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First published online 5 October 2005
doi: 10.1242/dev.02059

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Tead proteins activate the Foxa2 enhancer in the node in cooperation with a second factor

Atsushi Sawada¹, Yuriko Nishizaki^2,*, Hiroko Sato¹, Yukari Yada¹, Rika Nakayama³, Shinji Yamamoto¹, Noriyuki Nishioka¹, Hisato Kondoh² and Hiroshi Sasaki^1,

¹ Laboratory for Embryonic Induction, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan
² Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan
³ Laboratory for Animal Resources and Genetic Engineering (LARGE), RIKEN Center for Developmental Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan

View larger version (100K): [in a new window]   Fig. 1. Activity of the core element of the Foxa2 node/notochord enhancer in transgenic mouse embryos. (A) Schematic representation of the mouse Foxa2 enhancer. The node/notochord enhancer is located upstream of the transcription initiation site (yellow square). CS3 (red circle) is essential for enhancer activity. A 27-bp DNA fragment straddling CS3 is the core element (CE) of the enhancer. (B-J) Distribution of ß-galactosidase activity in transgenic mouse embryos that express LacZ under the control of eight copies of the CE. Whole-mount staining of LS (B), EB (C), LHF (D,E), E8.5 (G) and E9.0 (H) stage embryos. Sagittal section of an LHF embryo crossing through the node (F), and cross-sections of an E8.5 embryo (I,F). Approximate positions of sections shown in panels I and F are indicated in panel G by i and j, respectively. ame, axial mesoendoderm; ec, ectoderm; en, endoderm; h, heart; hf, headfold; hm, head mesenchyme; m, mesoderm; mb, midbrain; n, node; nc, notochord; nf, neural fold; np, neural plate; ov, otic vesicle; pm, presomitic mesoderm; pp, prechordal plate; ps, primitive streak; s, somite; tb, tailbud.

View larger version (24K): [in a new window]   Fig. 2. Activation of the node/notochord enhancer and CE in Wnt/ß-catenin treated P19 cells. Expression of reporters containing the enhancer (A) or 8 copies of CE (8xCE) (B) at 48 hours after co-transfection of Wnt expression plasmids. Expression of reporters containing the enhancer (C) or 8xCE (D) at 48 hours after co-transfection of stabilized-ß-catenin expression plasmid. (E) Timecourse of reporter activation after co-transfection with stabilized ß-catenin expression plasmid. Luciferase activities were normalized to the activity of 8xCE reporter without ß-catenin at each time point. In Figs 2, 3, and 4, the results of luciferase assays represent the average of two samples with standard deviations.

View larger version (61K): [in a new window]   Fig. 3. Activation of CE in ß-catenin-treated cells and node/notochord enhancer activity require two separate sequences within the CE. (A) Sequences of wild-type (WT) and mutated CEs (M1-5 and ins4/5). In the mutated CEs, the altered or inserted nucleotides are shown in red lower case. The nucleotides required for the activation are summarized in wild type sequence and denoted with red. (B) Expression of reporters containing wild-type and mutated 8xCEs 48 hours after co-transfection of the stabilized ß-catenin expression plasmid. (C) Transgenic embryos containing wild-type enhancer express ß-galactosidase in the node and notochord, reflecting endogenous Foxa2 expression accompanied by ectopic expression in the primitive streak region (n=9/11). (D,F) Transgenic embryos with mutated enhancers lacking activity in ß-catenin-expressing P19 cells did not show ß-galactosidase expression in the node or notochord (D: M1-2; n=0/8, F: M4; n=0/10). (E,G) Transgenic embryos with mutated enhancers with activity in P19 cells retained ß-galactosidase expression in the node and notochord (E: M3; n=3/7, G: M5; n=6/8). n, node; nc, notochord; ps, primitive streak.

View larger version (80K): [in a new window]   Fig. 4. Tead proteins bind the CE in a sequence-specific manner. (A) Gel mobility shift assay showing that Tead2 and Tead4 bind to the CE. Wild-type CE was used as a probe. Positions of the Tead-DNA complexes are indicated by arrows. Combination of protein and competitor are indicated above each lane. Sequences of the probe and competitors are described in Fig. 3A. (B) Sequences of the competitors used in panel C. Sequences of NF-kB-BS and GT-IIC were adapted from those described previously (Davidson et al., 1988; Fujita et al., 1992). Altered residues in CE/NF-kB and CE/GT-IIC are indicated in red lower case. Core recognition sequences of Rel and Tead are indicated by boxes. Position of the M4 mutation that is essential for NE activity is shown in red in the CE sequence. (C) Comparison of DNA-binding activities of Rel and Tead. Combinations of proteins and competitors are indicated above each lane. Positions of protein-DNA complexes are indicated on the right. (D) Transgenic embryos carrying a mutant enhancer in which CE was altered to CE/GT-IIC retained gene expression in the node and notochord (n=6/7). (E) Transgenic embryos carrying a mutant enhancer in which CE was altered to CE/NF-kB lost gene expression in the node/notochord (n=0/4). n, node; nc, notochord.

View larger version (74K): [in a new window]   Fig. 5. Tead and Yap65 are widely expressed during mouse embryogenesis. Whole-mount in situ hybridization of Tead1 (A,G), Tead2 (B,H,J), Tead3 (D), Tead4 (E) and Yap65 (F) in mid- to late-streak (A-F), early head-fold (G,H) and E8.5 (J) stage embryos. (C) A section of a whole-mount stained embryo shown in panel B. Approximate position is indicated by the line in panel B. (I,K) In situ hybridization of Tead2 on sectioned embryos. Approximate positions of sections are indicated in panels H and J.

View larger version (27K): [in a new window]   Fig. 6. Tead-Yap complex activates the CE in P19 cells. (A) Transfection assay showing activation of 8xCE reporter by co-transfection of Tead and Yap expression vectors. The reporter is indicated in the upper left corner of each panel. Combinations of Tead and Yap effectors are indicated below the panel. (B) The Tead binding site is required for activation of the CE by Tead-Yap complex. Mutation of the Tead-binding site (M4) abolished activation by Tead-Yap. (C) Transcriptional activity of Tead is not increased in ß-catenin-expressed P19 cells. By contrast to the activation of 8xCE, the authentic Tead-binding site (8xGT-IIC) was not activated in ß-catenin-expressed cells.

View larger version (64K): [in a new window]   Fig. 7. Tead regulates NE enhancer activity and notochord development in vivo. Electroporation of control (A,B) and Tead-EnR expression plasmid (C,D) in enhancer-LacZ transgenic mouse embryos. Representative cross-sections of embryos in A and C are shown in B and D, respectively. Green and red represent NE enhancer activity and electroporated cells, respectively. (A,B) Control electroporation did not affect NE expression. (C,D) Tead-EnR repressed NE activity and notochord development. en, endoderm; nc, notochord; nt, neural tube; s, somite. (E) Model of Foxa2 regulation in the node and notochord. In the node, a Wnt signal promotes formation of the Tead-POT complex on the core element of the enhancer. Once the cells migrate from node to notochord, POT disappears from the CE, and Tead together with other transcription factors (gray objects) continue the activation of the enhancer.

View larger version (143K): [in a new window]   Fig. 8. Tead regulates foxa2 in the zebrafish shield. Expression of foxa2 (A-C) or ntl (D) in shield-stage embryos injected with various RNAs. Injection of EGFP RNA (100 pg) did not alter Foxa2 expression. Injection of VP16-Tead (B; 100 pg) expanded Foxa2 expression (n=102/125), while injection of Tead-EnR (C,D; 25 pg) reduced Foxa2 expression (C; n=26/36) without affecting expression of ntl (D; n=13/13).