Ectopic expression of Egr/TNFα gives increased ROS and is suppressed by increased SOD activity.

<p><b>(A)</b> Ectopic expression of GFP and Egr/TNFα with <i>hh-GAL4</i> in the posterior portion of wing discs of wandering third instar larvae, GFP shows the region of ectopic expression (outlined by dotted line). <b>(B)</b> Punctate CellRox staining revealed increased ROS levels at posterior edge of disc. <b>(C)</b> Merged image with GFP in green and CellRox in red. <b>(D)</b> Control eye phenotype (<i>GMR>EV</i>) showing regular ordered arrays of ommatidial units on the surface of the adult eye <b>(E-F)</b> Ectopic expression of SOD1 or SOD2 alone has no effect. <b>(G)</b>. Ectopic expression of Egr/TNFα in the eye (GMR><i>egr</i>^<i>+w</i><i>>EV</i>) results in a decrease in eye size and disruption to ommatidial patterning. <b>(H-I)</b> The ectopic Egr/TNFα phenotype is obviously suppressed by increased levels of SOD1 or SOD2.</p

Altered WWOX modifies ectopic Egr/TNFα-mediated eye phenotype.

<p><b>(A)</b> Ectopic expression of Egr/TNFα (GMR><i>egr</i>^<i>+w</i><i>>empty vector (EV)</i>) results in a decrease in eye size and disruption to ommatidial patterning. <b>(B)</b> The ectopic Egr/TNFα phenotype is completely suppressed by decreased levels of TNFR (GMR><i>egr</i>^<i>+w</i><i>>wengen/TNFR-IR</i>). <b>(C)</b> Decreased expression of WWOX by RNAi knockdown (<i>GMR>egr</i>^<i>+w</i><i>>WWOX-IR</i>^<i>#1</i>) results in suppression of the rough eye phenotype. <b>(D)</b> Quantification of increased eye size with independent WWOX knockdown constructs (<i>GMR>egr</i>^<i>+w</i><i>>WWOX-IR</i>^<i>#1</i> and <i>GMR>egr</i>^<i>+w</i><i>>WWOX-IR</i>^<i>#2</i><i>)</i>. <b>(E)</b> Increased expression of WWOX by ectopic expression of the WWOX cDNA (GMR><i>egr</i>^<i>+w</i><i>>WWOX-cDNA</i>) resulted in an enhancement of the Eiger/TNFα phenotype. <b>(F)</b> Quantification of decreased eye size with independent ectopic expression constructs for WWOX (GMR><i>egr</i>^<i>+w</i><i>>WWOX-ORF</i> and GMR><i>egr</i>^<i>+w</i><i>>WWOX-cDNA)</i>. Significance indicated by **** = p <0.0001, * = p<0.05 and ns = not significant.</p

Ectopic Egr/TNFα has no obvious effect on cytoplasmically localized WWOX.

<p><b>(A)</b> Ectopic expression of GFP and WWOX in the posterior portion of wing discs of wandering third instar larvae <i>(hh>GFP>WWOX)</i>, GFP shows the region of ectopic expression. <b>(B)</b> WWOX staining reveals expression localized to areas complementary to the DAPI stained nuclei shown in <b>(C)</b> with the merged image shown in <b>(D)</b>. <b>(E-H)</b> Ectopic expression of WWOX in the presence of ectopic Egr/TNFα <i>(hh>GFP>WWOX>egr</i>^<i>+w</i><i>)</i> also results in cytoplasmic localisation of WWOX. Nuclei/DAPI staining shown in blue and WWOX staining in magenta. Boxed regions shown in A and E correspond to regions that are enlarged in B-D and F-H respectively.</p

WWOX is required for elimination of <i>Scribbled (Scrib)</i> mutant clones.

<p><b>(A-A’)</b> Clones of cells mutant for <i>Scrib</i> generated in the eye using the MARCM system are positively labelled with GFP. <b>(B-B’)</b> Elav staining reveals absence of differentiated photoreceptors within a portion of <i>Scrib</i> mutant clones. <b>(C-C’)</b> Merged image showing GFP in green and Elav in magenta. <b>(D-D’)</b> GFP expressing <i>Scrib</i> mutant clones with decreased WWOX expression <i>(Scrib</i>^<i>1</i>; <i>WWOX-IR)</i>. <b>(E-E’)</b> Elav staining reveals absence of differentiated photoreceptors within a portion of the <i>Scrib</i>^<i>1</i>;<i>WWOX-IR</i> mutant clones. <b>(F-F’)</b> Merged image showing GFP in green and Elav in magenta. Red boxes indicate the regions that are enlarged in D’-F’. <b>(G)</b> Quantification of the proportion of GFP expressing cells showed a significant increase when WWOX expression was decreased in the <i>Scrib</i> mutant clonal tissue compared to the <i>Scrib</i> mutant clones alone. <b>(H)</b> Quantification of total area of the eye disc containing <i>Scrib</i> clones with and without WWOX knockdown revealed no significant difference. <b>(I)</b> Clones of cells mutant for <i>Scrib</i> generated in the eye using the MARCM system result in a mild adult rough eye phenotype. <b>(J)</b> Decreased WWOX expression in the <i>Scrib</i> mutant clones gave a stronger phenotype with a decreased eye size, significant disruption to ommatidial patterning and the presence of necrotic lesions. <b>(K)</b> Quantification of the overall size of the adult eyes showed a significant decrease when WWOX levels were knocked down in <i>Scrib</i> mutant clones. <b>(L)</b> Quantification of percentage of adult fly eye showing necrotic spots of different sizes: Small (550–3000 pixels), Medium (3000–5500 pixels) or Large (>5500 pixels). Genotypes used in these experiments: <i>Scrib</i>^<i>1</i> = <i>(ey-FLP1</i>, <i>UAS-mCD8-GFP;;tub-GAL4 FRT82B tub-GAL80/ FRT82B</i>, <i>Scrib</i>¹<i>)</i>, <i>Scrib</i>^<i>1</i>;<i>WWOX-IR</i> = <i>(ey-FLP1</i>, <i>UAS-mCD8-GFP; UAS-WWOX-IR#2/+;tub-GAL4 FRT82B tub-GAL80/ FRT82B</i>, <i>Scrib</i>¹<i>)</i>. Significance indicated by * = p<0.05 and ** = p<0.005, ns = not significant.</p

WWOX modifies Caspase3 staining in wing pouch in response to ectopic Egr/TNFα.

<p><b>(A)</b> Ectopic expression of GFP and Eiger/TNFα with <i>hh-GAL4</i> in the posterior portion of wing discs of wandering third instar larvae, GFP showing the regions of ectopic expression. <b>(B)</b> Caspase 3 staining reveals high levels of apoptosis in the central wing pouch region as well as in two distinct regions extending towards the anterior. <b>(C)</b> Decreased WWOX expression results in a decrease in area of GFP expression. <b>(D)</b> Decreased WWOX expression results in a decreased region of Caspase 3 staining. <b>(E)</b> Increased WWOX expression results in an increase in area of GFP expression. <b>(F)</b> Increased WWOX expression results in a increased region of Caspase 3 staining. <b>(G)</b> Quantification of the area of Caspase 3 staining as a proportion of the area of the whole disc for individual wing discs of each genotype. Significance indicated by * = p<0.05, ** = p<0.005.</p

Qualitative effects of ectopic Egr/TNFα and increased apoptosis in the larval imaginal wing disc.

<p><b>(A)</b> Control disc showing ectopic expression in the posterior region of wing discs using <i>hh-GAL4</i> visualized by co-expression of GFP. <b>(B)</b> Ci staining of control discs show staining of the anterior compartment and is complementary to the region of GFP expression. <b>(C)</b> Caspase 3 staining revealed low levels of apoptosis in control imaginal wing discs. <b>(D)</b> Merged image where GFP is green, Caspase 3 staining is magenta and Ci is yellow. <b>(E-E’)</b> Ectopic expression of Eiger/TNFα resulted in a significant decrease in disc size and disruption to the pattern of GFP expression with punctate staining in the central wing pouch region. <b>(F-F’)</b> Staining of the anterior compartment with Ci reveals expression beyond the boundary and overlapping with the region of GFP expression. <b>(G-G’)</b> Caspase 3 staining reveals high levels of staining in the central wing pouch region and in two distinct regions extending towards to anterior portion of the disc (indicated with the red asterisks). <b>(H-H’)</b> Merged image (GFP is green, Caspase 3 is magenta and Ci is yellow). In all images the dotted line outlines the regions of GFP expression corresponding to the posterior region of the discs. Red boxes indicate the regions that are enlarged in E’-H’.</p

Model for the conserved role of WWOX in TNFα-mediated apoptosis <i>in vivo</i> in <i>D</i>. <i>melanogaster</i>.

<p>The function of WWOX in promoting cell death in response to Egr/TNFα signalling (through Wengen/TNFR) is mediated by ROS and Caspase 3. This can be modulated though expression of SOD enzymes that act to limit cellular levels of ROS and is also responsive to the metabolic status of cells.</p

Ubiquitous CUG and CAG RNA repeat expression leads to distinct localisation in muscle nuclei.

<p>Images from cryosections hybridised with fluorescent probes to detect repeat RNA. Images are representative of observations from multiple animals and independent transgenic lines for each repeat. <b>A</b>, Schematic of the repeat expression construct, indicating the location of the repeat and probe. <b>B-E</b>, Repeat transcripts detected with a complementary repeat probe. <b>B</b>, Progeny carrying the <i>da-GAL4</i> driver alone show no signal in the nucleus. <b>C</b>, Expression of <i>4xrCUG_∼100 [line 2]</i> leads to multiple foci throughout the nucleus. <b>D, E</b>, Expression of <i>4xrCAG_∼100 [line 1]</i>, or <i>4xrCAA_∼100 [line 1]</i> leads to between one and four sites of RNA concentration (arrowheads). <b>F</b>, Schematic of the construct giving repeat expression within the context of the GFP transcript, in this case RNA is detected using a probe against the GFP sequence. <b>G-K</b>, Repeat RNA localisation when expressed within a GFP transcript. <b>G</b>, No signal is observed using the GFP complementary probe against control EV progeny. <b>H</b>, <i>4xrCUG_∼100-GFP</i> expression leads to a similar pattern of foci as in <b>C</b>. <b>I, J</b>, Expression of 4x<i>rCAG_∼100-GFP</i> or <i>4xrCAA_∼100-GFP</i> leads to a similar pattern as in <b>D</b> and <b>E</b>. <b>K</b>, Expression of a single copy of GFP, not containing a repeat, leads to the formation of a single similar site of RNA concentration.</p

Tergite phenotype caused by ubiquitous expression of CAG or CUG repeat RNA in <i>Drosophila</i>.

<p><b>A</b>, wild-type flies show a regular arrangement of tergite bands along the dorsal abdomen (arrows). <b>B</b>, An example of the disrupted phenotype, whereby tergites do not fuse at all (white arrowheads), or fuse only partially (grey arrowheads). <b>C</b>, Phenotype severity was scored on a scale of 1–4, images show typical examples from each category, where category 1 is like wildtype; category 2, tergites disrupted but not split; category 3, one tergite split; and category 4, two or more tergites split. <b>D-E</b>, Graphs showing the proportion of progeny within each scoring category. <b>D</b>, <i>da-GAL4</i> driven expression of the EV control line (n = 161) and, <b>E</b>, <i>da-GAL4</i> driven expression of <i>4xrCAA_∼100</i> [line 1] (n = 148) gives no phenotype with almost all progeny like wild-type. <b>F</b>, <i>da-GAL4</i> driven expression of <i>4xrCUG_∼100 [line 1]</i> (n = 271) or <b>G</b>, <i>4xrCAG_∼100 [line 2]</i> (n = 343) gives a tergite disruption phenotype. <b>H,</b> Schematic (not to scale) showing the location of histoblast cells (black). Histoblasts proliferate and migrate to form the tergite bands (arrows). <b>I</b>, Expression within the histoblasts using <i>T155-GAL4</i> gives wild-type tergites in EV control progeny (n = 56). <b>J</b>, <i>T155-GAL4</i> expression of <i>4xrCAG_∼100</i> [line 1] (n = 25) gives a mild tergite phenotype, ***p<0.001 comparing the proportion with a phenotype in <b>I</b> and <b>J</b>.</p

Reducing Mbl levels does not enhance the tergite phenotype.

<p><b>A-D</b>, Proportion of progeny within each phenotype category when repeat expression is driven by <i>da-GAL4</i> alone (left column), or in the presence of the <i>mbl^E27</i> mutant allele such that Mbl levels are reduced (right column). <b>A</b>, Wild-type control, <b>B</b>, <i>4xrCAA_∼100 [line 1]</i>, <b>C</b>, <i>4xrCUG_∼100 [line 1]</i>, <b>D</b>, <i>4xrCAG_∼100 [line 2]</i>. <b>E, F</b>, Total proportion for each genotype that shows any phenotype (‘any phenotype’ - category 2, 3 and 4) and the proportion with a strong phenotype (‘strong phenotype’ – category 3 and 4). <b>E</b>, <i>4xrCUG_∼100 [line 1]</i> shows a reduction in the proportion with any phenotype, but no change in the proportion with a strong phenotype with reduced Mbl levels. <b>F</b>, No significant effect was observed with <i>4xrCAG_∼100 [line 2]</i>. Comparisons were made using Fisher’s exact test, with significant results indicated above the proportion, where *p<0.05.</p