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FIGURE 5.
Overexpression of ets1 attenuates BMP signal downstream of Smad1/5/8 phosphorylation and represses id3 through binding to its promoter. A–C, in situ hybridization analysis of the expression of the indicated markers in embryos injected with ets1 mRNA (sox3, 87%, 13 of 15; epi-keratin (epiker), 75%, 12 of 16; zic1, 100%, 22 of 22). An asterisk indicates the injected side. D, neural markers sox3, sox2, and ncam were induced by ets1 overexpression in an animal cap assay. E–G, expression of sizzled was examined in embryos injected with bmp4 mRNA (89%, 32 of 36) alone or together with ets1 mRNA (68%, 27 of 40). H, luciferase assay was performed to assay BMP signaling. chordin (chd) was used as a control. *, p < 0.05 between indicated group and the group only treated with BMP4. I, Western blot showing the level of phosphorylated Smad1/5/8 (p-Smad1/5/8) and total Smad1/5/8 in stage 16 embryos overexpressing ets1. Endogenous tubulin was used as a loading control. J, co-immunoprecipitation was performed using lysates from embryos injected with either ets1-HA, smad1-Myc separately, or both. K and L, expression of id3 in control (K) and ets1-injected embryos (L; 65%, 22 of 34) was examined by in situ hybridization. M–P, expression of foxd3 and snail2 in embryos injected with either the mixture of ets1 and gfp (M, 69%, 24 of 35; O, 61%, 22 of 36) or the mixture of ets1 and id3 (N, 69%, 31 of 45; P, 60%, 29 of 48). Q–R′, cell apoptosis was detected by TUNEL assay (black spots) in embryos with one side injection of ets1 mRNA (100%, 5 of 5). LacZ staining (light blue) was used to indicate the injected side. Q, control embryos; R, ets1-injected embryo; R′, high magnification of framed region in R. S–V, hairy2-GR mRNA (500 pg/embryos) was injected into embryos at the two-cell stage alone or together with ets1 (500 pg/embryos), and DEX was used to treat embryos from stage 13 to stage17. The expression of msx1 was examined using in situ hybridization. Although hairy2 slightly promoted msx1 expression (T, 88%, 23 of 26), co-expression with ets1 enhanced msx1 expression (U, 95%, 19 of 20; V, 96%, 25 of 26). W, the predicated Ets1 binding site in the id3 promoter of X. laevis and the primers used in ChIP. W′, schematic diagram illustrating the predicted SMAD1 and ETS1 binding sites in the human ID3 promoter and the primers used in the ChIP assay. X, ChIP was performed using antibodies against ETS1 in HEK293T cells, and the precipitated DNA fragments were amplified using primer pair 1 (P1) or the GAPDH primer pair, respectively. Endogenous ETS1 can bind to the predicted binding site. The GAPDH primer pair was used as the control. Y and Z, ChIP was done in X. laevis embryos injected with 500 pg of ets1-FLAG. Semiquantitative (Y) and real time PCR (Z) were performed using primer pair 3 (P3) or primer pair control (Pc) (Table 2). WE, uninjected whole embryo; AC, uninjected animal caps; RT−, without reverse transcriptase; con, control; IB, immunoblot. Error bars represent S.D. |
