1. Drosophila Schip1 Links Expanded and Tao-1 to Regulate Hippo Signaling
Hyung-Lok Chung, George J. Augustine, Kwang-Wook Choi 
Developmental Cell 
Volume 36, Issue 5, Pages (March 2016)
DOI: /j.devcel
Copyright © 2016 Elsevier Inc. Terms and Conditions

2. Figure 1 Loss of Schip1 Causes Overproliferation in the Eye
(A) Schematic alignment of Schip1 and human SCHIP1.
(B) Schematic structure of the Schip1 gene. Schip1GS12074 has a P element inserted in the second exon. Schip149 has a 794-bp deletion by imprecise excision of GS12074 P element.
(C and D) Scanning electron microscopy images of adult eyes. (C) w1118 control shows the normal eye. (D) An eye with Schip149 mutant clones shows an enlargement with folded surface, indicating overgrowth.
(E and F) Eye discs stained with the GFP clone marker. (E) Sizes of Schip1+ clones (GFP−) are similar to those of GFP+/GFP+ twin-spot clones (cells with brighter green). (F) Sizes of Schip149 mutant clones (−/−) are much bigger than GFP+ twin-spot clones (+/+). (+/−) indicates heterozygous cells.
(G) Quantification of the ratio of GFP− area/total area (%) for Schip1+ and Schip149 clones (n = 5; error bars represent ±SEM. ∗∗∗p < 0.001, t test).
(H and I) Schip1 mutant clones in mid-pupal eyes show increased number of interommatidial cells. (H) Schip1+ clones. (I) Schip149 clones. Arrows indicate the regions where interommatidial cells are increased.
Scale bars, 20 μm (H and I).
(J–K′) Effects of Schip1 mutant clones on BrdU and PH3 levels in eye discs. (J and J′) Schip1 mutant clones posterior to the SMW show ectopic BrdU staining. (J′) shows only red channel. (K and K′) Schip1 mutant clones posterior to the SMW show ectopic PH3 staining. (K′) shows only red channel. Marked area in (J′) and (K′) indicates the mutant clones posterior to the SMW. Positions of the SMW and the morphogenetic furrow (MF) are indicated by arrows.
Scale bars, 50 μm (E, F, J–K′).
Developmental Cell  , DOI: ( /j.devcel )
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3. Figure 2 Schip1 Regulates Hpo Pathway Target Genes
(A and B) Schip149 mutant clones show higher levels of Diap1 and Cyclin E in eye discs. (A–A″) Diap1 staining in Schip149 mutant clones. (A) Merge, (A′) GFP, (A″) Diap1. (B–B″) Cyclin E staining in Schip149 mutant clones. (B) Merge, (B′) GFP, (B″) Cyclin E. The areas marked by dotted lines show clear increases in the level of Cyclin E (B).
(C–C″) Ex staining in Schip149 mutant clones of wing disc. (C) Merge, (C′) GFP, (C″) Ex. Marked areas shows clear increases in the Ex level.
(D–D″) Knockdown of Schip1 by dpp-Gal4 induces ectopic expression of the ex-lacZ reporter in the anterior-posterior boundary region compared with dpp>GFP control. (D) dpp>GFP; ex-lacZ, (D′) GFP, (D″) dpp>Schip1 RNAi; ex-lacZ.
(E and F) Upregulation of Crb level in Schip1 clones. (E–E″) Crb staining in Schip149 mutant clones of wing disc. (E) Merge, (E) GFP, (E″) Crb. Marked areas show clear increases in the Crb level. (F–F″) crb-lacZ is normally induced along the dorsoventral boundary region (arrows). Schip1 knockdown by en-Gal4 induces ectopic expression of crb-lacZ in the posterior compartment (asterisks). (F) Merge, (F′) GFP, (F″) crb-lacZ.
(G and H) Upregulation of Crb by Schip1 RNAi was suppressed by knockdown of Yki. (G–G″) Crb level is increased by Schip1 RNAi. (G) Merge, (G′) GFP, (G″) Crb. (H–H″) yki RNAi suppressed the increase of Crb caused by Schip1 RNAi. (H) Merge, (H′) GFP, (H″) Crb. The areas marked by dotted lines show the boundary between posterior and anterior part of wing discs.
Scale bars, 20 μm.
Developmental Cell  , DOI: ( /j.devcel )
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4. Figure 3
Schip1 Inhibits Yki Activity by Regulating the Level of Yki Phosphorylation and Subcellular Localization
(A–A″) The pattern of Yki is changed in Schip149 mutant clones. (A) Merge, (A′) GFP, (A″) Yki. Dashed lines show the regions where the localization of Yki is clearly altered in wing disc.
(B–B″) Confocal analysis at higher magnification shows altered Yki localization. (B) Merge, (B′ and B″) Yki. Dashed lines in (B–B″) indicate the clone boundary between GFP-positive (Schip1+/+ and Schip1+/−) and GFP-negative cells (Schip1−/−). Several cells with brighter GFP staining in (B) are twin-spot cells. In (B′), approximate positions of nuclei filled with Yki are indicated by dotted circles within the clone.
(C and C′) Yki luciferase assays show that Schip1 can suppress the transcriptional activity of Yki. (C) Transcriptional activity of Yki measured by relative luciferase ratios in S2 cells transfected with the Gal4 DNA binding domain with Yki (Gal4DBD-Yki), UAS-luciferase, and plasmids expressing MBP as a negative control. (C′) Transcriptional activity of Yki measured by relative luciferase ratios in S2 cells transfected with Gal4DBD-Yki, UAS-luciferase, and plasmids expressing Hpo, Ex and Schip1. The transcriptional Yki activity is suppressed by Schip1.
(D) The expression level of phosphorylated Yki at S168 residue is increased by Schip1 overexpression, although the total amount of Yki was not noticeably affected.
All error bars represent SD (n = 5). Scale bars, 20 μm (A and B).
Developmental Cell  , DOI: ( /j.devcel )
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5. Figure 4 Genetic Interaction of Schip1 with Wts and Ex
(A–C) Overexpression of Wts rescues increased cell proliferation by knockdown of Schip1 in the posterior wing disc (en>Schip1 RNAi). (A) en Gal4/+, (B) en>Schip1 RNAi/+, (C) en>Schip1 RNAi, wts. Dotted lines indicate the anterior-posterior boundaries based on en>GFP (not shown). Anterior is to the left. Scale bars, 50 μm.
(A′–C′) Increased adult wing of en>Schip1 RNAi is rescued by Wts overexpression. (A′) en Gal4/+, (B′) en>Schip1 RNAi/+, (C′) en>Schip1 RNAi, wts. Red dashed lines indicate the wild-type wing size. Scale bars, 100 μm.
(D–F) Overexpression of Ex or Schip1 suppresses the defects of GMR>crbintra. (D) GMR>crbintra/+, (E) GMR>crbintra>ex, (F) GMR>crbintra>Schip1.
(G–I) Knockdown of Schip1 strongly rescues the small eye phenotype of Ex overexpression. (G) GMR Gal4/+, (H) GMR>ex/+, (I) GMR>ex>Schip1 RNAi.
(J–L) Overexpression of Schip1 partially rescues the small eye phenotype of Ex knockdown. (J) ey Gal4/+, (K) ey>ex RNAi, (L) ey>ex RNAi>Schip1.
Developmental Cell  , DOI: ( /j.devcel )
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6. Figure 5 Schip1 Co-localizes and Physically Interacts with Ex
(A and A′) Schip1 antibody staining is greatly reduced in Schip149 mutant clones (GFP-negative region marked by dashed line). (A) Merge, (A′) Schip1. (A″) Western blot analysis of adult tissue extracts of w1118 and Schip149/+ heterozygote. The Schip1 level is reduced in heterozygotes.
(B–E) Overlapping localization of Ex and Schip1 in wing imaginal discs of w1118. (B) Merge of Ex (green) and Schip1 (red) at a low magnification. (C–E) Z-section view at a high magnification of the box region in (B). (C) Ex, (D) Schip1, (E) merge. Note that some Ex staining is located more apical than Schip1. (C′–E′) Horizontal sections. (C′) Ex, (D′) Schip1, (E′) Merge.
(F–H) Schip1 directly interacts with FERM domain of Mer. (F) GST pull-down between MBP-Mer and GST-Schip1. (G) GST pull-down between GST-Schip1, MBP-Mer (1–314), and MBP-Mer (314–635). (H) CoIP between Schip1 and Mer.
(I–K) Schip1 directly binds to the FERM domain of Ex. (I) GST pull-down between Schip1 and Ex. (J) GST pull-down between Ex (N) (N-terminal FERM domain) and Schip1. (K) CoIP between Ex and Schip1.
(L–O) Localization of Schip1 is altered by knockdown or overexpression of Ex in wing disc. Green staining indicates GFP expression by ptc-Gal4 or en-Gal4. (L) The apical section of ptc>ex RNAi>GFP. The arrow shows a reduced Schip1 level in the ptc domain where ex RNAi is targeted. (M) Basal section of ptc>ex RNAi>GFP. The arrow shows Schip1 accumulation in the ptc domain. (N) Some Schip1 staining was mislocalized to the basal region in the posterior part of en>ex RNAi>GFP wing disc. Top panels: Z-section images. Bottom panels: Basal section images show a higher level of Schip1 in the posterior region. Apical sections show no significant difference in the posterior region (not shown). (O) Overexpression of Ex increases Schip1 in the apical region. Z sections are shown on the top. The arrow indicates the apical region where ectopic Schip1 is recruited by Ex overexpression. Red dashed lines indicate the approximate position of the apical basal boundary, and white dashed lines show the boundary between posterior and anterior part of wing disc (N and O).
Scale bars, 20 μm.
Developmental Cell  , DOI: ( /j.devcel )
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7. Figure 6
Schip1 Is Required for Hpo Activity by Promoting Phosphorylation and Proper Localization of Hpo
(A–A″) Loss of Schip1 decreases the phosphorylated Hippo (P-Hpo) level but increases the level of Hpo. (A) Anti-Hpo staining of Western blot of protein extracts from wing discs containing Schip149 mutant clones. (A′) Quantification of relative level of P-Hpo band intensity (n = 5). (A″) Quantification of relative Hpo band intensity for Schip1+ clones versus Schip149 mutant clones (n = 5). Error bars represent ±SEM. ∗∗∗p < 0.001; 0.001 < ∗∗p < 0.01.
(B–B″) Overexpression of Schip1 increases the P-Hpo level but decreases the level of Hpo. (B′) Quantification of relative level of P-Hpo band intensity (n = 5). (B″) Quantification of relative Hpo band intensity for Schip1+ clones versus Schip149 mutant clones (n = 5). Error bars represent ±SEM. ∗∗∗p < 0.001; 0.001 < ∗∗p < 0.01.
(C) c100p100 centrifugation assay. Schip1 overexpression increases the Hpo level in the membrane fraction. Schip1 RNAi shows the opposite effects.
(D–G′) Immunostaining of Hpo in Schip149 mutant clones of developing eye and wing imaginal disc. (D–D″) Hpo staining in Schip149 mutant clones in eye disc. (D) Merge, (D′) GFP, (D″) Hpo. (E–E″) Hpo staining in Schip149 mutant clones in wing disc. (E) Merge, (E′) GFP, (E″) Hpo. (F and F′) High magnification of Schip149 mutant clones (the boxed area in E). (F) Merge, (F′) Hpo. (G and G′) Z-stack image of Hpo staining in the Schip149 mutant clone in eye disc (G) and wing disc (G′).
Scale bars, 20 μm.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2016 Elsevier Inc. Terms and Conditions

8. Figure 7
Tao-1 Kinase Functions Downstream of Schip1 through Physical Association
(A–E) Effects of Tao-1 and Schip1 on Hpo expression in wing disc. GFP staining indicates the posterior compartment by en>Gal4. (A) Reduced Hpo staining by en>Tao-1 Flag. (B) Reduced Hpo staining by en>Schip1. (C) Enhanced Hpo staining by en>Schip1 RNAi. (D) Tao-1 overexpression suppresses the effects of Schip1 RNAi in en>Schip1 RNAi>Tao-1 Flag (E) CoIP between Schip1 and Tao-1.
(F) GST pull-down between MBP-Tao-1 and GST-Schip1.
(G) c100p100 centrifugation assay. Schip1 increases Tao-1 level in membrane fraction.
(H) Schip1 promotes the kinase activity of Tao-1 in S2 cell.
Scale bars, 20 μm (A–D).
Developmental Cell  , DOI: ( /j.devcel )
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9. Figure 8 Proposed Roles of Schip1 in Hippo Signaling
Several Hpo signaling components relevant to this study are shown schematically. Hpo together with Sav activates Wts. Activated Wts inhibits Yki activity by excluding Yki from the nucleus. Ex is associated with apical cell membrane. For active Hpo signaling, Ex directly interacts with Schip1 and recruits it to the apical membrane. Schip1 recruits Tao-1, thus leading to Hpo phosphorylation. Ex can also interact with Wts (Sun et al., 2015). It is unknown whether Schip1 regulates Ex-Wts interaction (question mark). Mer genetically acts upstream to Hpo but it can also function in a parallel pathway by directly recruiting Wts to the plasma membrane (Yin et al., 2013). Schip1 can bind Mer, and may also function in this parallel pathway (dotted arrow; see Discussion for details).
Developmental Cell  , DOI: ( /j.devcel )
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