Fig 1.
Effects of TOR inhibition on wing growth and Yki-driven cell proliferation.
A–C: Adult Drosophila wings. (A) A wing from a well-fed wild type fly. (B) Scaled-down wing produced by raising larvae on nutrient-poor food. (C) Scaled-down wing caused by blocking TOR signaling specifically in wing cells with nubbin.GAL4 UAS.TorTED. (D–G) Wing discs from late third instar larvae-bearing clones of mutant tissue outlined with dashed lines, and marked negatively (“black”) by absence of the GFP marker (green): (D) wild type (control) (E) TorΔP, (F) exe1, (G) exe1 TorΔP. Mutant clones were induced at the end of the first instar, 48±2 hr after egg laying and are associated with sibling “twin-spot” clones marked by two copies of the GFP marker (bright green) that serve as an internal control for the growth of w.t. tissue. Numbers denote mean clone size ratio compared to wt, and asterisks denote significances from t tests (* = p < 0.05, ** = p < 0.01, *** = p < 0.001, n. s. = not significant). In (G), the bottom italicised value is a comparison with the TorΔP genotype. Number of clones measured (n) = 32 (wt), 38 (ex), 51 (Tor), 36 (ex Tor). (H–K) Clones of the same genotypes as in (D–G) that coexpress p35 with GFP-NLS (generated using the MARCM technique [36]. Clones are positively labelled by GFP-NLS, and nuclei are counterstained with Hoechst (blue). (n) = 92 (wt+p35), 90 (ex+p35), 97 (Tor+p35), 79 (Ex Tor+p35). Numbers signify mean clone size ratio compared to wt+p35, and bottom italicised value in (K) with Tor+p35.
Fig 2.
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.
Fig 3.
TOR inhibition increases Yki nuclear accumulation.
(A,C,D) Wing discs expressing GFP-NLS in a stripe of cells under dpp.Gal4 control that either do (C,D, experimental), or do not (A, negative control), coexpress TorTED, TSC1, and TSC2. (B) Wing disc with wtsX1 clones (positive control), marked black by the absence of GFP. Discs are imaged at the level of nuclei and labelled for Yki (red), GFP (green), and DNA (Hoechst, blue). Coexpression of TorTED, TSC1, and TSC2 (C,D), like the loss of Wts (B), causes enhanced nuclear accumulation of Yki, apparent at this plane of focus as increased signal at low magnification (B,C) and by coincidence of the Yki and GFP-NLS signals at high magnification (D, e.g., arrowhead).
Fig 4.
TOR inhibition reduces Yki target gene expression.
Wing discs expressing GFP-NLS (green) under dpp.Gal4 control with (B, D, F, H), or without (A, C, E, G), coexpressing the TOR inhibitors TorTED, TSC1 and TSC2, as in Fig 3. TOR inhibition reduces the Yki targets DIAP1 protein (B), diap12B2C-lacZ (D) fj-lacZ (F) and ex-lacZ (H; kept for 8 hr at 29°C before dissection, to increase the level of TOR inhibition), all labelled in red (nuclei counterstained with Hoechst, blue).
Fig 5.
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).
Fig 6.
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).
Fig 7.
Integrating Yki-dependent wing growth with InR/TOR pathway activity.
(A) Under normal physiological conditions (left panel), Yki shuttles between the nucleus and cytoplasm in response to phosphorylation (P) by Wts, which targets Yki to cytosolic tethers. Wing disc intrinsic signals (e.g., the morphogens Dpp and Wg) drive wing growth by down-regulating Wts activity, allowing a small proportion of Yki to escape phosphorylation-dependent tethering, enter the nucleus, and bind to its growth-promoting target genes in complex with Sd. Inhibiting the InR/TOR pathway results in up-regulation or activation of a putative NSF that sequesters unphosphorylated Yki in the nucleus and impedes binding of the Yki-Sd complex to its target genes, reducing Yki-Sd dependent growth. (B) Proposed integration of InR/TOR and Wts/Yki signaling to scale wing growth. In well-fed animals (left panel), TOR activation by wing disc extrinsic, nutrient-dependent signals facilitates Yki-dependent tissue growth by two parallel means: (i) by inhibiting NSF and thereby potentiating Yki nuclear access and target gene expression, and (ii), by up-regulating cell physiological functions such as dS6-Kinase and possibly many others, to match growth potential to the level of Yki target gene activity. Reductions in InR/TOR activity diminish both outputs, leading to reduced cell proliferation and tissue growth. (C) Excess growth caused by superphysiological activation of TOR is offset by Yki-independent down-regulation of the antiapoptotic factors DIAP1 and bantam, promoting cell death and safeguarding the developing wing against hyperplasia.