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First published online 12 April 2006
doi: 10.1242/dev.02356

Development 133, 1871-1880 (2006)
Published by The Company of Biologists 2006
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vps25 mosaics display non-autonomous cell survival and overgrowth, and autonomous apoptosis

Hans-Martin Herz1, Zhihong Chen2, Heather Scherr2, Melinda Lackey2, Clare Bolduc2 and Andreas Bergmann2,*

1 University of Heidelberg/ZMBH, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany.
2 Department of Biochemistry and Molecular Biology, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard - Unit 1000, Houston, TX 77030, USA.


Figure 1
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  		Fig. 1. Isolation and characterization of K2 and N55 alleles. Genotypes are indicated in each panel. FRT, FRT42D. (A) The unmodified GMR-hid ey-FLP (GheF) eye-ablation phenotype. (B,C) K2 (B) and N55 (C) alleles are moderate suppressors of the GheF-eye phenotype. (D-F) Adult eyes of ey-FLP-induced mosaics of wild-type (D), and K2 (E) and N55 (F). K2 and N55 mosaic eyes lack mutant clones (marked by the absence of red pigment) and are overgrown. (G-I) Eye-antennal discs from third instar larvae of K2 (H) and N55 mosaics (I) are overgrown and disorganized compared with wild type (G). Scale bar: 100 µm. (J-L) Non-autonomous suppression of GMR-hidw- in K2 and N55 mosaics. (J) Control: in ark mosaics, largely ark mutant clones (marked by an absence of eye pigment) suppress GMR-hidw-. (K,L) In K2 (K) and N55 (L) mosaics, the suppression of GMR-hidw- is mediated by wild-type tissue (red pigment). K2 and N55 clones do not contribute.

 

Figure 2
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  		Fig. 2. TUNEL and BrdU analysis of N55 mosaics. (A-C) Increased TUNEL-positive cell death in N55 mutant clones (genotype: ey-FLP; FRT42D N55/FRT42D P[ubi-GFP]) of eye-antennal imaginal discs from third instar larvae. N55 clones are marked by the absence of GFP. (D-F) BrdU labeling of N55 mosaic wing imaginal discs. Note the increased proliferation in tissue adjacent to N55 clones (hs-FLP; FRT42D N55/FRT42D P[ubi-GFP]). (G-I) BrdU labeling of wild-type (wt) clones (hs-FLP; FRT42D +/FRT42D P[ubi-GFP]) in wing imaginal discs serves as a control to D-F. BrdU incorporation is homogeneous inside and outside the clones. (J) Schematic outline of the Vps25 protein and the molecular lesions in Vps25K2 and Vps25N55. WHA and WHB are winged-helix domains A and B.

 

Figure 3
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  		Fig. 3. Accumulation of ubiquitin, N and Dl in vps25 clones, and increased pSTAT immunoreactivity adjacent to vps25 clones. (A-C) Accumulation of ubiquitin in vps25 clones (genotype: hs-FLP UAS-GFP; FRT42D vps25N55/FRT42D tub-Gal80; tub-Gal4). Note vps25 clones are positively marked with GFP using the MARCM technique (Lee and Luo, 2001Go). (D-F) Accumulation of N in GFP-marked vps25 clones (genotype as in A-C). (G-I) Accumulation of Dl in GFP-marked vps25 clones (genotype as in A-C). (J-L) Increased N activity in GFP-marked vps25 clones (hs-FLP UAS-GFP; E[spl]m8 2.61-lacZ FRT42D vps25N55/FRT42D tub-Gal80; tub-Gal4). (M-O) Increased STAT activity inside and outside of vps25 clones (ey-FLP; FRT42D vps25N55/FRT42D P[ubi-GFP]) by anti-pSTAT labeling. Clones are marked by the absence of GFP.

 

Figure 4
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  		Fig. 4. N is required for non-autonomous proliferation of vps25 mosaics, but not for apoptosis. All vps25 clones expressing NDN (vps25/NDN) were labeled with GFP using MARCM. (A-I) vps25/NDN clones have reduced levels of pSTAT (A-C), non-autonomous cell proliferation (BrdU labeling in D-F), and Dl (G-I). The strong BrdU signal in E,F is due to the second mitotic wave. (J-L) Caspase-3*-positive apoptosis is unchanged in vps25/NDN clones. Genotype in A-L: hs-FLP UAS-GFP; FRT42D vps25N55 UAS-NDN/FRT42D tub-Gal80; tub-Gal4. (M) The unmodified GMR-hid GMR-Gal4 eye-ablation phenotype. (N) Overexpression of upd is not sufficient to suppress GMR-hid GMR-Gal4. Genotype: GMR-hid GMR-Gal4/UAS-upd.

 

Figure 5
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  		Fig. 5. Non-autonomous increase of Diap1 protein levels, and evidence of two cell death pathways. (A-C) Increased levels of Hid protein in vps25 clones (genotype: ey-FLP; FRT42D vps25N55/FRT42D P[ubi-GFP]) of third instar eye discs. Clones are marked by the absence of GFP. (D-F) Diap1 protein levels are reduced in, and increased adjacent to, vps25 clones (genotype as in A-C). (G-I) BrdU labeling of GFP-marked vps25/Diap1 mosaics (hs-FLP UAS-GFP/UAS-Diap1; FRT42D vps25N55/FRT42D tub-Gal80; tub-Gal4). Overexpression of Diap1 does not block non-autonomous proliferation. (J-L) Caspase-3*-labeling of GFP-marked vps25N55/Diap1 mosaics. Caspase-3* is completely blocked. Genotype as in G-I. Scale bar: 100 µm. (M-O) Caspase-3*-labeling of GFP-marked vps25 ark double mutant mosaics. Caspase-3*-dependent cell death is detectable in vps25 ark clones (hs-FLP UAS-GFP; FRT42D vps25N55 arkH16/FRT42D tub-Gal80; tub-Gal4). Scale bar: 100 µm. (P) Adult eyes of ey-FLP-induced mosaics of vps25 ark double mutants are severely overgrown and folded. Note that vps25 ark clones (ey-FLP; FRT42D vps25N55 arkH16/FRT42D P[w+]) are absent (marked by lack of red pigment).

 

Figure 6
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  		Fig. 6. JNK contributes to the apoptotic phenotype of vps25 clones. (A-C) Increased JNK activity in vps25 clones (genotype: ey-FLP; FRT42D vps25N55/FRT42D P[ubi-GFP]) shown by anti-pJNK labeling. Clones are marked by absence of GFP. (D-F) pJNK labeling of GFP-marked vps25/Diap1 mosaics (hs-FLP UAS-GFP/UAS-Diap1; FRT42D vps25N55/FRT42D tub-Gal80; tub-Gal4). (G-I) Caspase-3*-labeling of GFP-marked vps25/Puc mosaics (hs-FLP UAS-GFP; FRT42D vps25N55/FRT42D tub-Gal80; tub-Gal4/UAS-puc). Scale bar: 100 µm. (J-L) Caspase-3*-labeling of GFP-marked vps25 ark/Puc mosaics (hs-FLP UAS-GFP; FRT42D vps25N55 arkG8/FRT42D tub-Gal80; tub-Gal4/UAS-puc). Note, the clones marked with GFP are severely enlarged. Scale bar: 100 µm.

 

Figure 7
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  		Fig. 7. Increased Hippo signaling, but not cell competition, controls apoptosis in vps25 clones. (A-C) Caspase-3*-labeling of vps25 mosaics (genotype: ey-FLP; FRT42D vps25N55/FRT42D arm-lacZ M(2)) in a Minute (M) background. Caspase-3* activity is unaffected. Clones are marked by the absence of ß-Gal staining. (D-F) Caspase-3*-labeling of GFP-marked vps25/dMyc mosaics (hs-FLP UAS-GFP; FRT42 vps25N55/FRT42D tub-Gal80; tub-Gal4/UAS-dMyc). Caspase-3*-activity is unaffected. (G-I) Expanded (Ex) labeling of GFP-marked vps25 mosaics (hs-FLP UAS-GFP; FRT42D vps25N55/FRT42D tub-Gal80; tub-Gal4). Ex protein levels are reduced in vps25 clones (arrow), indicating increased Hippo activity. (J-L) Caspase-3*-labeling of GFP-marked vps25 hippo double mutant mosaics (hs-FLP UAS-GFP; FRT42D vps25N55 hippo3D/FRT42D tub-Gal80; tub-Gal4). Caspase-3* activity is blocked.

 

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© The Company of Biologists Ltd 2006