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First published online 12 November 2008
doi: 10.1242/dev.027151

Development 135, 4059-4069 (2008)
Published by The Company of Biologists 2008
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Mammalian Tead proteins regulate cell proliferation and contact inhibition as transcriptional mediators of Hippo signaling

Mitsunori Ota and Hiroshi Sasaki*

Laboratory for Embryonic Induction, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan.


Figure 1
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  		Fig. 1. Regulation of Tead activity by cell density and Hippo signaling. (A-F) Subcellular distribution of Yap1 and Tead proteins in NIH3T3 cells. Distribution of Yap1 (Yap; A,B) and Tead1 (C,D) proteins, and incorporation of BrdU (A',B') at low (A,C) or high (B,D) cell density. (E,F) Signal intensities of Yap1 (E, n=27) and Tead1 (F, n=24) proteins in the nuclei. (G) Schematic representation of the reporter plasmid monitoring the transcriptional activity of endogenous Tead proteins. (H,I) Regulation of Tead activity by cell density (H) or Hippo signaling components (I). (J-M) Reduction of nuclear Yap1 and Tead1 by Lats2. Distribution of Yap1 (J) and Tead1 (K) proteins. Lats2-overexpressing cells were labeled with EGFP expression (green) in J' and K'. Signal intensities of Yap1 (L, n=14) and Tead1 (M, n=16) proteins in the nuclei of Lats2-expressing cells. Empty vector-expressing cells were used as controls. The graphs show averages with standard errors. An asterisk indicates that the difference is statistically significant (P<0.05). Scale bars: in A, 50 µm for A-D; in J, 50 µm for J,K.

 

Figure 2
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  		Fig. 2. Regulation of proliferation, EMT and cell death by Tead and Yap1. (A) Growth curve of Yap1-overexpressing NIH3T3 cells. Control in A,D,F-J indicates cells infected with the empty virus. (B) Schematic representation of structures of modified Tead proteins. (C) Growth curve of modified Tead2-expressing cells. (D) Growth curve of Tead1-VP16- and Tead4-VP16-expressing cells. (E) Effects of modified Tead2 and Yap1 on transcriptional activity of Tead. (F) Effects of altered Tead activities on cell morphology. (G,H) Effects of modified Tead2 and Yap1 on growth of MTD1A cells in matrigel. Morphology (G) and size (H) of colonies. The bar graph shows an average of 50 colonies with standard errors. Asterisks indicate that the differences from control were statistically significant (P<0.05). (I,J) Effects of Tead2-VP16 and Yap1 on Taxol-induced apoptosis. Distribution of DNA amounts (I) and ratio of sub-G1 cells (J). Scale bars: 200 µm in F,G (left); 500 µm in G (right).

 

Figure 3
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  		Fig. 3. Correlation of Tead-co-activator activity and growth-promoting activity of mutant Yap1 proteins. (A) Schematic representation of structures of modified Yap1 proteins. (B) Absence of interactions between Yap1-{Delta}TeadBD and Tead1/2. White arrowheads indicate co-precipitated Tead1 and Tead2. Black arrowheads indicate non-specific bands. (C) Effects of modified Yap1 proteins on transcriptional activity of Tead proteins. (D-F) Growth curves of modified Yap1-expressing NIH3T3 cells. Effects of {Delta}WW and {Delta}TeadBD (D), S112A (E), and {Delta}AD and dnYap1 (F). Control indicates cells infected with the empty virus.

 

Figure 4
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  		Fig. 4. Transformation of NIH3T3 cells by Tead2-VP16 and Yap1. (A-D'') Effects on cell morphology in culture dish. NIH3T3 cells were infected with empty virus (A) or viruses expressing full-length Tead2 (B), Tead2-VP16 (C) or Yap1 (D). Left (A-D) and middle (A'-D') panels are the phase-contrast and the fluorescent images of the same area. Scale bar in A: 200 µm for A-D and A'-D'. Right panels (A''-D'') are low magnification images of the cells stained with Leishman stain. Scale bar in A'': 5 mm for A''-D''. (E-H) Tumor formation assay. Representative mice transplanted with NIH3T3 cells infected with empty virus (E), Tead2-VP16 virus (F) or Yap1 virus (G). (H) Summary of two independent experiments. The tumor was counted on 100th day after subcutaneous injection of cells. n=(number of tumor formation)/(number of transplantation).

 

Figure 5
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  		Fig. 5. Comparison of genes regulated by Yap1, Tead2-VP16, Tead2-EnR and cell density. (A)Yap1 and high cell density affect gene expression in an opposing manner. (B) Tead2-VP16 and high cell density affect gene expression in an opposing manner. (C) Yap1 and Tead2-VP16 affect gene expression in the same manner. (D) Quantitative RT-PCR confirmation of Yap1- and Tead2-VP16-regulated genes. Control indicates cells infected with the empty virus. (E) Quantitative RT-PCR showing the altered expression of the two Yap1- and Tead2-VP16-regulated genes in Tead1-/-;Tead2-/- and Yap1-/- embryos. Asterisks in D and E indicate that the differences from the control are statistically significant (P<0.05). (F) Tead2-EnR and high cell density affect gene expression in the same manner.

 

Figure 6
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  		Fig. 6. Distribution of Tead1 and Yap1 proteins in developing mouse embryos. (A-L) Distribution of Tead1 (A-F) and Yap1 (G-L) proteins in E8.5 (A,B,G,H) and E10.5 (C-F,I-L) embryos. (B,D-F,H,J-L) Enlarged images of boxed areas in A,C,G,I, respectively. Abbreviations: fg, foregut; ht, heart; lpm, lateral plate mesoderm; nc, notochord; nt, neural tube. Scale bars in A: 100 µm for A,G; in C, 250 µm for C,I; in B, 50 µm for B,H; in D, 50 µm for D-F,J-L.

 

Figure 7
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  		Fig. 7. Reduced proliferation of myocardium in Tead1-/- embryos. (A-D) Distribution of BrdU-labeled cells in wild-type (A,B) and Tead1-/- (C,D) embryos at E9.5. (B,D) Enlarged images of the boxed areas in A and C. (E) Expression of Tead1 proteins in E9.5 heart. Endocardium lacks Tead1 expression. Abbreviations: ec, endocardium; fg, foregut; ht, heart; mc, myocardium; nc, notochord; nt, neural tube. Scale bars: in A, 100 µm for A,C; in B, 50 µm for B,D,E.

 

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