-dependent PCD.

Previous reports have shown that JH also elicits the expression of several 20E response genes, including E75 in Drosophila S2 cells(Dubrovsky et al., 2004), E74B in late third instar larval organs(Beckstead et al., 2007) and Kr-H1 in the abdominal integuments of prepupae or pupae(Minakuchi et al., 2008). The above 2D-DIGE/MS and qPCR analyses revealed that JH elicited the expression of several 20E response genes, including Fbp1, Lsp2 and Kr-H1(Fig. 4; see Fig. S2 in the supplementary material). Furthermore, the qPCR analysis demonstrated that the 20E-triggered transcriptional cascade was reduced in the fat body of the JH-deficient animals at WPP and was enhanced in the fat body of the JH-overexpressing animals at EW (data not shown). In contrast to the caspase genes Dronc and Drice(Fig. 4A-B), but similar to other genes in the 20E-triggered transcriptional cascade, the death activator genes reaper (Fig. 5A)and Hid (Fig. 5B), as well as the autophagy genes Atg8A(Fig. 5C) and Atg8B(Fig. 5D), were downregulated in the fat body of JH-deficient animals at WPP, and Hid and Atg8A were upregulated in the JH-overexpressing animals at EW. The mRNA level of Iap1, another important gene involved in preventing PCD, was not altered in the fat body of the JH-deficient and JH-overexpressing animals (data not shown). Based on the above data, we conclude that JH does not suppress the 20E-triggered transcriptional cascade in preventing caspase-dependent PCD in the fat body of JH-deficient animals.

To further support the hypothesis that JH antagonizes 20E-induced caspase-dependent PCD in the fat body, we genetically manipulated the 20E signal in the JH-deficient line Aug21>; UAS-grim. The JH-deficient larvae were highly sensitive to 20E treatment. When second instar larvae were fed on the yeast mixture containing 1 μg/μl 20E, only∼10% of the JH-deficient animals pupariated and the rest died. However,under these conditions, ∼60% of the three control lines pupariated precociously and the pupae were smaller(Fig. 6A,B). Decreasing the 20E signal partially rescued the JH-deficient pupae. Using EcR RNAi to disrupt 20E signaling, heat shock-induced expression of EcR dsRNA causes a high degree of lethality in hs-EcR-RNAi animals(Lam and Thummel, 2000). When reared without heat-shock induction, ∼10% of Aug21>::hs-EcR-RNAi; UAS-grim(hs-EcR-RNAi on the JH-deficient background) animals survived to the pre-adult stage (Fig. 6C) and less than half of those emerged as adults(Fig. 6C′). These genetic experiments confirmed the hypothesis that JH antagonizes 20E-induced PCD to regulate larval-pupal metamorphosis in Drosophila(Fig. 6D).

As concluded above, JH does not suppress the 20E-triggered transcriptional cascade to prevent caspase-dependent PCD in the fat body. Since MET and GCE play a crucial role in JH action and lie upstream in the JH signal transduction pathway in Drosophila(Riddiford, 2008), we studied whether Met and gce mediate the prevention of PCD by JH. Most of the Met/gce-deficient Met^w3/Y; Act>/UAS-gceRNAianimals die during the pupal-adult transition (T.G.W., unpublished). Surprisingly, precocious and enhanced PCD was never observed in the larval fat body of the Met/gce-deficient animals (data not shown). Moreover, the Met/gce-deficient animals showed a similar lethality phenotype to that of the global JH-overexpressing Act>; UAS-jhamt animals, which also die during the pupal-adult transition(Niwa et al., 2008).

Previous studies have shown that global overexpression of Met in Met^w3::UAS-Met/Y; Act> causes high mortality during larval life (Barry et al.,2008). Similarly, nearly all Met^w3::UAS-Met/Y; Adh> animals, in which Met is overexpressed specifically in the fat body, died during larval life. Less than 10% of the Met-overexpressing animals were able to survive to the EW stage and ∼1% pupariated but never emerged as adults. Moreover, larval development of the Met-overexpressing animals was greatly delayed and their body weight dramatically reduced(Fig. 7A). Once the Met-overexpressing larvae reached the EW stage, larval fat body cells began to dissociate (Fig. 7B)from each other and underwent dramatic apoptosis(Fig. 7C). The developmental defects of the Met-overexpressing animals are similar to, but much stronger than, those of the JH-deficient animals (Figs 1 and 2). The phenotypic and genetic data for these animals strongly suggest that JH counteracts MET and GCE.

We then studied whether Met induces PCD via increasing the mRNA levels of the caspases Dronc and Drice. In comparison to male w¹¹¹⁸ animals at WPP, the mRNA levels of Dronc (Fig. 8A) and Drice (Fig. 8B) were downregulated in the fat body of the Met/gce-deficient animals and the downregulation was less significant in the Met mutant Met^w3/Y.

Because it is difficult to collect sufficient larval fat body from Met^w3::UAS-Met/Y; Act> or Met^w3::UAS-Met/Y; Adh>, we used Met^w3::UAS-Met/Y; FB>, in which Met is also specifically overexpressed in the fat body. Met^w3::UAS-Met/Y; FB> exhibited better survival and less significant developmental defects than Met^w3::UAS-Met/Y; Adh>. In comparison to male w¹¹¹⁸ animals at WPP, Dronc(Fig. 8C) and Drice(Fig. 8D) were dramatically upregulated in the fat body of the Met-overexpressing animals. Importantly, application of methoprene (1 μg/μl) at the EW stage significantly downregulated Dronc(Fig. 8C) and Drice(Fig. 8D) at WPP. However,application of methoprene to Met^w3/Y; Act>/UAS-gceRNAi had no significant effects on Dronc and Drice at WPP (data not shown). Together, these experiments demonstrated that JH is epistatic to MET and GCE.

We then investigated whether overexpression of Dronc in the fat body causes lethality and PCD. Approximately 95% of Adh>; UAS-Dronc animals, in which Dronc is overexpressed in the fat body, died during different larval stages(Fig. 8E), with significant apoptosis at EW (Fig. 8F). The remaining animals died at the pupal stage.

Altogether, the data in this paper demonstrate that JH counteracts the bHLH-PAS transcription factors MET and GCE to prevent caspase-dependent PCD in Drosophila (Fig. 8G).

The status quo action of JH has been well documented in several insect orders, particularly in Coleoptera, Orthoptera and Lepidoptera, in which JH treatment causes supernumerary larval molting and JH deficiency triggers precocious metamorphosis (Riddiford et al., 2003). However, as JH does not cause supernumerary larval molting in flies (Srivastava and Gilbert, 1968; Wilson and Fabian, 1986; Riddiford and Ashburner,1991), evidence for the status quo action of JH in Drosophila has remained elusive. From past studies and from the experimental data presented here, we conclude that the status quo hypothesis does indeed apply to JH action in Drosophila. First, although JH application during the final larval instar or during the prepupal stage has little effect on the differentiation of adult head and thoracic epidermis in Drosophila, it does prevent normal adult differentiation of the abdominal epidermis. After JH treatment, a second pupal, rather than an adult,abdominal cuticle is formed in Diptera (Srivastava and Gilbert, 1968; Zhou and Riddiford, 2002). Second, JH or a JH agonist applied to Drosophila at the onset of metamorphosis results in lethality during pupal-adult metamorphosis (Madhaven,1973). Similarly, global overexpression of jhamt results in severe defects during the pupal-adult transition and eventually death(Niwa et al., 2008). Third, CA ablation leading to JH deficiency caused precocious and enhanced fat body PCD(Fig. 2). Fourth, JH deficiency resulted in pupal lethality (Fig. 1A) and delayed larval development (see Fig. S1 in the supplementary material), although JH deficiency was not sufficient to cause precocious metamorphosis. The composite data demonstrate that JH in Drosophila does have status quo actions on the abdominal epidermis during pupal-adult metamorphosis and on the fat body during larval-pupal metamorphosis. We conclude that the status quo action of JH in Drosophila is functionally important, but more subtle than that in Coleoptera, Orthoptera and Lepidoptera. However, it is not clear whether JH is essential for embryonic and earlier larval development because the CA cells are not completely ablated in the JH-deficient animals until the EW stage. To address this question, it would be necessary to generate a mutant (i.e. of jhamt) that interrupts JH but not the farnesyl pyrophosphate biosynthesis pathway.

The insect fat body is analogous to vertebrate adipose tissue and liver and functions as a major organ for nutrient storage and energy metabolism(Hoshizaki, 2005; Liu et al., 2009). In response to 20E pulses, Drosophila larval organs undergo a developmental remodeling process during metamorphosis(Ward et al., 2003). Blocking the 20E signal specifically in the fat body during the larval-pupal transition(Lsp2>; UAS-EcR^DN) prevented the fat body from undergoing PCD (our unpublished data) and cell dissociation(Cherbas et al., 2003).

The experimental data in this paper demonstrated that JH prevents caspase-dependent PCD in the fat body during the larval-pupal transition in Drosophila. First, JH deficiency in Aug21>, UAS-grimresulted in the fat body undergoing precocious and enhanced PCD and cell dissociation (Fig. 2). Precocious and enhanced apoptosis appeared as early as L3D1 in the JH-deficient animals (Fig. 2A,B). Methoprene application on L3D1 was able to rescue ∼40%of the pupae to adults, but it failed to rescue post-EW(Fig. 1D). Second, 2D-DIGE/MS and qPCR analyses indicated that the fat body in the JH-deficient animals has multiple developmental defects. The upregulation of the caspase genes Dronc and Drice (Fig. 4A-B) should account for the PCD in the fat body, as overexpression of Dronc in the fat body causes PCD, cell dissociation, and thus lethality (Fig. 8E,F; data not shown). As demonstrated previously, overexpression of Dronc (Dorstyn et al.,1999; Lee et al.,2000) or Drice(Kilpatrick et al., 2005) in cells and tissues is sufficient to cause caspase-dependent PCD. Third, the 20E-triggered transcriptional cascade in the fat body was downregulated in the JH-deficient animals (Fig. 5),indicating that JH does not suppress the 20E-triggered transcriptional cascade in preventing caspase-dependent PCD in the fat body.

The antagonizing effect of JH on 20E-induced PCD in the fat body was further confirmed in the JH-deficient animals by 20E treatment and RNA interference of EcR (Fig. 6). One might expect that perfect timing, titer and receptor response of JH and 20E are required to ensure accurate PCD in a tissue- and stage-specific manner during Drosophila metamorphosis(Ward et al., 2003). In the JH-deficient animals, the upregulation of Dronc and Driceresulted in precocious and enhanced PCD, such that the JH-deficient animals are committed to die during the larval-pupal transition(Fig. 1A). This hypothesis was strengthened by overexpression of Dronc specifically in the fat body,which caused larval lethality (Fig. 8E). Taken together, we conclude that JH antagonizes 20E-induced caspase-dependent PCD in controlling fat body remodeling and larval-pupal metamorphosis in Drosophila (Fig. 6D).

Based on the phenotypes and gene expression profiles in the four fly lines used, we conclude that JH counteracts MET and GCE to prevent caspase-dependent PCD (Fig. 8G). First, the Met-overexpressing animals died during larval life(Barry et al., 2008)(Fig. 7A), with precocious and enhanced PCD and cell dissociation in the fat body(Fig. 7B,C; data not shown). Dramatic upregulation of Dronc and Drice was observed when Met was specifically overexpressed in the fat body and this upregulation was significantly decreased by methoprene application(Fig. 8C,D) demonstrating that JH is epistatic to MET and GCE. Moreover, the Dronc-overexpressing animals (Fig. 8E,F) exhibited similar phenotypes to the Met-overexpressing animals. Second, in the fat body of the JH-deficient animals, PCD(Fig. 2) and the expression of Dronc and Drice (Fig. 4A-B) were upregulated but not as significantly as in the Met-overexpressing animals. This might explain why the JH-deficient animals did not die until early pupal life(Fig. 1E). Third, both the global JH-overexpressing animals (Niwa et al., 2008) and the Met/gce-deficient animals(T.G.W., unpublished) died during the pupal-adult transition. In these animals, Dronc and Drice were downregulated and caspase-dependent PCD was decreased in the fat body(Fig. 8A,B; our unpublished data), implying that these animals died from a lack of caspase-dependent PCD. Weak mutants of Dronc (Xu et al.,2005) and Drice mutants(Muro et al., 2006) die during pupal life, showing that caspase-dependent PCD is essential for Drosophila metamorphosis. In addition, we also observed that methoprene application at the onset of metamorphosis results in delayed fat body remodeling (our unpublished data).

In the future, it will be crucial to elucidate the detailed molecular mechanism of how JH counteracts MET and GCE to prevent caspase-dependent PCD. In Drosophila S2 cells, the transcriptional activity of MET is dependent on the JH concentration (Miura et al., 2005) and both MET-MET and MET-GCE interactions can be greatly diminished by JH (Godlewski et al., 2006). The bHLH-PAS transcription factors typically function as hetero- or homodimers (Gu et al.,2000). If MET/GCE is the JHR, the transcriptional activities of the dimerized MET/GCE and the JH-MET/GCE complex should differ. In other words, the dimerized MET/GCE should induce transcription of Dronc and Drice and, in turn, JH binding to form the JH-MET/GCE complex should reduce this induction. Although, to our knowledge, there are no examples in the literature in which a receptor, without ligand, acts as a transcriptional activator and the transcriptional activity of the receptor is diminished when the ligand is bound, we could speculate that the JHR is a unique hormone receptor and perhaps that is the reason why it has yet to be isolated and characterized. Unfortunately, the two experiments described above(Miura et al., 2005; Godlewski et al., 2006) were conducted in Drosophila S2 cells, where the possibility of an endogenous JHR could not be eliminated. Although MET/GCE is definitely a key component in the JH signal transduction pathway, whether MET/GCE is the bona fide JHR remains conjecture.

It is very likely that MET cross-talks with EcR-USP via a large molecular complex (Li et al., 2007). One can hypothesize that MET promotes 20E action in the absence of JH and suppresses 20E action in the presence of JH, a model which we favor. Drosophila FKBP39 (FK506-BP1) could be a key component in this complex because it physically interacts with MET, EcR and USP, and binds the D. melanogaster JH response element 1(Li et al., 2007). Moreover, Drosophila FKBP39 inhibits 20E-induced autophagy(Juhász et al., 2007). Further analysis of the complex will be crucial to precisely define the molecular mechanism of cross-talk between the action of JH and 20E.

In summary, we conclude that JH counteracts MET and GCE to prevent caspase-dependent PCD in controlling fat body remodeling and larval-pupal metamorphosis in Drosophila. The Drosophila fat body has provided an excellent model for studying the long-standing question of JH signal transduction. To finally settle the question of the bona fide JHR and to understand the precisely defined molecular mechanism of JH action requires further research at a variety of levels in several species of insects that can be genetically manipulated, such as Drosophila, Bombyx and Tribolium.