Genetic interactions between <i>tay</i>, <i>Mkp3</i> and <i>Erk</i>.

<p>(A–H) Genetic interactions between <i>tay</i> and <i>Erk</i>. (A–B) The over-expression of Tay does not supress the phenotype of ectopic veins caused by the expression of Erk^sem (<i>sal^PEv-Gal4/UAS-Erk^sem</i>; A) in <i>EP-866/+; sal^EPv-Gal4/UAS-Erk^sem</i> flies (B). (C–D) Control wild type wings grown at 29°C (C) and <i>638-Gal4/UAS-tay-i; UAS-GFP/+</i> wing showing ectopic veins in inter-vein regions and reduced size (D). (E–F) The ectopic vein phenotype of <i>638-Gal4/+; UAS-Erk-HA/+</i> flies (E) is enhanced when the expression of <i>tay</i> is reduced in <i>638-Gal4/UAS-tay-i; UAS-Erk-HA/+</i> flies (F). (G–H) The ectopic vein phenotype of <i>638-Gal4/+; UAS-Erk^sem-HA/+</i> wings (G) is strongly increased when the expression of <i>tay</i> is reduced in <i>638-Gal4/UAS-tay-i; UAS-Erk^sem-HA/+</i> flies (H). (I-P) Genetic interactions between <i>tay</i> and <i>Mkp3</i>. (I–J) The loss of veins phenotype of <i>sal^EPv-Gal4/UAS-Mkp3</i> wings (I) is not modified when Tay is over-expressed in <i>EP-866</i>/+; <i>sal^EPv-Gal4/UAS-Mkp3</i> wings (J). (K–L) The ectopic veins phenotype of <i>638-Gal4/UAS-tay-i; UAS-GFP/+</i> wings (K) is increased when the expression of <i>Mkp3</i> is also reduced in <i>638-Gal4/UAS-tay-i; UAS-Mkp3-i/+</i> flies (L). (M–N) The phenotype of ectopic veins of <i>638-Gal4/+; UAS-tay-i/+</i> wings (M) is increased in heterozygous <i>Mkp3</i> flies (<i>638-Gal4/UAS-tay-i; Mkp3^M76-R2b/+</i>; N). (O–P) The phenotype of loss of veins and strong wing size reduction produced by the over-expression of Tay in the entire wing blade and hinge (<i>EP-866; nub-Gal4/+; Mkp3^M76-R2b/+</i>; P) is not modified in homozygous <i>Mkp3</i> background (<i>EP-866; nub-Gal4/+; Mkp3^M76-R2b/Mkp3^M76-R2b</i>; Q). Control <i>Mkp3^M76-R2b</i> homozygous wings are shown in panel O, and control heterozygous <i>Mkp3^M76-R2b</i>/+ wings are indistinguishable from wild type.</p

Effects of Tay over-expression in the activation of Erk.

<p>(A–D) Accumulation of dP-Erk in late third instar wing discs over-expressing Erk-HA. (A) Control <i>sal^EPv-Gal4/UAS-Erk-HA</i>; <i>UAS-GFP/+</i> wing disc. (B) <i>sal^EPv-Gal4/UAS-Erk-HA</i>; <i>UAS-tay.FL-Flag/+</i> wing disc. (C) <i>sal^EPv-Gal4/UAS-Erk-HA</i>; <i>UAS-Ras^V12/+</i> wing disc. (D) <i>sal^EPv-Gal4/UAS-Erk-HA</i>; <i>UAS-Ras^V12/UAS-tay.FL-Flag</i> wing disc. (E–F) Accumulation of dP-Erk in late third instar wing discs over-expressing Erk^sem-HA. (E) Control <i>sal^EPv-Gal4/UAS-Erk^sem-HA</i>; <i>UAS-GFP/+</i> wing disc. (F) <i>sal^EPv-Gal4/UAS-Erk^sem-HA</i>; <i>UAS-tay.FL-Flag/+</i> wing disc. (G) <i>sal^EPv-Gal4/UAS-Erk^sem-HA</i>; <i>UAS-Ras^V12/+</i> wing disc. (H) <i>sal^EPv-Gal4/UAS-Erk^sem-HA</i>; <i>UAS-Ras^V12/UAS-tay.FL-Flag</i> wing disc. (I–L) Wings of <i>sal^EPv-Gal4/UAS-Erk^sem-HA</i>; <i>UAS-GFP/+</i> (I), <i>sal^EPv-Gal4/UAS-Erk^sem-HA</i>; <i>UAS-tay.FL-Flag/+</i> (J), <i>sal^EPv-Gal4/UAS-Erk^sem-HA</i>; <i>UAS-Ras^V12/+</i> (K) and <i>sal^EPv-Gal4/UAS-Erk^sem-HA</i>; <i>UAS-Ras^V12/UAS-tay.FL-Flag</i> (L) genotypes.</p

Effects of Tay on EGFR signalling.

<p>(A–D) Wild type wing (A) and late third instar wing discs showing the expression of dP-Erk (B), Delta (Dl, C) and <i>argos-lacZ</i> (D). (E–H) <i>EP-866; sal^EPv-Gal4/sal^EPv-Gal4</i> wing (E) showing reduced size and partial loss of the L2, L3 and L4 veins. Late third instar wing disc of the same genotype showing dP-Erk (F), Delta (G) and <i>argos-lacZ</i> (H) expression. Only the expression of dP-Erk, Delta and <i>argos-lacZ</i> in the L5 vein, which is outside the domain of <i>sal^EPv-Gal4</i>, is present. (I–J′) Wing of <i>UAS-tay-i; ap-Gal4/UAS-GFP</i> genotype (I) and corresponding third instar wing disc showing the expression of dP-ERK (J–J′). Note the difference in expression levels between dorsal (labelled in green in J) and ventral inter vein cells. J′ corresponds to the red channel of J. (K) Expression of <i>argos-lacZ</i> in wing disc of <i>UAS-tay-i/638-Gal4</i> genotype. Note that the reduction of <i>tay</i> expression in the entire wing blade produces ectopic expression of <i>argos-lacZ</i>, mainly in the L3/L4 intervein and around the L5 vein (white arrowheads). (L–L′) Phenotype of Ras^V12 over-expression (<i>sal^EPv-Gal4 UAS-Ras^V12/UAS-GFP</i>; L), and accumulation of dP-Erk (red; L′) in <i>sal^EPv-Gal4 UAS-Ras^V12/UAS-GFP</i> wing disc. (M–M′) The expression of Tay reduces the phenotype of <i>Ras^V12</i> wings in <i>EP-866/+; sal^EPv-Gal4/UAS-Ras^V12</i> flies (M), and also reduces the accumulation of dP-Erk (red; M′) in <i>EP-866/+; sal^EPv-Gal4/UAS-Ras^V12</i> wing discs. Tangential sections through the length of the wing epithelium are shown to the right of L′ and M′.</p

Subcelullar localization and phenotypes of human AUTS2 in flies.

<p>(A) Sequence conservation between human AUTS2 (<i>H. sapiens</i>; amino acids 485–736), Zebra fish AUTS2 (<i>D. renio</i>; amino acids 209–400) and Tay (<i>D. melanogaster</i>; amino acids 1760–1921). The colour code indicates the physicochemical properties of each amino acid (Jalview software) and the bars below each amino acid the identity in two or three species. (B–B′) Subcellular localization of AUTS2 (Flag red in B, white in B′) in wing discs of <i>sal^EPv-Gal4/+; UAS-hAUTS2-Flag/+</i>. Below and to the right are the corresponding horizontal and transversal sections of the disc. The expression of F-actin is in green and To-Pro in blue. B′ is the red channel of B showing the accumulation of AUTS2. (C–C′) Higher magnification of the dorsal region of a <i>sal^EPv-Gal4/+; UAS-hAUTS2-Flag/+</i> disc, showing the expression of AUTS2 (red in C, white in C″) and To-Pro (blue). The single red channel is shown in C′. (D–I) The phenotype of ectopic veins produced by the expression of AUTS2 in the entire wing blade and hinge (<i>nub-Gal4/UAS-hAUTS2-Flag</i>; D) is abolished when the expression of AUTS2 in accompanied with a reduction of Erk expression in <i>nub-Gal4/UAS-hAUTS2-Flag; UAS-Erk-i/+</i> flies (F), producing a loss of vein phenotype very similar to <i>nub-Gal4/UAS-Erk-i</i> flies (E). (G–I) Expression of AUTS2 in <i>sal^EPv-Gal4/UAS-hAUTS2-Flag</i> flies (G) and expression of Erk^sem in <i>sal^EPv-Gal4/UAS-Erk^sem-HA</i> flies (H) causes weak phenotypes of ectopic veins that are enhanced when AUTS2 and Erk^sem are expressed together in <i>sal^EPv-Gal4/UAS-Erk^sem-HA; UAS-hAUTS2-Flag</i> flies (I). (J–J′) Third instar wing disc showing the expression of dP-Erk (red in J, white in J′) in <i>ap-Gal4/UAS-hAUTS2-Flag</i> wing discs. Note the difference in expression levels between dorsal (labelled in green in J) and ventral cells. J′ corresponds to the red channel of J.</p

Tay interacts with Erk and Mkp3.

<p>(A–B) Interactions between Tay and Mkp3 (A) and between Tay and Erk (B). Embryo extracts were immunoprecipitated with anti-Flag (upper lines in A and B), anti-Myc (lower lines in A) and anti-HA (lower lines in B). Immunoprecipitates (IPs) and lysates (Inputs) were immunoblotted with anti-Myc (detecting Mkp3-Myc protein), anti-HA (detecting Erk^sem-HA protein) and anti-Tay (we could not detect neither endogenous Tay nor Tay-Flag protein). In control IPs carried out with <i>da-Gal4/UAS-tay.FL-Flag</i> (T) and <i>da-Gal4/UAS-Mkp3-Myc</i> (M) embryos in A, and <i>da-Gal4/UAS-tay.FL-Flag</i> (T) and <i>da-Gal4/UAS-Erk^sem-HA</i> (E) embryos in B, we did not detect any Tay, Mkp3 or Erk proteins. In Co-IPs experiments carried out with <i>da-Gal4/UAS-tay.FL-Flag; UAS-Mkp3-Myc/+</i> embryos (T+M) in A and <i>da-Gal4/UAS-tay.FL-Flag; UAS-Erk^sem-HA/+</i> (T+E) embryos in B, we detected Mkp3-Myc protein in A and Erk^sem-HA protein in B when the extracts were immunoprecipitated with anti-Flag (black arrows). (C) Pull down assay showing the interactions Tay-Mkp3 and Tay-Erk. Tay protein was in vitro translated and radiolabelled with Met^S35. Tay was detected when it was incubated with Mkp3-GST protein and Erk-GST protein, but was not detected in control pulldowns with GST. (D–D″) Subcellular localization of Tay (Flag, white in D–D″) in wing imaginal discs of <i>sal^EPv-Gal4/UAS-tay.FL-Flag</i> (D), <i>sal^EPv-Gal4/UAS-tay.1-Flag</i> (D′) and <i>sal^EPv-Gal4/UAS-tay.2-Flag</i> (D″) and their corresponding wings. (E) Schematic representation of Tay (Tay.FL) and the N-terminal (Tay.1) and C-terminal (Tay.2) forms. (F–G) Interactions between Tay.2 and Mkp3 (F) and Tay.2 and Erk (G). Embryo extracts were immunoprecipitated with anti-Flag (upper lines in F and G), anti-Myc (lower lines in F) and anti-HA (lower lines in G). Immunoprecipitates (IPs) and lysates (Inputs) were immunoblotted with anti-Myc (detecting Mkp3-Myc protein), anti-HA (detecting Erk^sem-HA protein) and anti-Tay (detecting Tay.2-Flag protein). In control IPs carried out with <i>da-Gal4/UAS-tay.2-Flag</i> (T) and <i>da-Gal4/UAS-Mkp3-Myc</i> (M) embryos in F, and with <i>da-Gal4/UAS-tay.2-Flag</i> (T) and <i>da-Gal4/UAS-Erk^sem-HA</i> (E) embryos in G, we did not detect any Tay.2, Mkp3 or Erk protein. In Co-IPs experiments carried out with <i>da-Gal4/UAS-tay.2-Flag; UAS-Mkp3-Myc/+</i> embryos (T+M) in F, we did not detect Mkp3-Myc protein when the extracts were immunoprecipitated with anti-Flag. In Co-IPs experiments carried out with <i>da-Gal4/UAS-tay.2-Flag; UAS-Erk^sem-HA/+</i> embryos (T+E) in G, we detected Erk^sem-HA protein when the extracts were immunoprecipitated with anti-Flag (black arrow). (H–I′) Accumulation of Tay in wing discs over-expressing Erk, Erk^sem, Ras^V12 and Mkp3. The over-expression is limited to the dorsal compartment of <i>ap-Gal4/UAS-Erk-HA</i> (H) and <i>ap-Gal4/UAS-Erk^sem-HA</i> (H′) discs, or to the central domain of the wing blade in <i>sal^EPv-Gal4/UAS-Ras^V12</i> (I) and <i>sal^EPv-Gal4/UAS-Mkp3-Myc</i> (I′) discs. Note the increased levels of Tay in dorsal compartments over-expressing Erk-HA and Erk^sem-HA (H–H′).</p

Genetic interactions between Tay and EGFR signalling.

<p>(A–C) The reduction of Tay (<i>sal^EPv-Gal4/UAS-tay-i</i>; A) increases the phenotype of ectopic veins caused by Ras^V12 over-expression (<i>sal^EPv-Gal4/UAS-GFP; UAS-Ras^V12/+</i>; B) in <i>sal^EPv-Gal4/UAS-tay-i; UAS-Ras^V12/+</i> (C).). (D–F) Interaction of <i>tay</i> and <i>rhomboid</i>. The reduction of Tay (<i>638-Gal4/UAS-tay-i</i>; D) and the increase in <i>rhomboid</i> expression (<i>638-Gal4/+; UAS-rho/+</i>; E) cause ectopic veins, and this phenotype is strongly enhanced in the double combination (<i>638-Gal4/UAS-tay-i; UAS-rho/+</i>; F). (G–I) Interaction of Tay and EGFR. The expression of Tay (<i>EP-866/+; sal^EPv-Gal4/+</i>; G) enhances the phenotype of EGFR loss (<i>sal^EPv-Gal4/UAS-EGFRi</i>; H) in <i>EP-866; sal^EPv-Gal4/+; UAS- EGFRi/+</i> flies (I). Interaction of Tay and EGFR. The reduction of <i>tay</i> expression does not modify the strong loss of veins phenotype caused by the expression of a dominant negative form of EGFR (<i>638-Gal4/+; UAS-EGFR^DN/+</i>; K) in <i>638-Gal4/UAS-tay-i; UAS-EGFR^DN/+</i> flies (L). A wild type wing is shown in J.</p

Loss-of-function analysis of <i>tay</i>.

<p>(A) Wild type wing grown at 29°C. (B) Wing size reduction, extra veins and loss of wing margin in <i>638-Gal4/+; UAS-tay-i/+</i> wings at 29°C. (C–D) The weak phenotype of <i>638-Gal4/+; UAS-tay-i/+</i> wings at 25°C (C) is increased in flies heterozygous for a <i>tay</i> deficiency (<i>638-Gal4/Df(1)tay; UAS-tay-i/+</i>; D). (E–G) Examples of mitotic recombination clones in flies of <i>f^36a Df(1)tay FRT18A/FRT18A UbiGFP; hsFLP/+</i> genotype. In E, example of a dorso-ventral clone located between the L3 and L4 veins (E′ and E″ are higher magnifications of the dorsal and ventral wing surfaces, respectively). In F example of a dorsal clone located anterior to the L2 vein. This clone (F′) differentiates vein cells and induces vein differentiation in the ventral wing surface (F″). In G example of a ventral clone located between the L3 and L4 veins that differentiate an ectopic vein in the ventral surface (G″) and induces vein differentiation in the dorsal surface (G′).</p

Genetic characterization of the <i>EP-866</i> insertion and expression analysis of <i>tay</i>.

<p>(A) Wild type wing (<i>wt</i>) indicating the position of the longitudinal veins L2–L5. (B–D) Combinations of <i>EP-866</i> with Gal4 drivers expressed in the entire wing blade and hinge (<i>EP-866/+; nub-Gal4/+</i>; B), in the central region of the wing blade (<i>EP-866; sal^EPv-Gal4/+</i>; C) and in the pupal wing veins (<i>EP-866/+; shv-Gal4/+</i>; D). (E) Schematic representation of the genomic region around the insertion site of <i>EP-866</i>, showing the position and orientation of <i>tay</i>, <i>CG9066</i>, <i>CG15916</i> and <i>shibir</i>e coding regions. (F–G) Rescue of the <i>EP-866/Gal4</i> phenotype by expression of RNA interference directed against <i>tay</i> (<i>UAS-tay-i</i>). In both cases, <i>EP-866; sal^EPv-Gal4/+; UAS-tay-i/+</i> (F) and <i>EP-866/+; shv-Gal4/UAS-tay-i</i> (G) the phenotype is rescued (compare F with C and G with D). (H–H′) Expression of Tay (red) and GFP (green) in <i>ap-Gal4 UAS-GFP/+; UAS-tay-i/+</i> third instar wing discs. The red channel (H′) shows the strong reduction in Tay protein levels. (I–J) <i>in situ</i> hybridization with a <i>tay</i> probe in a late third instar wing disc (I) and in a pupal wing 28 h after puparium formation (J). (K–L) High magnification pictures of a salivary gland (K) and the peripodial membrane of the wing disc (L) showing the accumulation of Tay (red channel) in the cell nucleus. The expression of FasIII (green) shows the contour of the cells. (M–N″) Expression of Tay (red), F-actin (green) and To-Pro (blue) in a third instar wing disc. (N–N″) Orthogonal section of the disc showed in M showing the individual channels for Tay (N′) and To-Pro (N″). (O–O′) Expression of Tay (red) in <i>EP-866; sal^EPv-Gal4/+; UAS-GFP/+</i> discs, showing nuclear localization of Tay in cells over-expressing the protein. The expression of GFP is in green (O).</p

Supplemental Figures and figure legends 1 to 6 from An <i>in vivo</i> genetic screen in <i>Drosophila</i> identifies the orthologue of human cancer/testis gene <i>SPO11</i> among a network of targets to inhibit <i>lethal(3)malignant brain tumour</i> growth

Using transgenic RNAi technology, we have screened over 4.000 genes to identify targets to inhibit malignant growth caused by the loss of function of <i>lethal(3)malignant brain tumour</i> (mbt) in <i>Drosophila in vivo</i>. We have identified 131 targets, which belong to a wide range of gene ontologies. Most of these target genes are not significantly overexpressed in mbt tumours hence showing that, rather counterintuitively, tumour-linked overexpression is not a good predictor of functional requirement. Moreover, we have found that most of the genes upregulated in mbt tumours remain overexpressed in tumour-suppressed double-mutant conditions, hence revealing that most of the tumour transcriptome signature is not necessarily correlated with malignant growth. One of the identified target genes is <i>meiotic W68</i> (<i>mei-W68</i>), the <i>Drosophila</i> orthologue of the human Cancer Testes gene <i>Sporulation-specific protein 11</i> (<i>SPO11</i>), the enzyme that catalyses the formation of meiotic double-strand breaks. We show that <i>Drosophila mei-W68/SPO11</i> drives oncogenesis by causing DNA damage in a somatic tissue, hence providing the first instance in which a <i>SPO11</i> orthologue is unequivocally shown to have a pro-tumoural role. Altogether, the results from this screen point to the possibility of investigating the function of human cancer relevant genes in a tractable experimental model organism like <i>Drosophila.</i

Table S2. Results from the high-content screen. from An <i>in vivo</i> genetic screen in <i>Drosophila</i> identifies the orthologue of human cancer/testis gene <i>SPO11</i> among a network of targets to inhibit <i>lethal(3)malignant brain tumour</i> growth

The table contains all the information regarding the high-content screen including the list of all the RNAi lines screened, the corresponding VDRC UAS-RNAi line ID, FlyBase ID, CG name, Symbol, and the behaviour in the screen assay. Lines that performed as mbt tumor suppressors only in the first in both first and second rounds of screen are labeled orange and green, respectively. Only the latter (green) were tagged as confirmed mbt-SPRs. Lines that lead to larval lethality or larval brains smaller than wild type are labeled brown and purple, respectively