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The nuclear receptor DHR3 modulates dS6 kinase-dependent growth in Drosophila.

Montagne J, Lecerf C, Parvy JP, Bennion JM, Radimerski T, Ruhf ML, Zilbermann F, Vouilloz N, Stocker H, Hafen E, Kozma SC, Thomas G - PLoS Genet. (2010)

Bottom Line: S6 kinases (S6Ks) act to integrate nutrient and insulin signaling pathways and, as such, function as positive effectors in cell growth and organismal development.Consistent with these findings, we have identified an endogenous DHR3 isoform that lacks the DBD.These results provide the first molecular link between the dS6K pathway, critical in controlling nutrient-dependent growth, and that of DHR3, a major mediator of ecdysone signaling, which, acting together, coordinate metamorphosis.

View Article: PubMed Central - PubMed

Affiliation: Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland. montagne@cgm.cnrs-gif.fr

ABSTRACT
S6 kinases (S6Ks) act to integrate nutrient and insulin signaling pathways and, as such, function as positive effectors in cell growth and organismal development. However, they also have been shown to play a key role in limiting insulin signaling and in mediating the autophagic response. To identify novel regulators of S6K signaling, we have used a Drosophila-based, sensitized, gain-of-function genetic screen. Unexpectedly, one of the strongest enhancers to emerge from this screen was the nuclear receptor (NR), Drosophila hormone receptor 3 (DHR3), a critical constituent in the coordination of Drosophila metamorphosis. Here we demonstrate that DHR3, through dS6K, also acts to regulate cell-autonomous growth. Moreover, we show that the ligand-binding domain (LBD) of DHR3 is essential for mediating this response. Consistent with these findings, we have identified an endogenous DHR3 isoform that lacks the DBD. These results provide the first molecular link between the dS6K pathway, critical in controlling nutrient-dependent growth, and that of DHR3, a major mediator of ecdysone signaling, which, acting together, coordinate metamorphosis.

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Immunodetection of DHR3 proteins.(A–F) Wing imaginal discs stained with: (A, C, D, F) antibody to DHR3, and (B, E) antibody to GFP. (A, B) Overexpression of DHR3-RS and USA-GFP in the posterior compartment was induced using an engrailed-Gal4 driver. (C) and (D) ap-Gal4 drives overexpression of DHR3-RS and DHR3-EP, respectively. (E, F) Flip-out clone (induced 3 days prior to dissection) in a prepupal wing imaginal disc expressing UAS-DHR3-RNAi and UAS-GFP. (E) Decrease in DHR3 staining in a flip-out clone labeled by GFP (F), indicating that DHR3 is expressed in this tissue. (G) Western blot analysis of DHR3 in larval protein extracts from control (Co) and heat shock-induced UAS lines expressing DHR3-RA (RA), DHR3-RB (RB), and DHR3-RS (RS) transcripts, or the DHR3-EPs (E1 and E2). (H) Western blot of the endogenous DHR3 protein in late third-instar larvae (L3), prepupae (pp), and prepupae expressing an RNAi to DHR3 (RNAipp). In (G, H), the arrows at the left indicate the position of the DBD-containing (high) and DBD-lacking (low) DHR3 proteins.
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pgen-1000937-g004: Immunodetection of DHR3 proteins.(A–F) Wing imaginal discs stained with: (A, C, D, F) antibody to DHR3, and (B, E) antibody to GFP. (A, B) Overexpression of DHR3-RS and USA-GFP in the posterior compartment was induced using an engrailed-Gal4 driver. (C) and (D) ap-Gal4 drives overexpression of DHR3-RS and DHR3-EP, respectively. (E, F) Flip-out clone (induced 3 days prior to dissection) in a prepupal wing imaginal disc expressing UAS-DHR3-RNAi and UAS-GFP. (E) Decrease in DHR3 staining in a flip-out clone labeled by GFP (F), indicating that DHR3 is expressed in this tissue. (G) Western blot analysis of DHR3 in larval protein extracts from control (Co) and heat shock-induced UAS lines expressing DHR3-RA (RA), DHR3-RB (RB), and DHR3-RS (RS) transcripts, or the DHR3-EPs (E1 and E2). (H) Western blot of the endogenous DHR3 protein in late third-instar larvae (L3), prepupae (pp), and prepupae expressing an RNAi to DHR3 (RNAipp). In (G, H), the arrows at the left indicate the position of the DBD-containing (high) and DBD-lacking (low) DHR3 proteins.

Mentions: To determine whether the endogenous isoform DHR3-PS, lacking the DBD, is expressed in vivo, a rabbit antiserum to DHR3 was produced using peptides that correspond to sequences downstream of the first AUG following the DBD coding sequence (Figure S2). Expression of the UAS–DHR3-RS (Figure 3A), was induced in the posterior wing-disc compartment using the engrailed-Gal4 (en-Gal4) driver. This line also harbored a UAS-GFP, activated by the en-Gal4 driver leading to the production of GFP, which allowed for double immunostaining. The results of this experiment revealed co-localization of GFP and DHR3-PS expressions (Figure 4A and 4B). Likewise, when induced by the ap-Gal4 driver, both the DHR3-RS and the DHR3-EP lines exhibited increased immunostaining within the dorsal wing-disc compartment, which was much stronger for DHR3-RS than for DHR3-EP (compare Figure 4C and 4D). Because DHR3-RS, but not DHR3-EP, provoked the bent-down wing phenotype when induced alone by ap-Gal4 (compare Figure 1D with Figure 3D), these results are consistent with the ability of DHR3-RS to induce growth in a dosage-dependent manner. To determine whether we could also detect endogenous DHR3, flip-out clones directing DHR3-RNAi expression were generated, and a UAS-GFP was used to positively label these clones [49]. The staining observed in prepupal discs was strongly reduced in flip-out clones (Figure 4E and 4F), with remnant staining most likely reflecting incomplete depletion of DHR3 expression. Clones displaying a decrease in specific staining could be detected in all imaginal discs from prepupae (data not shown), indicating that DHR3 is widely represented at this stage of development. In addition, weak staining could be detected in both the imaginal discs and the fat body from mid-third-instar larvae (data not shown), suggesting the presence of low levels of DHR3 at this stage. Thus, endogenous DHR3 is detectable in prepupae, but also likely present at low levels in larval tissues.


The nuclear receptor DHR3 modulates dS6 kinase-dependent growth in Drosophila.

Montagne J, Lecerf C, Parvy JP, Bennion JM, Radimerski T, Ruhf ML, Zilbermann F, Vouilloz N, Stocker H, Hafen E, Kozma SC, Thomas G - PLoS Genet. (2010)

Immunodetection of DHR3 proteins.(A–F) Wing imaginal discs stained with: (A, C, D, F) antibody to DHR3, and (B, E) antibody to GFP. (A, B) Overexpression of DHR3-RS and USA-GFP in the posterior compartment was induced using an engrailed-Gal4 driver. (C) and (D) ap-Gal4 drives overexpression of DHR3-RS and DHR3-EP, respectively. (E, F) Flip-out clone (induced 3 days prior to dissection) in a prepupal wing imaginal disc expressing UAS-DHR3-RNAi and UAS-GFP. (E) Decrease in DHR3 staining in a flip-out clone labeled by GFP (F), indicating that DHR3 is expressed in this tissue. (G) Western blot analysis of DHR3 in larval protein extracts from control (Co) and heat shock-induced UAS lines expressing DHR3-RA (RA), DHR3-RB (RB), and DHR3-RS (RS) transcripts, or the DHR3-EPs (E1 and E2). (H) Western blot of the endogenous DHR3 protein in late third-instar larvae (L3), prepupae (pp), and prepupae expressing an RNAi to DHR3 (RNAipp). In (G, H), the arrows at the left indicate the position of the DBD-containing (high) and DBD-lacking (low) DHR3 proteins.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2865512&req=5

pgen-1000937-g004: Immunodetection of DHR3 proteins.(A–F) Wing imaginal discs stained with: (A, C, D, F) antibody to DHR3, and (B, E) antibody to GFP. (A, B) Overexpression of DHR3-RS and USA-GFP in the posterior compartment was induced using an engrailed-Gal4 driver. (C) and (D) ap-Gal4 drives overexpression of DHR3-RS and DHR3-EP, respectively. (E, F) Flip-out clone (induced 3 days prior to dissection) in a prepupal wing imaginal disc expressing UAS-DHR3-RNAi and UAS-GFP. (E) Decrease in DHR3 staining in a flip-out clone labeled by GFP (F), indicating that DHR3 is expressed in this tissue. (G) Western blot analysis of DHR3 in larval protein extracts from control (Co) and heat shock-induced UAS lines expressing DHR3-RA (RA), DHR3-RB (RB), and DHR3-RS (RS) transcripts, or the DHR3-EPs (E1 and E2). (H) Western blot of the endogenous DHR3 protein in late third-instar larvae (L3), prepupae (pp), and prepupae expressing an RNAi to DHR3 (RNAipp). In (G, H), the arrows at the left indicate the position of the DBD-containing (high) and DBD-lacking (low) DHR3 proteins.
Mentions: To determine whether the endogenous isoform DHR3-PS, lacking the DBD, is expressed in vivo, a rabbit antiserum to DHR3 was produced using peptides that correspond to sequences downstream of the first AUG following the DBD coding sequence (Figure S2). Expression of the UAS–DHR3-RS (Figure 3A), was induced in the posterior wing-disc compartment using the engrailed-Gal4 (en-Gal4) driver. This line also harbored a UAS-GFP, activated by the en-Gal4 driver leading to the production of GFP, which allowed for double immunostaining. The results of this experiment revealed co-localization of GFP and DHR3-PS expressions (Figure 4A and 4B). Likewise, when induced by the ap-Gal4 driver, both the DHR3-RS and the DHR3-EP lines exhibited increased immunostaining within the dorsal wing-disc compartment, which was much stronger for DHR3-RS than for DHR3-EP (compare Figure 4C and 4D). Because DHR3-RS, but not DHR3-EP, provoked the bent-down wing phenotype when induced alone by ap-Gal4 (compare Figure 1D with Figure 3D), these results are consistent with the ability of DHR3-RS to induce growth in a dosage-dependent manner. To determine whether we could also detect endogenous DHR3, flip-out clones directing DHR3-RNAi expression were generated, and a UAS-GFP was used to positively label these clones [49]. The staining observed in prepupal discs was strongly reduced in flip-out clones (Figure 4E and 4F), with remnant staining most likely reflecting incomplete depletion of DHR3 expression. Clones displaying a decrease in specific staining could be detected in all imaginal discs from prepupae (data not shown), indicating that DHR3 is widely represented at this stage of development. In addition, weak staining could be detected in both the imaginal discs and the fat body from mid-third-instar larvae (data not shown), suggesting the presence of low levels of DHR3 at this stage. Thus, endogenous DHR3 is detectable in prepupae, but also likely present at low levels in larval tissues.

Bottom Line: S6 kinases (S6Ks) act to integrate nutrient and insulin signaling pathways and, as such, function as positive effectors in cell growth and organismal development.Consistent with these findings, we have identified an endogenous DHR3 isoform that lacks the DBD.These results provide the first molecular link between the dS6K pathway, critical in controlling nutrient-dependent growth, and that of DHR3, a major mediator of ecdysone signaling, which, acting together, coordinate metamorphosis.

View Article: PubMed Central - PubMed

Affiliation: Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland. montagne@cgm.cnrs-gif.fr

ABSTRACT
S6 kinases (S6Ks) act to integrate nutrient and insulin signaling pathways and, as such, function as positive effectors in cell growth and organismal development. However, they also have been shown to play a key role in limiting insulin signaling and in mediating the autophagic response. To identify novel regulators of S6K signaling, we have used a Drosophila-based, sensitized, gain-of-function genetic screen. Unexpectedly, one of the strongest enhancers to emerge from this screen was the nuclear receptor (NR), Drosophila hormone receptor 3 (DHR3), a critical constituent in the coordination of Drosophila metamorphosis. Here we demonstrate that DHR3, through dS6K, also acts to regulate cell-autonomous growth. Moreover, we show that the ligand-binding domain (LBD) of DHR3 is essential for mediating this response. Consistent with these findings, we have identified an endogenous DHR3 isoform that lacks the DBD. These results provide the first molecular link between the dS6K pathway, critical in controlling nutrient-dependent growth, and that of DHR3, a major mediator of ecdysone signaling, which, acting together, coordinate metamorphosis.

Show MeSH
Related in: MedlinePlus