Volume 9, Issue 5, Pages 711-720 (November 2005) Tumor Suppressor Properties of the ESCRT-II Complex Component Vps25 in Drosophila  Barry J. Thompson, Juliette Mathieu, Hsin-Ho Sung, Eva Loeser, Pernille Rørth, Stephen M. Cohen  Developmental Cell  Volume 9, Issue 5, Pages 711-720 (November 2005) DOI: 10.1016/j.devcel.2005.09.020 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 vps25 Mutant Clones Cause Ectopic Outgrowths in Multiple Tissues The FLP/FRT system was used to generate clones of cells homozygous for a control chromosome or the vps25 mutant chromosome during development. (A) In the eye, the homozygous cells can be identified by the orange eye color encoded by the white+ gene carried on the PiggyBac transposon. Note that this control insertion exhibits normal clone and tissue growth. (B) In contrast, vps25 mutant clones occupy only a tiny proportion of the eye, but they induce overgrowth of the eye as a whole. (C) Proximal region of adult wing containing clones of a control chromosome. (D) vps25 mutant clones in the wing caused outgrowth from the proximal region of the dorsal wing surface. (E) Adult leg containing clones of a control chromosome. (F) vps25 mutant clones in the leg caused outgrowths from the ventral side. These phenotypes are similar to those caused by induction of ectopic DV axis organizers. Developmental Cell 2005 9, 711-720DOI: (10.1016/j.devcel.2005.09.020) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 vps25 Mutant Clones Induce Ectopic DV Organizer Activity in Eye, Wing, and Leg Imaginal Discs The FLP/FRT system was used to generate clones of cells homozygous for a control chromosome or the vps25 mutant chromosome during development. Clones are marked by the absence of GFP, and wild-type “twin spot” sister clones are marked by bright GFP fluorescence (green). Discs are stained with DAPI (blue), and the expression of secreted signals that mediate organizer activity is examined (αUpd, αWg, or dpp-lacZ; red). (A) A wild-type third instar eye imaginal disc containing control clones. In the eye imaginal disc, the DV organizer is established by Notch signaling that induces expression of the secreted signal Upd. By the end of the third larval instar, Upd is no longer expressed, unless Notch signaling is ectopically activated (Chao et al., 2004). (B) In clones mutant for vps25, Upd is ectopically expressed, mimicking activated Notch signaling. (C) Enlargement of (B). (D) Wild-type third instar eye imaginal disc containing control clones. In the wing imaginal disc, Notch signaling induces Wingless (Wg) along the DV boundary. (E) Wg expression is ectopically activated in vps25 mutant clones, again indicating that these clones have activated Notch signaling. (F) When small clones are made that can survive in the wing pouch, presumably due to perdurance of Vps25 protein, the mutant cells continue to express Wingless, but only in those cells close to the DV boundary, indicating a degree of ligand dependence. (G) In the leg disc, an antagonistic Dpp-Wg regulatory loop exists such that Dpp is expressed dorsally. (H) Ectopic activation of Dpp signaling in ventral cells represses Wg, leading to ectopic expression of the Dpp ligand. vps25 mutant clones located ventrally also repress Wg (not shown) and upregulate expression of the Dpp ligand, observed with a dpp-lacZ transgene, mimicking clones expressing activated Tkv. (I) In contrast, in wing discs the dpp-lacZ transgene is expressed along the A-P boundary in response to Hh signaling, but dpp-lacZ is not a target of Dpp signaling. (J) Consequently, dpp-lacZ is not activated in vps25 mutant clones in wing discs. These results indicate that Notch and Dpp receptor signaling is activated in vps25 mutant cells and is responsible for generating ectopic DV organizer activity and consequent tissue outgrowths. Note that in the (A and D) controls, clones and twin spots are similar in size. In contrast, (B, C, E, and F) vps25 mutant clones are smaller than twin spots and are often eliminated, particularly in the (E) wing pouch. DAPI (blue) marks nuclei and shows that vps25 mutant cells often exhibit pyknotic nuclei, indicative of apoptosis. Developmental Cell 2005 9, 711-720DOI: (10.1016/j.devcel.2005.09.020) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 Notch, Tkv, and Other Ubiquitylated Proteins Accumulate in Endosomes of vps25 Mutant Cells (A–H) In the (A–D) postmitotic cells of the eye disc and in the (E–H) endoreplicating follicle cells of the ovary, clones mutant for vps25 (marked by the absence of GFP, green) accumulate Notch (αN, red), Delta (αDl, red), the Dpp receptor Thickveins (αTkv, red), and ubiquitylated proteins (αUb, blue) in large, cytoplasmic puncta. (H) These puncta colocalize with a GFP-2xFYVE domain fusion protein (FYVE-GFP) that marks endosomes (in this case, expression of FYVE-GFP identifies the clone). These classical MVB sorting defects are similar to, or stronger than, those previously shown for Drosophila hrs mutants (Jekely and Rorth, 2003). Developmental Cell 2005 9, 711-720DOI: (10.1016/j.devcel.2005.09.020) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 RNAi of Vps25, Vps22, and Vps32, but Not Hrs, Activates Notch Signaling in Cultured Cells The effect upon Notch signaling of RNAi-knockdown of three core ESCRT components (Vps25, Vps22, and Vps32) and the ancillary factor Hrs was compared. Notch signaling (measured as relative luciferase activity, RLA) was assayed by using a luciferase reporter (NRE) that is moderately activated upon transfection of the Notch receptor (FL Notch). (A–D) (A–C) RNAi of the three core ESCRT components increased Notch signaling, while (D) RNAi of Hrs decreased signaling. These effects are a specific consequence of activated Notch signaling, as they depend strictly on the presence of FL Notch and upon functional binding sites for the nuclear effector of Notch, Su(H), in the NRE reporter (which are absent in a mutant reporter, mut). (E and F) In addition, when γ-secretase activity is reduced by addition to the culture medium of the presenilin inhibitor DAPT (10 μM), Notch signaling is strongly reduced, indicating that the signaling we observe in these cells occurs by the normal presenilin-dependent cleavage mechanism. Experiments were performed in triplicate and were repeated several times with comparable results. Error bars show standard deviation from one representative experiment. Developmental Cell 2005 9, 711-720DOI: (10.1016/j.devcel.2005.09.020) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 5 Blocking Apoptosis Unleashes the Oncogenic Potential of vps25 Mutant Cells (A) A pair of wild-type eye discs stained with phalloidin (green) and for the marker of neural differentiation, Elav (blue). (B) The MARCM system was used to express p35 and RFP (red) in vps25 mutant clones, induced in a pair of eye discs by using eyeless-FLP as a source of FLP recombinase. Clones survive and populate a large proportion of the discs. In addition, the discs show massive overgrowth in three dimensions. Note that use of this system to express p35 in wild-type cells produces discs that are indistinguishable from wild-type (not shown). (C) A pair of wild-type eye discs stained with DAPI (blue), expressing GFP (green) and Elav (red). (D) A pair of eye discs in which eyeless-FLP was used to generate clones of vps25 mutant cells surrounded by neighbors that are heterozygous for a Minute mutation, which causes neighboring cells to compete poorly. vps25 mutant clones survive and occupy a large region of the discs. Furthermore, the discs again show massive overgrowth in three dimensions and, in this case, have fused to the brain (marked by Elav). Note that when wild-type clones are given a competitive advantage by using the Minute technique, the disc as a whole does not overgrow and resembles a wild-type disc (not shown). Developmental Cell 2005 9, 711-720DOI: (10.1016/j.devcel.2005.09.020) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 6 Blocking Apoptosis Causes Tumorous Overgrowths in Eye, Wing, and Leg Imaginal Discs (A) Wild-type eye disc stained with phalloidin (green) and for the marker of neural differentiation, Elav (blue). (B and C) (B) Eye disc containing clones of vps25 mutant cells expressing p35 and RFP. (C) Enlargement of (B). Note that mutant cells in the eye also fail to express the marker of neural differentiation, Elav. (D) Wild-type wing disc stained with phalloidin (green) and for Wingless (Wg, blue). (E) Wing disc containing clones of vps25 mutant cells expressing p35 and RFP. (F) Enlargement of (E), shown with DAPI staining. (G) Wild-type leg disc (left) and haltere disc (lower right) stained with phalloidin (green) and for Wingless (Wg, blue). (H) Leg (left) and haltere (right) discs containing clones of vps25 mutant cells expressing p35 and RFP. (I) Enlargement of (H), shown with DAPI staining. In all cases, the presence of vps25 mutant clones expressing p35 causes large tumorous overgrowths. The mutant cells lose their epithelial organization and pile upon one another to form three-dimensional masses. However, surrounding wild-type cells remain epithelial (e.g., [F]). Developmental Cell 2005 9, 711-720DOI: (10.1016/j.devcel.2005.09.020) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 7 Alleviating Competition Allows Tumorous Overgrowth of vps25 Mutant Clones (A) Eye disc in which the eyeless-FLP technique was used to induce clones of wild-type cells surrounded by Minute/+ heterozygous cells (marked by expression of GFP, green), which have a competitive disadvantage. Discs were stained with phalloidin (red) and for Elav (blue). (B) Enlargement of (A), showing DAPI staining (blue). (C) Eye disc in which the eyeless-FLP technique was used to induce clones of vps25 mutant cells surrounded by Minute/+ heterozygous cells (expressing GFP, green). Absence of competition from neighbors allows the vps25 mutant cells to survive and proliferate to occupy a large region of the overgrown disc. Note that the mutant cells lose their epithelial organization. The disc is stained with phalloidin (red) and for Elav (blue), which remains only in brain tissue that has fused to the mutant eye disc. (D) Enlargement of (C), showing DAPI staining (blue). (E) Eye discs expressing p35 and RFP in wild-type cells, induced by using the eyeless-FLP/MARCM technique, were transplanted into the abdomens of adult females. After 1 week, the discs always remain at the site of transplantation. (F) Eye discs expressing p35 and RFP in vps25 mutant cells, induced in the same manner, were found to migrate away from the site of transplantation after 1 week, in 2/15 cases. Developmental Cell 2005 9, 711-720DOI: (10.1016/j.devcel.2005.09.020) Copyright © 2005 Elsevier Inc. Terms and Conditions