1. Volume 22, Issue 1, Pages 206-217 (January 2018)
winged eye Induces Transdetermination of Drosophila Imaginal Disc by Acting in Concert with a Histone Methyltransferase, Su(var)3-9 
Keita Masuko, Naoyuki Fuse, Kanae Komaba, Tomonori Katsuyama, Rumi Nakajima, Hirofumi Furuhashi, Shoichiro Kurata 
Cell Reports 
Volume 22, Issue 1, Pages (January 2018)
DOI: /j.celrep
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2. Cell Reports 2018 22, 206-217DOI: (10.1016/j.celrep.2017.11.105)
Copyright © 2017 The Author(s) Terms and Conditions

3. Figure 1
Wge Overexpression Alters Histone Modifications in a BAH Domain-Dependent Manner
(A) Western blots of SG lysates for ey>GFP (control) and ey>wge. Applied doses were 4:2:1. The Wge proteins with tag (HA [hemagglutinin]-Wge) were detected using anti-HA antibody.
(B) Quantifications of (A). Fold change of relative signal intensities, when control signal is taken as 1, are shown. Data indicate mean ± SD of three independent experiments. ∗p < 0.05, by Student’s t test.
(C–F) Immunostaining of SG polytene chromosomes for ey>lacZ (control; C, H3K9Ac; E, H3K9me2) and ey>wge (D, H3K9Ac; F, H3K9me2). Scale bars, 10 μm.
(G and H) Higher magnification views of (F). Scale bars, 5 μm.
(G) Arrows indicate non-co-localized sites, and arrowheads indicate co-localized sites.
(H) Centromeric H3K9me2 signal is surrounded by a dotted line.
(I) Western blots of SG lysates for ey>GFP (control), ey>wge, and ey>wgeΔBAH. The Wge proteins with tag (FLAG-Wge or FLAG-WgeΔBAH) were detected using anti-FLAG antibody.
(J) Immunostaining of polytene chromosomes with ey>wgeΔBAH. Scale bar, 10 μm.
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4. Figure 2 Wge Is Preferentially Localized at the Nuclear Periphery
(A) Immunostaining of SG tissue for ey>wge.
(B–B”) Higher magnification views of (A).
(C) Higher magnification and merged view of (B)–(B”).
(D) Immunostaining of SG tissue for ey>wgeΔBAH.
(E–E”) Higher magnification views of (D).
(F) Higher magnification and merged view of (E)–(E”).
(G and G’) Immunostaining of eye imaginal disc for ey-flp Ay-GAL4, UAS-GFP, UAS-wge. Arrows indicate typical NP localization of Wge.
Scale bar, 10 μm. See also Figure S1.
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5. Figure 3
Su(var)3-9 Is Required for wge-Mediated Histone Modifications and Eye-to-Wing TD
(A–D) Immunostaining of SG tissue (control, A; ey>wge, B; Su(var)3-91/2, C; ey>wge, Su(var)3-91/2, D). ey>lacZ was used as control.
(E) Western blots of SG lysates for ey>GFP (cont), ey>wge (wge), and each of these under Su(var)3-91/2 background. Wge protein (FLAG-Wge) was detected using anti-FLAG antibody. Su(var)3-91/2 transheterozygotes express the Su(var)3-9 mutant proteins. MW, molecular weight.
(F) Western blots of immunoprecipitates using anti-FLAG antibody and nuclear extracts of S2 cultured cells in which FLAG-Wge expression was induced (+) or not induced (-). Anti-FLAG antibody detected FLAG-Wge protein (∼180 kDa) and a non-specific protein (marked with an asterisk).
(G and H) Immunostaining of SG cells (ey>wge, G, and ey>wge, Su(var)3-91/2, H).
(I and J) Eye-to-wing TD frequency in adult flies for Su(var)3-91/2 (I) and for Su(var)2054/+ (J). Ratios of the number of flies with an ectopic wing in the eye field to the total number of flies are indicated. In (J), ey-GAL4 females and Su(var)2054/UAS-GFP; UAS-wge males were crossed, and their progenies were observed. ∗p < 0.05, chi-square test.
(K–N) Representative eye phenotype of ey>+ (K), ey>wge (L), ey>wge, Su(var)3-91/2 (M) and ey>wge, Su(var)2054/+ (N) fly.
(O–Q) Su(var)3-91/2 transheterozygote (P) and Su(var)2054/+ heterozygote (Q) do not show any developmental defects in the eye or wing similar to wild-type (OregonR, O).
Scale bars, 10 μm.
See also Figures S2, S3, and S4.
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6. Figure 4
Su(var)3-9 Is Required for Wound-Induced TD of Leg Imaginal Discs
(A–H) X-GAL staining of imaginal discs for vgBE-lacZ (control) and vgBE-lacZ, Su(var)3-91/2. Larval prothoracic leg discs (A and E) and wing discs (B and F) are shown. To obtain leg disc fragments (posterior 3/4 and anterior 1/4 fragments), the leg disc was cut as indicated by the dotted line in (A). Some P3/4 fragments (C) or A 1/4 fragments (D) showed activation of vgBE-lacZ expression after in vivo culture, but Su(var)3-91/2 rarely showed such expression (G and H). Scale bars, 100 μm.
(I) Leg-to-wing TD frequency after in vivo culture. Ratios between the number of X-GAL-positive discs and the total number of discs are shown. ∗p < 0.05, chi-square test.
(J) Leg disc size after in vivo culture. Data represent mean ± SD. n.s., no significant difference.
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7. Figure 5
Transcriptional Profiling of Eye- and Wing-Specific Genes in wge-Mediated TD
(A) MA-plot for comparison of public RNA-seq data between eye discs and wing discs (Naval-Sánchez et al., 2013). Wing-enriched genes (green dots) and eye-enriched genes (red dots) were evaluated based on the criterion of FDR < Housekeeping genes and others are indicated by blue and black dots, respectively.
(B) Heatmap depicting relative expression of eye-EGs in microarray data. C1: ey-GAL4; C2: UAS-wge; wge: ey>wge; wgeSV: ey>wge, Su(var)3-91/2. The color key indicates high (red) and low (blue) expression.
(C) Heatmap showing wing-EGs and notable wing-specific genes (lower box).
In (B) and (C), 42 of 115 eye-EGs and 23 of 49 wing-EGs were not included because of weak signals (see Supplemental Experimental Procedures).
(D and E) Heatmap of (D) housekeeping genes and (E) antenna-specific genes.
(F) The graph shows fold enrichment of eye-EGs and wing-EGs in DEGs between controls and ey>wge.
(G–H”) Immunostaining of Wge-overexpressing clones induced in eye disc for hs-flp; Ay-GAL4 UAS-wge UAS-GFP eya-lacZ (control, G) and hs-flp; Ay-GAL4 UAS-wge UAS-GFP eya-lacZ; Su(var)3-91/2 (H). Wge-overexpressing clones are surrounded by white lines. Scale bar, 10 μm.
See also Figure S5, Tables S1, S2, and S3, and Supplemental Experimental Procedures.
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8. Figure 6
Transcriptional Profiling of Non-eye- and Wing-Specific Genes in wge-Mediated TD
(A) Heatmap depicting relative expression of TD-enriched genes (Klebes et al., 2005) in microarray data. Genes whose expression was dependent on Su(var)3-9 are noted in red.
(B) The pie chart shows the proportion of DEGs in ey>wge categorized according to the indicated features.
(C) The graph shows relative enrichment of each class of regeneration genes (Katsuyama et al., 2015) in DEGs. A red line represents no enrichment.
(D) Heatmap depicting class I and class III regeneration genes in DEGs. Genes whose expression was dependent on Su(var)3-9 are noted in red.
(E and F) Heatmap showing up-DEGs (E) and down-DEGs (F) in ey>wge. In all heatmaps, the abbreviations and color key are the same as in Figures 5B and 5C. The asterisk shows Down-DEGs that were downregulated in a Su(var)3-9-dependent manner.
See also Table S4.
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