E3 ubiquitin ligase Deltex facilitates the expansion of Wingless gradient and antagonizes Wingless signaling through a conserved mechanism of transcriptional effector Armadillo/β-catenin degradation

Vartika Sharma
Nalani Sachan
Bappi Sarkar
Mousumi Mutsuddi
Ashim Mukherjee author has email address

Department of Molecular and Human Genetics, Institute of Science, Banaras Hindu University, Varanasi-221005, Uttar Pradesh, India
Department of Integrative Biology and Physiology University of California Los Angeles California, USA
Department of Cell Biology, NYU Langone Medical Center, New York, USA

https://doi.org/10.7554/eLife.88466.2

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Figures and data

dx genetically interacts with Wg pathway components.
(A1-F3) Representative wings from males with the indicated genotypes. dx (A2) and dx¹⁵² (A3) hemizygote show extra vein material at the distal end of the wing compared to wild-type flies (w¹¹¹⁸) (A1). (B2, B3, and C2, C3) Both the dx allele show an enhancement of wing veinthickening along with wing notching in hemizygous combination with different alleles of wg (wg^CX3 and wg^CX4) heterozygotes. (D2, D3, and E2, E3) Different alleles of fz (fz¹ and fz^MB07478) in trans-heterozygous conditions show enhanced vein thickening and wing nicking phenotype with dx hemizygotes. (F2, F3) dx alleles show strong genetic interaction with the Wg target gene sens, wherea loss-of-function allele of sens (sens^E58) shows wing nicking phenotype in flies that are homozygous for dx alleles. (G) Graph showing the frequency of wing notching phenotypes observed in indicated genetic combinations (n=100). (H1-H4) Representative image of Cut in wing imaginal disc in different geneticcombinations. (H2) The expression of Cut in wg^CX4heterozygote is similar to the wild-type Cut expression (H1). (H3) dx¹⁵²hemizygotes show a reduction in Cut expression in the D/V boundary of the wing disc. (H4) Wing disc from dx¹⁵²/Y; wg^CX4/+ genotype show a further reduction in Cut expression when compared to dx¹⁵²/Y wing discs. Images in H1-H4 are representatives of 3 independent experiments (n=6). Scale bar: A1-F3: 200µm. H1-H4:50µm.

Loss of Dx reduces Wg Signaling gradient and target gene expression.
(A2) dx¹⁵² wing disc shows a narrow Wg expression gradient compared to wild-type third instar larval wing discs (A1). (A1” and A2”) show Wg-lacZ staining in the mentioned genotype. (B2) A constricted expression of Sens was observed in dx null discs (dx¹⁵²) (marked by arrowhead) compared to the control wild-type wing imaginal discs (B1). (C1) The expression of Vg in the wild-type disc. (C2) dx null discs showed a broadened Vg expression, marked by arrowheads. (A1’-C2’) Show the average fluorescence intensity of images in A1-C2. (D1-D2) Expression of the RNAi targeting Dx by en-GAL4 or ap-GAL4 results in morphological aberrations in the expressing regions. (E) Graph showing the percentage of flies showing respective phenotypes in the mentioned genotype (n=100). Images in A-C are representatives of 3 independent experiments (n=6). Scale Bar: A1-A2: 10µm. B1-C2: 50µm. D1-D2: 200µm.

Dx modulates the expression of the Wg target genes.
(A1-A3) Over-expression of Dx using posterior domain specific en-GAL4 results in a complete loss of Senseless (A2). Third instar larval wing discs of the indicated genotypes were immunostained to detect FLAG (green) and Senseless (red). (B1-B3) A reduction in expression of Cut was observed in the posterior domain of the wing disc upon Dx over-expression with en-GAL4. (C1-C3) Expression of Vestigial (Vg), on the contrary, was found to be expanded in the posterior compartment. Arrowheads indicate the expansion of Vg expression close to the A/P boundary of the wing imaginal disc (C2). (D1-E3) Over-expression of caspase inhibitor p35 together with Dx does not show any rescue in the expression of Senseless (D2) and Cut (E2) suggesting the event is independent of apoptosis. The white line marks the boundary of FLAG expression. Images in A-E are representatives of 3 independent experiments (n=6). Scale Bar: A1-E3:50 µm.

Over-expression of Dx expands the Wg diffusion gradient.
(A1-B4) Over-expression of Dx by A/P domain-specific ptc-GAL4 results in the broadening of the Wg diffusion domain (A2). Grayscale images are for better contrast (A3). (B2) High magnification picture shows increased Wg puncta at the A/P-D/V junction with more significant expression in the ventral portion of the disc (marked by arrowhead) (B2 and B3). The white line marks the Ptc domain, respectively. (C1-C3) Dx over-expression with en-GAL4 results in a diffused Wg expression in the posterior domain of the wing disc. (D1-D3) A marked increase in extracellular Wg (eWg) was observed upon Dx over-expression with en-GAL4. (E3) Representative image of wild type Wg expression. Images in A-D are representatives of 3 independent experiments (n=6). Scale Bar: A1-A4, C1-D3, and E: 50µm. B1-B4: 10µm.

Dx-mediated Wg Signaling is regulated by endocytosis.
(A1-A5) Dx (Blue) co-localizes with Rab5 (Green) and Wg (Red) in the same subcellular compartment. The arrows mark the co-localized spots. (A5) High magnification image of (A4). (C1) Over-expression of a Dominant Negative Rab5 with C96-Gal4 inhibits the endosomal fusion at the D/V boundary, enhancing the Wg diffusion gradient. (C2) Co-expression of Dx with Rab5DN shows a further enhancement of the Rab5DN phenotype in the Wg diffusion gradient. (B1-B2) Representative adult wing image of the mentioned genotype (n=50). (B1) Over-expression of Rab5DN results in wing tissue loss consistent with decreased Wg signaling. (B2) A further reduction in wing size is observed upon co-expressing Dx with Rab5DN. (B3) The graph shows the wing area percentage of the mentioned genotypes (n=6) ****P<.0001; unpaired t-test. (E1 and F1) Over-expression of Rab7 with C96-GAL4 renders no significant change in Wg expression. (E2 and F2) Rab7 when co-expressed with Dx enhances the Wg spreading. (D1 and D2) Representative adult wing of defined genotype (n=50). Note the notching phenotype in the wing upon over-expression of FLAG-Dx together with UAS-Rab7 (D2). (D3) The graph shows the percentage of flies showing notching phenotype (n=100). (G and H) Representative adult wing image of UAS-FLAG-Dx over-expressed with C96-GAL4. (H) Wg expression in UAS-FLAG-Dx>C96-GAL4 discs. Images in C and E are representatives of 3 independent experiments (n=6). Scale bar: A1-A4: 5µm. C1-C2 and E1-E2: 50µm. F1-F2: 20µm. B1-B2 and D1-D2: 200µm.

Dx modulates Wg Signal transduction.
(A1-C4) Dx over-expression with en-GAL4 in the wing imaginal disc results in a reduction of endogenous Armadillo throughout the disc with a more pronounced reduction at the D/V boundary. (B1-B3) Confocal Z stack Intensity profiling shows a reduced Arm level in the posterior compartment of the disc. (C1-C4) A higher magnification picture shows a significant overlap between FLAG-tagged Dx and endogenous Arm (marked by arrowhead). Note the reduction of Arm in the posterior compartment of the disc. (C5) Graph shows the average Arm fluorescence intensity (a.u., arbitrary units) (n=7). ****P<.0001; unpaired t-test. (D1-D3) Show the average FLAG and Arm fluorescence intensity profiles for the representative wing disc. (E1-E6) Representative adult wing of defined genotype (n=50). (E1-E2) Over-expression of the activated form of Arm (Arm^S10) with C96-GAL4 shows ectopic hairs more significantly along the wing margin of the adult wing. (E3-E4) Dx over-expression in the wing shows no ectopic hairs. (E5-E6) Co-expression of Dx with ArmS¹⁰ results in the suppression of Arm gain of function effect and a relative reduction in the number of ectopic bristles was observed. (E7) Graph shows the number of bristles in each combination. ****p<.0001; one-way ANOVA/Turkey’s multiple comparisons test. (F1-F6) Representative adult eye and eye imprint of defined genotype (n=10). (F1-F2) GMR-specific expression of an activated form of Arm produces a severely reduced eye with diffused ommatidia. (F3-F4) Dx over-expression renders no significant phenotype. (F5-F6) Arm over-expression phenotype gets dramatically suppressed when Dx was co-expressed in the same background. (F7) Graph represents the phenotypic score of the eye size (n=6). Images in A-C are representatives of 3 independent experiments (n=6). Scale Bar: A1-A3: 10µm. C1-C3: 2µm. E1-E6 and F1-F6: 200µm.

Dx degrades Arm by a proteasome-mediated mechanism.
(A, B) C o - immunoprecipitation of FLAG - Dx and Arm. Co-immunoprecipitation was carried out with lysate over-expressing Arm and FLAG-Dx using GMAR-GAL4. + symbol indicates the presence of lysate and the – symbol shows the absence of lysate. FLAG-Dx immunoprecipitated Arm was detected by anti-Arm antibody (A). Arm immunoprecipitated FLAG-Dx was detected by FLAG antibody. (C1-C3) Dx o v e r - e x p r e s s e d discs treated with MG132 for 3hrs showed a rescue in Arm expression and a more intense Arm staining in the posterior compartment was found after proteasome inhibitor treatment. (D1-D3) Confocal Z stack Intensity profiling shows a comparable Arm level in the disc’s posterior compartment. (E1-E3) A higher magnification picture shows no significant change in the expression of Arm in the posterior compartment of the disc suggesting the rescue in Arm degradation. (F) Graph shows the average Arm fluorescence intensity (a.u., arbitrary units) (n=7). ns; unpaired t-test. (G) Western blot analysis confirms the immunocytochemical studies where proteasome inhibitor MG132 increases Arm protein levels in Dx over-expressed tissue samples. (H) Graph G represents the intensity profilingof the Western blot. ***P<.001; unpaired t-test. Images in A-F are representatives of 3 independent experiments (n=6). Scale Bar: Scale Bar: A1-A4: 10µm. C1-C4: 2µm.

DTX1 physically interacts with β-catenin and facilitates its degradation.
(A) HA-tagged human DTX1 transfected in HEK-293 cell line was pulled down with an anti-HA antibody. Immunoblot was done with β-catenin. A prominent band at 92kDa corresponding to β-catenin post-MG132 treatment was observed. GAPDH was used as an internal control. (B) A rescue in β-catenin level post MG132 treatment was observed. GAPDH serves as an internal control. (C) Graph C represents the intensity profiling of the Western Blot in C. **P < 0.01, ***P<.001; one-way ANOVA/Turkey’s multiple comparisons test. Images in A-F are representatives of 3 independent experiments. (D) The cartoon explains the two plausible mechanisms of Wg regulation through Dx. Dx facilitates Wg gradient formation on one hand and on the other it targets Arm for its degradation thereby regulating the signaling output.

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