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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

TOR inhibition results in Yki nuclear seclusion and depends on the conserved Sd binding and WW domains but not the Wts phosphorylation sites of Yki.(A, B) Confocal sections of Act5C>CD2>GAL4 wing discs that either do (B, experimental) or do not (A, control) coexpress TorTED, TSC1, and TSC2 in most cells 8–10 hr after heat shock to excise the >CD2> stop cassette. Yki (red), CD2 (magenta), SdGFP (from a GFP knockin allele of sd; green) and DNA (blue) are shown at the level of nuclei. Control discs (expressing only GFP-NLS) have no effect on Yki localistion (A) in contrast to experimental discs coexpressing TORTED, TSC1, and TSC2, which show enhanced nuclear accumulation of Yki as indicated by increased signal at low magnification (B; arrowheads show clusters of Act5C>CD2>GAL4 cells in which the >CD2> stop cassette was not excised, which coincide with reduced Yki nuclear staining). (C, D) ChIP of Yki (C) or SdGFP (D; using the sdGFP allele and anti-GFP antibody) with mock IP (IgG) at the 2B2C diap1 enhancer and a control locus (PDH: pyruvate dehydrogenase) in control and experimental discs, as in (A,B). 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 = 4 independent experimental replicates. Yki and SdGFP are strongly enriched at 2B2C in control (w.t.) discs compared to PDH controls. Enrichment of both proteins is reduced in experimental (>TORTED/TSC1&TSC2) discs; (E) Conserved functional domains of Yki. (F–H) Wing discs uniformly expressing w.t. (F), P88L (G), or WW domain mutant (W292A P295A W361A P364A) (H) forms of GFP tagged Yki that also express TORTED, TSC1, and TSC2 in a stripe under dpp.Gal4 control (as in Figs 3 and 4; the dashed white lines indicate the A/P compartment boundary, which abuts the right edge of the dpp.Gal4 expressing stripe). The discs are imaged at the level of nuclei and show nuclear accumulation of the w.t. (F) but neither the P88L (G) or WW (H) mutant, forms of Yki, as indicated by signal intensity. (I, J) Confocal sections of wing discs taken at the level of nuclei to the assess the nuclear accumulation of wild-type Yki-GFP (I), as well as a mutant form of Yki-GFP (J), that carries both the P88L substitution (which blocks nuclear accumulation in response to TOR inhibition, G) as well as the S111A, S168A, and S250A (S3->A) substitutions (which obviate phosphorylation of Yki by Wts and cause otherwise wild-type Yki to accumulate in the nucleus). Both proteins are expressed in clones under the direct control of the Tubα1 promoter following Flp-out cassette excision of a Tubα1>DsRed>yki-GFP transgene: the clones are marked black by the absence of DsRed expression (magenta). Wild-type Yki-GFP (I) appears predominantly cytosolic, whereas YkiP88L S3->A-GFP (J) appears much more nuclear, as indicated by the difference in staining patterns imaged at the nuclear plane—largely absent in nuclei for (I) and relatively uniform for (J): hence, the P88L mutation, which blocks Yki nuclear accumulation in response to TOR inhibition does not preclude nuclear accumulation in the absence of phosphorylation by Wts. (K) YkiP88L-GFP expressing wing disc carrying clones of wts—clones (outlined with dashed white lines). Nuclear accumulation of the YkiP88L-GFP protein is elevated in the absence of phosphorylation by Wts, as indicated by increased GFP signal intensity imaged at the nuclear plane, corroborating the results in (I,J).
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pbio.1002274.g005: TOR inhibition results in Yki nuclear seclusion and depends on the conserved Sd binding and WW domains but not the Wts phosphorylation sites of Yki.(A, B) Confocal sections of Act5C>CD2>GAL4 wing discs that either do (B, experimental) or do not (A, control) coexpress TorTED, TSC1, and TSC2 in most cells 8–10 hr after heat shock to excise the >CD2> stop cassette. Yki (red), CD2 (magenta), SdGFP (from a GFP knockin allele of sd; green) and DNA (blue) are shown at the level of nuclei. Control discs (expressing only GFP-NLS) have no effect on Yki localistion (A) in contrast to experimental discs coexpressing TORTED, TSC1, and TSC2, which show enhanced nuclear accumulation of Yki as indicated by increased signal at low magnification (B; arrowheads show clusters of Act5C>CD2>GAL4 cells in which the >CD2> stop cassette was not excised, which coincide with reduced Yki nuclear staining). (C, D) ChIP of Yki (C) or SdGFP (D; using the sdGFP allele and anti-GFP antibody) with mock IP (IgG) at the 2B2C diap1 enhancer and a control locus (PDH: pyruvate dehydrogenase) in control and experimental discs, as in (A,B). 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 = 4 independent experimental replicates. Yki and SdGFP are strongly enriched at 2B2C in control (w.t.) discs compared to PDH controls. Enrichment of both proteins is reduced in experimental (>TORTED/TSC1&TSC2) discs; (E) Conserved functional domains of Yki. (F–H) Wing discs uniformly expressing w.t. (F), P88L (G), or WW domain mutant (W292A P295A W361A P364A) (H) forms of GFP tagged Yki that also express TORTED, TSC1, and TSC2 in a stripe under dpp.Gal4 control (as in Figs 3 and 4; the dashed white lines indicate the A/P compartment boundary, which abuts the right edge of the dpp.Gal4 expressing stripe). The discs are imaged at the level of nuclei and show nuclear accumulation of the w.t. (F) but neither the P88L (G) or WW (H) mutant, forms of Yki, as indicated by signal intensity. (I, J) Confocal sections of wing discs taken at the level of nuclei to the assess the nuclear accumulation of wild-type Yki-GFP (I), as well as a mutant form of Yki-GFP (J), that carries both the P88L substitution (which blocks nuclear accumulation in response to TOR inhibition, G) as well as the S111A, S168A, and S250A (S3->A) substitutions (which obviate phosphorylation of Yki by Wts and cause otherwise wild-type Yki to accumulate in the nucleus). Both proteins are expressed in clones under the direct control of the Tubα1 promoter following Flp-out cassette excision of a Tubα1>DsRed>yki-GFP transgene: the clones are marked black by the absence of DsRed expression (magenta). Wild-type Yki-GFP (I) appears predominantly cytosolic, whereas YkiP88L S3->A-GFP (J) appears much more nuclear, as indicated by the difference in staining patterns imaged at the nuclear plane—largely absent in nuclei for (I) and relatively uniform for (J): hence, the P88L mutation, which blocks Yki nuclear accumulation in response to TOR inhibition does not preclude nuclear accumulation in the absence of phosphorylation by Wts. (K) YkiP88L-GFP expressing wing disc carrying clones of wts—clones (outlined with dashed white lines). Nuclear accumulation of the YkiP88L-GFP protein is elevated in the absence of phosphorylation by Wts, as indicated by increased GFP signal intensity imaged at the nuclear plane, corroborating the results in (I,J).

Mentions: To test this possibility directly, we focused on diap1, the locus where the general mechanism of transcriptional control by Yki is best characterized, with a defined enhancer element, 2B2C, that is controlled exclusively by Yki in complex with Sd (henceforth Yki-Sd) [14,18,19,48]. We prepared chromatin from control and TOR-inhibited wing discs (the latter coexpressing TORTED+TSC1+TSC2 under the control of a Flp-out Act5C>CD2>Gal4 driver) and immune-precipitated using a Yki antibody [15]. To produce enough tissue despite TOR inhibition, discs were allowed to grow to a large size before a strong heat shock was used to excise the stop cassette within the Act5C>CD2>Gal4 driver and initiate Gal4 expression in nearly all cells (Fig 5A and 5B; loss of magenta CD2 marks the Act5C>Gal4 expressing tissue), with dissection of 200 discs per genotype 8–10 hr later (see Methods).


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

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

TOR inhibition results in Yki nuclear seclusion and depends on the conserved Sd binding and WW domains but not the Wts phosphorylation sites of Yki.(A, B) Confocal sections of Act5C>CD2>GAL4 wing discs that either do (B, experimental) or do not (A, control) coexpress TorTED, TSC1, and TSC2 in most cells 8–10 hr after heat shock to excise the >CD2> stop cassette. Yki (red), CD2 (magenta), SdGFP (from a GFP knockin allele of sd; green) and DNA (blue) are shown at the level of nuclei. Control discs (expressing only GFP-NLS) have no effect on Yki localistion (A) in contrast to experimental discs coexpressing TORTED, TSC1, and TSC2, which show enhanced nuclear accumulation of Yki as indicated by increased signal at low magnification (B; arrowheads show clusters of Act5C>CD2>GAL4 cells in which the >CD2> stop cassette was not excised, which coincide with reduced Yki nuclear staining). (C, D) ChIP of Yki (C) or SdGFP (D; using the sdGFP allele and anti-GFP antibody) with mock IP (IgG) at the 2B2C diap1 enhancer and a control locus (PDH: pyruvate dehydrogenase) in control and experimental discs, as in (A,B). 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 = 4 independent experimental replicates. Yki and SdGFP are strongly enriched at 2B2C in control (w.t.) discs compared to PDH controls. Enrichment of both proteins is reduced in experimental (>TORTED/TSC1&TSC2) discs; (E) Conserved functional domains of Yki. (F–H) Wing discs uniformly expressing w.t. (F), P88L (G), or WW domain mutant (W292A P295A W361A P364A) (H) forms of GFP tagged Yki that also express TORTED, TSC1, and TSC2 in a stripe under dpp.Gal4 control (as in Figs 3 and 4; the dashed white lines indicate the A/P compartment boundary, which abuts the right edge of the dpp.Gal4 expressing stripe). The discs are imaged at the level of nuclei and show nuclear accumulation of the w.t. (F) but neither the P88L (G) or WW (H) mutant, forms of Yki, as indicated by signal intensity. (I, J) Confocal sections of wing discs taken at the level of nuclei to the assess the nuclear accumulation of wild-type Yki-GFP (I), as well as a mutant form of Yki-GFP (J), that carries both the P88L substitution (which blocks nuclear accumulation in response to TOR inhibition, G) as well as the S111A, S168A, and S250A (S3->A) substitutions (which obviate phosphorylation of Yki by Wts and cause otherwise wild-type Yki to accumulate in the nucleus). Both proteins are expressed in clones under the direct control of the Tubα1 promoter following Flp-out cassette excision of a Tubα1>DsRed>yki-GFP transgene: the clones are marked black by the absence of DsRed expression (magenta). Wild-type Yki-GFP (I) appears predominantly cytosolic, whereas YkiP88L S3->A-GFP (J) appears much more nuclear, as indicated by the difference in staining patterns imaged at the nuclear plane—largely absent in nuclei for (I) and relatively uniform for (J): hence, the P88L mutation, which blocks Yki nuclear accumulation in response to TOR inhibition does not preclude nuclear accumulation in the absence of phosphorylation by Wts. (K) YkiP88L-GFP expressing wing disc carrying clones of wts—clones (outlined with dashed white lines). Nuclear accumulation of the YkiP88L-GFP protein is elevated in the absence of phosphorylation by Wts, as indicated by increased GFP signal intensity imaged at the nuclear plane, corroborating the results in (I,J).
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Related In: Results  -  Collection

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

pbio.1002274.g005: TOR inhibition results in Yki nuclear seclusion and depends on the conserved Sd binding and WW domains but not the Wts phosphorylation sites of Yki.(A, B) Confocal sections of Act5C>CD2>GAL4 wing discs that either do (B, experimental) or do not (A, control) coexpress TorTED, TSC1, and TSC2 in most cells 8–10 hr after heat shock to excise the >CD2> stop cassette. Yki (red), CD2 (magenta), SdGFP (from a GFP knockin allele of sd; green) and DNA (blue) are shown at the level of nuclei. Control discs (expressing only GFP-NLS) have no effect on Yki localistion (A) in contrast to experimental discs coexpressing TORTED, TSC1, and TSC2, which show enhanced nuclear accumulation of Yki as indicated by increased signal at low magnification (B; arrowheads show clusters of Act5C>CD2>GAL4 cells in which the >CD2> stop cassette was not excised, which coincide with reduced Yki nuclear staining). (C, D) ChIP of Yki (C) or SdGFP (D; using the sdGFP allele and anti-GFP antibody) with mock IP (IgG) at the 2B2C diap1 enhancer and a control locus (PDH: pyruvate dehydrogenase) in control and experimental discs, as in (A,B). 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 = 4 independent experimental replicates. Yki and SdGFP are strongly enriched at 2B2C in control (w.t.) discs compared to PDH controls. Enrichment of both proteins is reduced in experimental (>TORTED/TSC1&TSC2) discs; (E) Conserved functional domains of Yki. (F–H) Wing discs uniformly expressing w.t. (F), P88L (G), or WW domain mutant (W292A P295A W361A P364A) (H) forms of GFP tagged Yki that also express TORTED, TSC1, and TSC2 in a stripe under dpp.Gal4 control (as in Figs 3 and 4; the dashed white lines indicate the A/P compartment boundary, which abuts the right edge of the dpp.Gal4 expressing stripe). The discs are imaged at the level of nuclei and show nuclear accumulation of the w.t. (F) but neither the P88L (G) or WW (H) mutant, forms of Yki, as indicated by signal intensity. (I, J) Confocal sections of wing discs taken at the level of nuclei to the assess the nuclear accumulation of wild-type Yki-GFP (I), as well as a mutant form of Yki-GFP (J), that carries both the P88L substitution (which blocks nuclear accumulation in response to TOR inhibition, G) as well as the S111A, S168A, and S250A (S3->A) substitutions (which obviate phosphorylation of Yki by Wts and cause otherwise wild-type Yki to accumulate in the nucleus). Both proteins are expressed in clones under the direct control of the Tubα1 promoter following Flp-out cassette excision of a Tubα1>DsRed>yki-GFP transgene: the clones are marked black by the absence of DsRed expression (magenta). Wild-type Yki-GFP (I) appears predominantly cytosolic, whereas YkiP88L S3->A-GFP (J) appears much more nuclear, as indicated by the difference in staining patterns imaged at the nuclear plane—largely absent in nuclei for (I) and relatively uniform for (J): hence, the P88L mutation, which blocks Yki nuclear accumulation in response to TOR inhibition does not preclude nuclear accumulation in the absence of phosphorylation by Wts. (K) YkiP88L-GFP expressing wing disc carrying clones of wts—clones (outlined with dashed white lines). Nuclear accumulation of the YkiP88L-GFP protein is elevated in the absence of phosphorylation by Wts, as indicated by increased GFP signal intensity imaged at the nuclear plane, corroborating the results in (I,J).
Mentions: To test this possibility directly, we focused on diap1, the locus where the general mechanism of transcriptional control by Yki is best characterized, with a defined enhancer element, 2B2C, that is controlled exclusively by Yki in complex with Sd (henceforth Yki-Sd) [14,18,19,48]. We prepared chromatin from control and TOR-inhibited wing discs (the latter coexpressing TORTED+TSC1+TSC2 under the control of a Flp-out Act5C>CD2>Gal4 driver) and immune-precipitated using a Yki antibody [15]. To produce enough tissue despite TOR inhibition, discs were allowed to grow to a large size before a strong heat shock was used to excise the stop cassette within the Act5C>CD2>Gal4 driver and initiate Gal4 expression in nearly all cells (Fig 5A and 5B; loss of magenta CD2 marks the Act5C>Gal4 expressing tissue), with dissection of 200 discs per genotype 8–10 hr later (see Methods).

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