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. 2008 Mar;14(3):377-87.
doi: 10.1016/j.devcel.2008.01.006. Epub 2008 Feb 7.

The TEAD/TEF family of transcription factor Scalloped mediates Hippo signaling in organ size control

Lei Zhang  1 , Fangfang Ren, Qing Zhang, Yongbin Chen, Bing Wang, Jin Jiang
Affiliations

The TEAD/TEF family of transcription factor Scalloped mediates Hippo signaling in organ size control

Lei Zhang et al. Dev Cell. 2008 Mar.

Abstract

The Hippo (Hpo) signaling pathway governs cell growth, proliferation, and apoptosis by controlling key regulatory genes that execute these processes; however, the transcription factor of the pathway has remained elusive. Here we provide evidence that the TEAD/TEF family transcription factor Scalloped (Sd) acts together with the coactivator Yorkie (Yki) to regulate Hpo pathway-responsive genes. Sd and Yki form a transcriptional complex whose activity is inhibited by Hpo signaling. Sd overexpression enhances, whereas its inactivation suppresses, tissue overgrowth caused by Yki overexpression or tumor suppressor mutations in the Hpo pathway. Inactivation of Sd diminishes Hpo target gene expression and reduces organ size, whereas a constitutively active Sd promotes tissue overgrowth. Sd promotes Yki nuclear localization, whereas Hpo signaling retains Yki in the cytoplasm by phosphorylating Yki at S168. Finally, Sd recruits Yki to the enhancer of the pathway-responsive gene diap1, suggesting that diap1 is a direct transcriptional target of the Hpo pathway.

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Figures

Figure 1
Figure 1. Sd and Yki form a transcriptional complex regulated by Hpo signaling
(A) Diagram of Sd protein and deletion constructs. The TEA DNA binding domain is between aa 85–155. (B) S2 cells were transfected with the indicated Yki and Sd constructs, followed by immunoprecipitation and western blot analyses with the indicated antibodies. Arrows indicate IgG. (C) Diagram of the 3XSd2-luc reporter gene. Three copies of a DNA fragment containing tandem Sd binding sites (shaded sequence) from the dSRF enhancer (dSRF-A2-I) were placed upstream of the heat shock basal promoter (hs) followed by the luciferase coding sequence. (D) Sd and Yki act synergistically to activate the 3XSd2-luc reporter gene. S2 cells were transfected by the indicated Yki and Sd expression constructs plus the luciferase reporter gene, followed by the dual luciferase assay. F, N, and C indicate the full-length Sd, Sd-N, and Sd-C, respectively.
Figure 2
Figure 2. Sd synergizes with Yki to promote tissue growth and Hpo target gene expression
(A–C′) Side (A–C) or dorsal views (A′–C′) of wild type eye (A, A′), eyes expressing Yki (B, B′) or Yki + Sd (C, C′) with GMR-Gal4. (D–F″) BrdU incorporation (D, E, F), cycE (D′, E′, F′) and diap1 (D″, E″, F″) expression in wild type eye discs (D, D′, D″), or eye discs expressing Yki (E, E′, E″) or Yki +Sd (F, F′, F″). Arrowheads indicate the position of SMW.
Figure 3
Figure 3. Inactivation of Sd blocks tissue overgrowth induced by Yki or Hpo pathway mutations
(A–D) Adult eyes of GMR-Gal4 UAS-Yki (A), GMR-Gal4 UAS-Yki; UAS-Sd-RNAiN (B), GMR-Gal4 UAS-Yki; UAS-Sd-RNAiC (C), and GMR-Gal4 UAS-Yki; UAS-Sd-RNAiN +UAS-Sd-RNAiC (D). (E–J) Adult eyes containing hpo (E), sav (G), or, wts (I) mutant clones or corresponding mutant clones that express UAS-SdRNAiN and UAS-SdRNAiC (F, H, J). (K–P) cycE (K–L) and diap1 (M–N) expression, and BrdU incorporation (O–P) in eye discs expressing Yki (K, M, O) or Yki plus Sd RNAi transgenes (L, N, P) with GMR-Gal4. (Q–Q″) Adult eye (Q) or eye disc (Q′, Q″) with YkiM123 expressing clones. YkiM123 expressing cells were recognized by Myc (red) and GFP (green) staining in discs (Q″). (R–R″) Adult eye (R) or eye disc (R′, R″) with sd B clones expressing YkiM123. sd B mutant cells expressing YkiM123 were labeled by both Myc and GFP expression (R″).
Figure 4
Figure 4. Inactivation of Sd reduces organ size and downregulates Hpo target genes
(A–E) Adult eyes of wild type (A), or expressing two copies of UAS-SdRNAiN (B), two copies of UAS-SdRNAiC (C), UAS-SdRNAiN +UAS-SdRNAiC (D), or two copies of UAS-Yki-RNAiN (E) with ey-Gal4 in the presence of UAS-Dicer2. (F–G′, J–J′) Wing discs expressing UAS-GFP (F, F′), UAS-GFP +UAS-Sd-RNAiN + UAS-Sd-RNAiC (G, G′), or UAS-GFP plus two copies of UAS-Yki-RNAi (J, J′) with hh-Gal4 were immunostained to show the expression of Diap1(red) and GFP (green). GFP marked the P-compartment (arrows). (H–I′, K, K′) Wild type wing disc (H, H′), or wing discs expressing UAS-Sd-RNAiN +UAS-Sd-RNAiC (I, I′) or two copies of UAS-Yki-RNAi (K, K′) with hh-Gal4 were immunostained to show the expression of Ci (red) and ex-lacZ (blue). The A/P boundary is indicated by white lines based on Ci expression (I′, K′). Arrows indicate the P-compartment. (L–M′) Eye (L, L′) and wing (M. M′) discs containing flip-out clones expressing UAS-Sd-RNAiN +UAS-Sd-RNAiC with act>CD2>Gal4 were immunostained to show the expression of CD2 (red) and a bantam sensor (BS; green). Cells expressing Sd-RNAi transgenes were marked by the lack of CD2 expression (arrows).
Figure 5
Figure 5. An active form of Sd induces tissue overgrowth and activates Hpo target genes
(A–D) Wild type eye (A), or eyes expressing Sd-GA (B, C) or Sd (D) with GMR-Gal4. (E, F) Adult eyes expressing dMSTn (E) or dMSTn plus Sd-GA (F) with GMR-Gal4. (G-J). Wild type eye discs (G, I) or eye discs expressing Sd-GA with GMR-Gal4 (H, J) were immunostained to show the expression of CycE (G–H) or Diap1 (I–J). (K, K′) A wing disc expressing Sd-GA with hh-Gal4 was immunostained to show the expression of ex-lacZ (blue) and Ci (red). Ci marked the A-compartment.
Figure 6
Figure 6. Characterization of diap1 enhancers
(A) Deletion analysis of diap1 enhancer elements. The diap1 genomic region containing the first intron and two neighboring exons is shown at the top. A diap1-lacZ enhancer trap line, l(3)j5C8, is inserted at +3860 bp. diap1-GFP transgenes containing indicated genomic fragments are listed. (−): no discernible expression; (*): weaker expression. (B) ChIP analysis of the 1.8 kb diap1 enhancer. Individual Sd binding consensus sites are indicated by open or filled arrowheads (filled arrowheads indicate the binding sites with reversed orientation). The sequences of individual Sd binding consensus sites are shown to the right. E1 to E4 demarcate the regions amplified by PCR in the ChIP experiments. (C–E′) GFP expression in wing (C, D, E) or eye (C′, D′, E′) discs driven by diap1-GFP4.3 (C, C′), diap1-GFP3.5 (D, D′), or diap1-GFP1.8 (E, E′). (F–G′) Eye discs that contain diap1-GFP4.3 and flip-out clones expressing Yki (F, F′) or Sd-GA (G, G′) with act>CD2>Gal4 were immunostained to the expression of GFP (green) and CD2 (red). Cells expressing Yki or Sd-GA were marked by the lack of CD2 expression.
Figure 7
Figure 7. Hpo signaling regulates Yki nuclear localization and activity through phosphorylating S168
(A–B) S2 cells expressing Myc-Yki (A) or Myc-YkiM1 (B) with or without Fg-Hpo and/or HA-Sd were immunostained with anti-Myc (red) and anti-HA (green) antibodies. Nuclei were marked by DRAQ5 (blue) and demarcated by red dashed lines. Images in individual channels were shown as black and white in top and middle rows and merged images in bottom rows. (C–D″) High magnification view of wing discs expressing Fg-Yki either alone (C–C″) or together with HA-Sd (D–D″) using MS1096 and immunostained with anti-Flag (red) and anti-HA (green in D) antibodies. The nuclei were labeled by 7-AAD (green in C–C″). (E–F″) High magnification view of wing discs carrying wts clones and expressing Fg-Yki either alone (E–E″) or together with HA-Sd (F–F″). wts mutant cells were marked by the lack of GFP expression (green). (G–J) Adult eyes expressing Myc-Yki (G), Myc-YkiM1 (H), Myc-YkiM12 (I), or Myc-YkiM123 (J) with GMR-Gal4. Of note, a strong transgenic line for Myc-Yki is shown here. (K–N) High magnification view of wing discs expressing Myc-Yki (K), Myc-Yki plus HA-Sd (L), Myc-YkiM123 (M), or Myc-YkiM123 plus HA-Sd (N). (O) The Drosophila Hpo pathway. See text for details.
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