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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

Superphysiological TOR activity causes excess, Yki-independent growth that is offset by a negative feedback that down-regulates the anti apoptic factors DIAP and bantam.(A) Wing disc carrying a clone of Tsc1Q87X mutant cells (marked black by absence of GFP, green, and outlined with a dashed white line; counterstained with Hoechst, blue). Yki accumulation (red), imaged at the nuclear plane, is unchanged in the clone. (B) Yki nucleocytoplasmic distribution is similar in wild-type and Tsc1—discs. Effective separation of cell fractions was confirmed by Tubulin (cytoplasm) and Histone 3 (nucleus). (C, D) Wing discs carrying Tsc1Q87X mutant clones (marked and imaged as in A): expression of the Yki target genes fj-lacZ (C) and ex-lacZ (D) (red) is not affected. (E, F) Compared to wild type discs (E), expression of Rheb in a stripe under ptc.GAL4 control (F) causes a reduction in DIAP accumulation (red), as well as bantam micro-RNA activity, the latter indicated by relief of repression of a bantam-GFP sensor (green) [54]; peak activity of the ptc.Gal4 driver is indicated by expression of β-galactosidase from a UAS.lacZ transgene, magenta). (G) Protein extracts of wild type, Tsc1Q87X/Tsc1PA23, and homozygous wtsP2 (positive control) discs probed for DIAP1 protein reveal that DIAP1 is strongly reduced in Tsc1—discs. β-actin was used as a loading control. (H) Phospho-Yki S168 levels Tsc1—homozygous mutants discs are not elevated (and are in fact mildly reduced) compared to wild type control discs (total Yki and β-actin were used as loading controls; CIP treatment was used to ensure the correct product was being observed). (I)Tsc1—discs do not show a reduction in Yki enrichment at a Yki responsive enhancer in the bantam locus compared to wild-type discs. IgG mock IP and enrichment at the PDH (pyruvate dehydrogenase) locus were included as controls. (J, K)hdc-GAL4 wing discs that either do (K, experimental) or do not (J, control) express a UAS.rheb transgene, labelled for active caspase III (green): both discs are approximately the same size (counterstained with Hoechst, blue), but the experimental disc shows pronounced Caspase activity in contrast to the control. (L–N) Coexpressing a UAS.diap transgene together with UAS.rheb (L) prevents cell death caused by expression of UAS.rheb (K) and results in tissue hyperplasia, as indicated by the increase in disc size. Hyperplasia is further increased by the addition of bantam expression under the direct control of the Tubα1 promoter (M). (N) Quantification of disc sizes. 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 = 20 (wt), 24 (Rheb), 18 (diap1), 10 (tub-ban), 20 (rheb+diap1), 15 (rheb+tub-ban), 22 (rheb+diap1+tub-ban).
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pbio.1002274.g006: Superphysiological TOR activity causes excess, Yki-independent growth that is offset by a negative feedback that down-regulates the anti apoptic factors DIAP and bantam.(A) Wing disc carrying a clone of Tsc1Q87X mutant cells (marked black by absence of GFP, green, and outlined with a dashed white line; counterstained with Hoechst, blue). Yki accumulation (red), imaged at the nuclear plane, is unchanged in the clone. (B) Yki nucleocytoplasmic distribution is similar in wild-type and Tsc1—discs. Effective separation of cell fractions was confirmed by Tubulin (cytoplasm) and Histone 3 (nucleus). (C, D) Wing discs carrying Tsc1Q87X mutant clones (marked and imaged as in A): expression of the Yki target genes fj-lacZ (C) and ex-lacZ (D) (red) is not affected. (E, F) Compared to wild type discs (E), expression of Rheb in a stripe under ptc.GAL4 control (F) causes a reduction in DIAP accumulation (red), as well as bantam micro-RNA activity, the latter indicated by relief of repression of a bantam-GFP sensor (green) [54]; peak activity of the ptc.Gal4 driver is indicated by expression of β-galactosidase from a UAS.lacZ transgene, magenta). (G) Protein extracts of wild type, Tsc1Q87X/Tsc1PA23, and homozygous wtsP2 (positive control) discs probed for DIAP1 protein reveal that DIAP1 is strongly reduced in Tsc1—discs. β-actin was used as a loading control. (H) Phospho-Yki S168 levels Tsc1—homozygous mutants discs are not elevated (and are in fact mildly reduced) compared to wild type control discs (total Yki and β-actin were used as loading controls; CIP treatment was used to ensure the correct product was being observed). (I)Tsc1—discs do not show a reduction in Yki enrichment at a Yki responsive enhancer in the bantam locus compared to wild-type discs. IgG mock IP and enrichment at the PDH (pyruvate dehydrogenase) locus were included as controls. (J, K)hdc-GAL4 wing discs that either do (K, experimental) or do not (J, control) express a UAS.rheb transgene, labelled for active caspase III (green): both discs are approximately the same size (counterstained with Hoechst, blue), but the experimental disc shows pronounced Caspase activity in contrast to the control. (L–N) Coexpressing a UAS.diap transgene together with UAS.rheb (L) prevents cell death caused by expression of UAS.rheb (K) and results in tissue hyperplasia, as indicated by the increase in disc size. Hyperplasia is further increased by the addition of bantam expression under the direct control of the Tubα1 promoter (M). (N) Quantification of disc sizes. 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 = 20 (wt), 24 (Rheb), 18 (diap1), 10 (tub-ban), 20 (rheb+diap1), 15 (rheb+tub-ban), 22 (rheb+diap1+tub-ban).

Mentions: Our results describe how inhibiting TOR signaling reduces Yki function as part of the mechanism that might scale wing growth downwards in response to nutritional deprivation. To further elucidate how TOR and Yki might function together to control wing size, we asked whether superphysiological TOR activity might alter Yki localization and/or activity to scale wing size upwards. However, we were unable to detect any such changes in Yki using either of two well-established approaches to overactivate the InR/TOR signaling in vivo. Specifically, overactivating TOR by removing the negative regulator TSC1 in wing cells did not appear to alter the nuclear-cytoplasmic distribution of Yki as assayed either by immunofluorescence in clones of Tsc1—mutant cells (Fig 6A) or by quantitating the nuclear–cytoplasmic ratio of Yki in fractionated whole wing discs from entirely Tsc1—mutant larvae (Fig 6B). Similarly, we tested whether clonal removal of TSC1, or alternatively, clonal overexpression of the positive regulator Rheb, affects the expression of any of several Yki target genes, and again, observed no differences (Fig 6C and 6D; S9A and S9B Fig). Hence, superphysiological gains in TOR activity do not appear to influence either Yki localization or activity, suggesting that peak endogenous TOR signaling is sufficient to fully override Yki nuclear seclusion as well as any other InR/TOR-dependent constraints on Yki coactivator function.


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

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

Superphysiological TOR activity causes excess, Yki-independent growth that is offset by a negative feedback that down-regulates the anti apoptic factors DIAP and bantam.(A) Wing disc carrying a clone of Tsc1Q87X mutant cells (marked black by absence of GFP, green, and outlined with a dashed white line; counterstained with Hoechst, blue). Yki accumulation (red), imaged at the nuclear plane, is unchanged in the clone. (B) Yki nucleocytoplasmic distribution is similar in wild-type and Tsc1—discs. Effective separation of cell fractions was confirmed by Tubulin (cytoplasm) and Histone 3 (nucleus). (C, D) Wing discs carrying Tsc1Q87X mutant clones (marked and imaged as in A): expression of the Yki target genes fj-lacZ (C) and ex-lacZ (D) (red) is not affected. (E, F) Compared to wild type discs (E), expression of Rheb in a stripe under ptc.GAL4 control (F) causes a reduction in DIAP accumulation (red), as well as bantam micro-RNA activity, the latter indicated by relief of repression of a bantam-GFP sensor (green) [54]; peak activity of the ptc.Gal4 driver is indicated by expression of β-galactosidase from a UAS.lacZ transgene, magenta). (G) Protein extracts of wild type, Tsc1Q87X/Tsc1PA23, and homozygous wtsP2 (positive control) discs probed for DIAP1 protein reveal that DIAP1 is strongly reduced in Tsc1—discs. β-actin was used as a loading control. (H) Phospho-Yki S168 levels Tsc1—homozygous mutants discs are not elevated (and are in fact mildly reduced) compared to wild type control discs (total Yki and β-actin were used as loading controls; CIP treatment was used to ensure the correct product was being observed). (I)Tsc1—discs do not show a reduction in Yki enrichment at a Yki responsive enhancer in the bantam locus compared to wild-type discs. IgG mock IP and enrichment at the PDH (pyruvate dehydrogenase) locus were included as controls. (J, K)hdc-GAL4 wing discs that either do (K, experimental) or do not (J, control) express a UAS.rheb transgene, labelled for active caspase III (green): both discs are approximately the same size (counterstained with Hoechst, blue), but the experimental disc shows pronounced Caspase activity in contrast to the control. (L–N) Coexpressing a UAS.diap transgene together with UAS.rheb (L) prevents cell death caused by expression of UAS.rheb (K) and results in tissue hyperplasia, as indicated by the increase in disc size. Hyperplasia is further increased by the addition of bantam expression under the direct control of the Tubα1 promoter (M). (N) Quantification of disc sizes. 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 = 20 (wt), 24 (Rheb), 18 (diap1), 10 (tub-ban), 20 (rheb+diap1), 15 (rheb+tub-ban), 22 (rheb+diap1+tub-ban).
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pbio.1002274.g006: Superphysiological TOR activity causes excess, Yki-independent growth that is offset by a negative feedback that down-regulates the anti apoptic factors DIAP and bantam.(A) Wing disc carrying a clone of Tsc1Q87X mutant cells (marked black by absence of GFP, green, and outlined with a dashed white line; counterstained with Hoechst, blue). Yki accumulation (red), imaged at the nuclear plane, is unchanged in the clone. (B) Yki nucleocytoplasmic distribution is similar in wild-type and Tsc1—discs. Effective separation of cell fractions was confirmed by Tubulin (cytoplasm) and Histone 3 (nucleus). (C, D) Wing discs carrying Tsc1Q87X mutant clones (marked and imaged as in A): expression of the Yki target genes fj-lacZ (C) and ex-lacZ (D) (red) is not affected. (E, F) Compared to wild type discs (E), expression of Rheb in a stripe under ptc.GAL4 control (F) causes a reduction in DIAP accumulation (red), as well as bantam micro-RNA activity, the latter indicated by relief of repression of a bantam-GFP sensor (green) [54]; peak activity of the ptc.Gal4 driver is indicated by expression of β-galactosidase from a UAS.lacZ transgene, magenta). (G) Protein extracts of wild type, Tsc1Q87X/Tsc1PA23, and homozygous wtsP2 (positive control) discs probed for DIAP1 protein reveal that DIAP1 is strongly reduced in Tsc1—discs. β-actin was used as a loading control. (H) Phospho-Yki S168 levels Tsc1—homozygous mutants discs are not elevated (and are in fact mildly reduced) compared to wild type control discs (total Yki and β-actin were used as loading controls; CIP treatment was used to ensure the correct product was being observed). (I)Tsc1—discs do not show a reduction in Yki enrichment at a Yki responsive enhancer in the bantam locus compared to wild-type discs. IgG mock IP and enrichment at the PDH (pyruvate dehydrogenase) locus were included as controls. (J, K)hdc-GAL4 wing discs that either do (K, experimental) or do not (J, control) express a UAS.rheb transgene, labelled for active caspase III (green): both discs are approximately the same size (counterstained with Hoechst, blue), but the experimental disc shows pronounced Caspase activity in contrast to the control. (L–N) Coexpressing a UAS.diap transgene together with UAS.rheb (L) prevents cell death caused by expression of UAS.rheb (K) and results in tissue hyperplasia, as indicated by the increase in disc size. Hyperplasia is further increased by the addition of bantam expression under the direct control of the Tubα1 promoter (M). (N) Quantification of disc sizes. 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 = 20 (wt), 24 (Rheb), 18 (diap1), 10 (tub-ban), 20 (rheb+diap1), 15 (rheb+tub-ban), 22 (rheb+diap1+tub-ban).
Mentions: Our results describe how inhibiting TOR signaling reduces Yki function as part of the mechanism that might scale wing growth downwards in response to nutritional deprivation. To further elucidate how TOR and Yki might function together to control wing size, we asked whether superphysiological TOR activity might alter Yki localization and/or activity to scale wing size upwards. However, we were unable to detect any such changes in Yki using either of two well-established approaches to overactivate the InR/TOR signaling in vivo. Specifically, overactivating TOR by removing the negative regulator TSC1 in wing cells did not appear to alter the nuclear-cytoplasmic distribution of Yki as assayed either by immunofluorescence in clones of Tsc1—mutant cells (Fig 6A) or by quantitating the nuclear–cytoplasmic ratio of Yki in fractionated whole wing discs from entirely Tsc1—mutant larvae (Fig 6B). Similarly, we tested whether clonal removal of TSC1, or alternatively, clonal overexpression of the positive regulator Rheb, affects the expression of any of several Yki target genes, and again, observed no differences (Fig 6C and 6D; S9A and S9B Fig). Hence, superphysiological gains in TOR activity do not appear to influence either Yki localization or activity, suggesting that peak endogenous TOR signaling is sufficient to fully override Yki nuclear seclusion as well as any other InR/TOR-dependent constraints on Yki coactivator function.

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