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Scaling the Drosophila Wing: TOR-Dependent Target Gene Access by the Hippo Pathway Transducer Yorkie.

Parker J, Struhl G - PLoS Biol. (2015)

Bottom Line: Here, we show that the TOR pathway regulates Yki by a separate and novel mechanism in the Drosophila wing.Instead of controlling Yki nuclear access, TOR signaling governs Yki action after it reaches the nucleus by allowing it to gain access to its target genes.When TOR activity is inhibited, Yki accumulates in the nucleus but is sequestered from its normal growth-promoting target genes--a phenomenon we term "nuclear seclusion." Hence, we posit that in addition to its well-known role in stimulating cellular metabolism in response to nutrients, TOR also promotes wing growth by liberating Yki from nuclear seclusion, a parallel pathway that we propose contributes to the scaling of wing size with nutrient availability.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and Development, Columbia University, New York, New York, United States of America; Division of Biology, Imperial College London, London, United Kingdom.

ABSTRACT
Organ growth is controlled by patterning signals that operate locally (e.g., Wingless/Ints [Wnts], Bone Morphogenetic Proteins [BMPs], and Hedgehogs [Hhs]) and scaled by nutrient-dependent signals that act systemically (e.g., Insulin-like peptides [ILPs] transduced by the Target of Rapamycin [TOR] pathway). How cells integrate these distinct inputs to generate organs of the appropriate size and shape is largely unknown. The transcriptional coactivator Yorkie (Yki, a YES-Associated Protein, or YAP) acts downstream of patterning morphogens and other tissue-intrinsic signals to promote organ growth. Yki activity is regulated primarily by the Warts/Hippo (Wts/Hpo) tumour suppressor pathway, which impedes nuclear access of Yki by a cytoplasmic tethering mechanism. Here, we show that the TOR pathway regulates Yki by a separate and novel mechanism in the Drosophila wing. Instead of controlling Yki nuclear access, TOR signaling governs Yki action after it reaches the nucleus by allowing it to gain access to its target genes. When TOR activity is inhibited, Yki accumulates in the nucleus but is sequestered from its normal growth-promoting target genes--a phenomenon we term "nuclear seclusion." Hence, we posit that in addition to its well-known role in stimulating cellular metabolism in response to nutrients, TOR also promotes wing growth by liberating Yki from nuclear seclusion, a parallel pathway that we propose contributes to the scaling of wing size with nutrient availability.

No MeSH data available.


Related in: MedlinePlus

Yki activity does not promote growth by up-regulating InR/TOR signaling.(A–C) Wing discs labelled for phospho-Akt S505, an indicator of InR pathway activity, bearing mutant clones marked black by absence of GFP (green); w.t. twin spots are marked by bright green. (A) pten1 (positive control), (B) exe1, and (C) wtsX1 (experimentals, having elevated Yki activity owing to reduced or absent phosphorylation by Wts). Phospho-Akt S505 is not increased in (B) and (C), in contrast to (A). (D) Western blot of protein extracts derived from late third instar wing discs of the genotypes shown, labelled for phospho-Akt S505 (Total Akt and β-actin were used as loading controls; pten1/ptendj189 was used as a positive control; the reduction in S505 staining in the Tsc1Q87X/Tsc1PA23 lane is due to feedback of TOR activation onto Akt phosphorylation [39]). In contrast to reduced Pten, loss of either Ex or Wts does not cause an increase in pAkt S505. (E) Blot of same genotypes as in (D), labelled for phospho-S6 Kinase T398, an indicator of TOR pathway activity (β-Tubulin was used as a loading control, and runs as two species). As observed for phospho-Akt S505, Phospho-S6 Kinase T398 levels are elevated by loss of Pten and Tsc activity, but not by loss of either Ex or Wts activity. (F–I): Wing discs from late third instar larvae-bearing MARCM clones expressing UAS.p35 (labelled positively with GFP-NLS, green; nuclei are counterstained with Hoechst, blue). The genotypes of clones are (F) UAS.p35, (G) ykib5+UAS.p35, (H) ykib5+ UAS.p35+UAS.Dp110, and (I) ykib5+ UAS.p35+UAS.Rheb. J–L: Quantification of clones sizes, cell numbers, and cell sizes from genotypes in F–I. Error bars are Standard Error of the Mean and asterisks denote significances from t tests (* = p < 0.05, ** = p < 0.01, *** = p <0.001, n. s. = not significant). n = 42 (p35), 49 (ykib5+p35), 76 (ykib5+p35+Dp110), 76 (ykib5+p35+Rheb). Expression of either UAS.Dp110 or UAS.Rheb results in an increase in the size of yki mutant clones caused by an increase in cell size but not cell number.
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pbio.1002274.g002: Yki activity does not promote growth by up-regulating InR/TOR signaling.(A–C) Wing discs labelled for phospho-Akt S505, an indicator of InR pathway activity, bearing mutant clones marked black by absence of GFP (green); w.t. twin spots are marked by bright green. (A) pten1 (positive control), (B) exe1, and (C) wtsX1 (experimentals, having elevated Yki activity owing to reduced or absent phosphorylation by Wts). Phospho-Akt S505 is not increased in (B) and (C), in contrast to (A). (D) Western blot of protein extracts derived from late third instar wing discs of the genotypes shown, labelled for phospho-Akt S505 (Total Akt and β-actin were used as loading controls; pten1/ptendj189 was used as a positive control; the reduction in S505 staining in the Tsc1Q87X/Tsc1PA23 lane is due to feedback of TOR activation onto Akt phosphorylation [39]). In contrast to reduced Pten, loss of either Ex or Wts does not cause an increase in pAkt S505. (E) Blot of same genotypes as in (D), labelled for phospho-S6 Kinase T398, an indicator of TOR pathway activity (β-Tubulin was used as a loading control, and runs as two species). As observed for phospho-Akt S505, Phospho-S6 Kinase T398 levels are elevated by loss of Pten and Tsc activity, but not by loss of either Ex or Wts activity. (F–I): Wing discs from late third instar larvae-bearing MARCM clones expressing UAS.p35 (labelled positively with GFP-NLS, green; nuclei are counterstained with Hoechst, blue). The genotypes of clones are (F) UAS.p35, (G) ykib5+UAS.p35, (H) ykib5+ UAS.p35+UAS.Dp110, and (I) ykib5+ UAS.p35+UAS.Rheb. J–L: Quantification of clones sizes, cell numbers, and cell sizes from genotypes in F–I. Error bars are Standard Error of the Mean and asterisks denote significances from t tests (* = p < 0.05, ** = p < 0.01, *** = p <0.001, n. s. = not significant). n = 42 (p35), 49 (ykib5+p35), 76 (ykib5+p35+Dp110), 76 (ykib5+p35+Rheb). Expression of either UAS.Dp110 or UAS.Rheb results in an increase in the size of yki mutant clones caused by an increase in cell size but not cell number.

Mentions: Why is Yki-driven growth limited by the level of TOR activity? TOR might be required upstream to facilitate Yki activity, or in parallel to trigger other, independent growth-related processes. Alternatively, TOR might be required downstream, with Yki promoting growth at least in part by elevating InR/TOR pathway activity. To test this latter possibility, we assayed whether conditions that abnormally increase Yki activity (loss of either Ex or Wts) have a corresponding effect on the levels of phospho-Akt (pAkt-S505), which are normally elevated by enhanced InR signaling (e.g., by removal of the InR pathway inhibitor PTEN; Fig 2A). We find that pAkt-S505 levels are normal in ex—or wts—clones (Fig 2B and 2C), as well as in protein extracts from the overgrown wing discs of homozygous ex—or wts—larvae (Fig 2D). Likewise, the levels of phosphorylated S6-Kinase (pS6K-T398), a readout of TOR activity, were not affected in ex—or wts—discs (Fig 2E). We therefore infer that Wts/Hpo regulated Yki activity does not stimulate either InR or TOR pathway activity in the wing, consistent with results from Drosophila cell culture [15]. This finding argues against a downstream role of InR/TOR signaling in mediating Yki-driven growth.


Scaling the Drosophila Wing: TOR-Dependent Target Gene Access by the Hippo Pathway Transducer Yorkie.

Parker J, Struhl G - PLoS Biol. (2015)

Yki activity does not promote growth by up-regulating InR/TOR signaling.(A–C) Wing discs labelled for phospho-Akt S505, an indicator of InR pathway activity, bearing mutant clones marked black by absence of GFP (green); w.t. twin spots are marked by bright green. (A) pten1 (positive control), (B) exe1, and (C) wtsX1 (experimentals, having elevated Yki activity owing to reduced or absent phosphorylation by Wts). Phospho-Akt S505 is not increased in (B) and (C), in contrast to (A). (D) Western blot of protein extracts derived from late third instar wing discs of the genotypes shown, labelled for phospho-Akt S505 (Total Akt and β-actin were used as loading controls; pten1/ptendj189 was used as a positive control; the reduction in S505 staining in the Tsc1Q87X/Tsc1PA23 lane is due to feedback of TOR activation onto Akt phosphorylation [39]). In contrast to reduced Pten, loss of either Ex or Wts does not cause an increase in pAkt S505. (E) Blot of same genotypes as in (D), labelled for phospho-S6 Kinase T398, an indicator of TOR pathway activity (β-Tubulin was used as a loading control, and runs as two species). As observed for phospho-Akt S505, Phospho-S6 Kinase T398 levels are elevated by loss of Pten and Tsc activity, but not by loss of either Ex or Wts activity. (F–I): Wing discs from late third instar larvae-bearing MARCM clones expressing UAS.p35 (labelled positively with GFP-NLS, green; nuclei are counterstained with Hoechst, blue). The genotypes of clones are (F) UAS.p35, (G) ykib5+UAS.p35, (H) ykib5+ UAS.p35+UAS.Dp110, and (I) ykib5+ UAS.p35+UAS.Rheb. J–L: Quantification of clones sizes, cell numbers, and cell sizes from genotypes in F–I. Error bars are Standard Error of the Mean and asterisks denote significances from t tests (* = p < 0.05, ** = p < 0.01, *** = p <0.001, n. s. = not significant). n = 42 (p35), 49 (ykib5+p35), 76 (ykib5+p35+Dp110), 76 (ykib5+p35+Rheb). Expression of either UAS.Dp110 or UAS.Rheb results in an increase in the size of yki mutant clones caused by an increase in cell size but not cell number.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4608745&req=5

pbio.1002274.g002: Yki activity does not promote growth by up-regulating InR/TOR signaling.(A–C) Wing discs labelled for phospho-Akt S505, an indicator of InR pathway activity, bearing mutant clones marked black by absence of GFP (green); w.t. twin spots are marked by bright green. (A) pten1 (positive control), (B) exe1, and (C) wtsX1 (experimentals, having elevated Yki activity owing to reduced or absent phosphorylation by Wts). Phospho-Akt S505 is not increased in (B) and (C), in contrast to (A). (D) Western blot of protein extracts derived from late third instar wing discs of the genotypes shown, labelled for phospho-Akt S505 (Total Akt and β-actin were used as loading controls; pten1/ptendj189 was used as a positive control; the reduction in S505 staining in the Tsc1Q87X/Tsc1PA23 lane is due to feedback of TOR activation onto Akt phosphorylation [39]). In contrast to reduced Pten, loss of either Ex or Wts does not cause an increase in pAkt S505. (E) Blot of same genotypes as in (D), labelled for phospho-S6 Kinase T398, an indicator of TOR pathway activity (β-Tubulin was used as a loading control, and runs as two species). As observed for phospho-Akt S505, Phospho-S6 Kinase T398 levels are elevated by loss of Pten and Tsc activity, but not by loss of either Ex or Wts activity. (F–I): Wing discs from late third instar larvae-bearing MARCM clones expressing UAS.p35 (labelled positively with GFP-NLS, green; nuclei are counterstained with Hoechst, blue). The genotypes of clones are (F) UAS.p35, (G) ykib5+UAS.p35, (H) ykib5+ UAS.p35+UAS.Dp110, and (I) ykib5+ UAS.p35+UAS.Rheb. J–L: Quantification of clones sizes, cell numbers, and cell sizes from genotypes in F–I. Error bars are Standard Error of the Mean and asterisks denote significances from t tests (* = p < 0.05, ** = p < 0.01, *** = p <0.001, n. s. = not significant). n = 42 (p35), 49 (ykib5+p35), 76 (ykib5+p35+Dp110), 76 (ykib5+p35+Rheb). Expression of either UAS.Dp110 or UAS.Rheb results in an increase in the size of yki mutant clones caused by an increase in cell size but not cell number.
Mentions: Why is Yki-driven growth limited by the level of TOR activity? TOR might be required upstream to facilitate Yki activity, or in parallel to trigger other, independent growth-related processes. Alternatively, TOR might be required downstream, with Yki promoting growth at least in part by elevating InR/TOR pathway activity. To test this latter possibility, we assayed whether conditions that abnormally increase Yki activity (loss of either Ex or Wts) have a corresponding effect on the levels of phospho-Akt (pAkt-S505), which are normally elevated by enhanced InR signaling (e.g., by removal of the InR pathway inhibitor PTEN; Fig 2A). We find that pAkt-S505 levels are normal in ex—or wts—clones (Fig 2B and 2C), as well as in protein extracts from the overgrown wing discs of homozygous ex—or wts—larvae (Fig 2D). Likewise, the levels of phosphorylated S6-Kinase (pS6K-T398), a readout of TOR activity, were not affected in ex—or wts—discs (Fig 2E). We therefore infer that Wts/Hpo regulated Yki activity does not stimulate either InR or TOR pathway activity in the wing, consistent with results from Drosophila cell culture [15]. This finding argues against a downstream role of InR/TOR signaling in mediating Yki-driven growth.

Bottom Line: Here, we show that the TOR pathway regulates Yki by a separate and novel mechanism in the Drosophila wing.Instead of controlling Yki nuclear access, TOR signaling governs Yki action after it reaches the nucleus by allowing it to gain access to its target genes.When TOR activity is inhibited, Yki accumulates in the nucleus but is sequestered from its normal growth-promoting target genes--a phenomenon we term "nuclear seclusion." Hence, we posit that in addition to its well-known role in stimulating cellular metabolism in response to nutrients, TOR also promotes wing growth by liberating Yki from nuclear seclusion, a parallel pathway that we propose contributes to the scaling of wing size with nutrient availability.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and Development, Columbia University, New York, New York, United States of America; Division of Biology, Imperial College London, London, United Kingdom.

ABSTRACT
Organ growth is controlled by patterning signals that operate locally (e.g., Wingless/Ints [Wnts], Bone Morphogenetic Proteins [BMPs], and Hedgehogs [Hhs]) and scaled by nutrient-dependent signals that act systemically (e.g., Insulin-like peptides [ILPs] transduced by the Target of Rapamycin [TOR] pathway). How cells integrate these distinct inputs to generate organs of the appropriate size and shape is largely unknown. The transcriptional coactivator Yorkie (Yki, a YES-Associated Protein, or YAP) acts downstream of patterning morphogens and other tissue-intrinsic signals to promote organ growth. Yki activity is regulated primarily by the Warts/Hippo (Wts/Hpo) tumour suppressor pathway, which impedes nuclear access of Yki by a cytoplasmic tethering mechanism. Here, we show that the TOR pathway regulates Yki by a separate and novel mechanism in the Drosophila wing. Instead of controlling Yki nuclear access, TOR signaling governs Yki action after it reaches the nucleus by allowing it to gain access to its target genes. When TOR activity is inhibited, Yki accumulates in the nucleus but is sequestered from its normal growth-promoting target genes--a phenomenon we term "nuclear seclusion." Hence, we posit that in addition to its well-known role in stimulating cellular metabolism in response to nutrients, TOR also promotes wing growth by liberating Yki from nuclear seclusion, a parallel pathway that we propose contributes to the scaling of wing size with nutrient availability.

No MeSH data available.


Related in: MedlinePlus