Fig. 7

ets1 and mef bind to the lmo2 promoter; ets1 activates the lmo2 promoter through EBS2. (A) EMSA of wild-type ets1 and C-terminal truncated ets1ΔN binding to oligo wtEBS1. Lane 1 contains labeled probe without lysate. Several nonspecific bands appear when labeled probe is incubated with unprogrammed lysate (lane 2). Neither version of ets1 can bind to labeled wtEBS1 (lanes 3 and 4). The second EMSA was performed with wild-type (wtEBS2,3) and mutant probes (e2-, e3-). Lane 1 contains labeled probe without lysate. A nonspecific band appears when labeled probe is incubated with unprogrammed lysate (lane 2). Wild-type ets1 exhibits minimal binding (lane 3), while ets1ΔN binds robustly (lane 4). ets1ΔN is used in lanes 4–10. Competition was performed with 50x and 500x excess of unlabeled wtEBS2,3 (lanes 5 and 6) as well as unlabeled mutant competitors (lanes 7–10). Lanes 7 and 8 show that the e2- mutant oligo is unable to compete away binding, which demonstrates that ets1ΔN specifically binds to EBS2. Alternatively, lanes 9 and 10 show that there is no specific binding to EBS3. The third EMSA shows mef binding to oligo wtEBS2,3 (lane 3). Lanes 4 and 5 show that mef specifically binds to EBS2 and lanes 6 and 7 show that binding to EBS3 can be competed off. (B) Shown in the first row of this panel are LGb embryos. ets1-injected LGb embryos exhibit a widened EGFP domain, diffuse paraxial mesoderm, and tailbud expression. In the second row, increased ICM as well as diffuse expression of EGFP are seen in injected LGb embryos at 22 hpf. In the third row, ets1 overexpression expands the lmo2 domain into the paraxial mesoderm. (C) Shown in the first row are LG174 stable transgenic embryos; within this group, ets1-injected embryos have higher levels of EGFP expression globally and locally in the stripes. ets1 activates EGFP expression in the yolk syncytium when co-injected with wild-type reporter construct LG174, but only has minimal activity when co-injected with LG174e2-, suggesting that ets1 activates the promoter through the binding of EBS2 in vivo. (D) Morpholino knockdown of ets1 (ets1 splice-MO) does not affect transgene expression. These morphant embryos have a severe reduction in ets1 as measured by whole mount in situ hybridization, demonstrating morpholino efficacy.