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. 2013 Sep 1;140(17):3691–3702. doi: 10.1242/dev.088328

Fig. 4.

Fig. 4.

The transcription factor cMyb is crucial for the enhancer activity of P7eNPBE. (A) Position size and sequence of the enhancer P7eNPBEΔ2 in comparison with P7Ur8 and P7eNPBE. (B-C′) Putative DNA-binding sites of four selected TFs shown in colored boxes (cMyb, light blue; Klf4 pink; ETS1, dark blue; Zeb1, red). Two examples of mutated areas are indicated, with yellow for early unaltered (B, arrowheads) and gray for abrogated (C,C′, arrows) reporter expression. (D-G′) These TFs were electroporated to assess effect on P7eNPBE enhancer activity. (D) Overexpression of Klf4 (red) led to normal P7eNPBE-EGFP expression in neural folds and neural plate border (arrowheads) and to exclusion from the NNE (arrows). (E-G′) cMyb (red) overexpression led to reduced P7eNPBE-EGFP signal in the neural folds (E-E′, arrow) with residual salt and pepper patterns, and expansion in the NNE (E,E′, open arrowheads). (F-G′) Sections of a similarly treated and staged embryo showing P7eNPBE expression in the NNE (open arrowheads, F,F′) and neural plate (arrowheads, G). P7eNPBE-EGFP is not ectopically activated in the mesoderm (double arrowheads G) and is much reduced in the neural folds, but displays a ‘salt and pepper’ pattern, with some cells lacking (arrows), and others presenting, expression (arrowheads) (compare with Fig. 2C,D). (H) EMSA performed using a 20 bp probe (bold sequence in A) containing the wild-type cMyb-binding site or the M2 mutated version. When cMyb is added, it binds to the wild-type probe, generating a clear shift (lane 2 ∼38% bound probe), which is severely reduced when cold unlabeled wild-type probe is added (lane 3, ∼2%); instead, adding the same excess amount of cold unlabeled mutated M2 probe still displays a robust shift (lane 4, ∼31%); however, only 8% of the labeled mutated-M2 probe is bound by cMyb (lane 6). (I) cMyb is expressed in the early ectoderm of chick embryos prior to Pax7. Whole mount in situ hybridization on HH3-5 chick embryos. Dotted lines at HH5 indicate sections shown on the left. m, mesoderm; np, neural plate; npb, neural plate border; nne, non-neural ectoderm; nf, neural folds.