Identification of Biniou binding sites in the hand visceral enhancer. (A) Gel mobility shift assays with the HV-element as probe. Competition experiments using a 20- 200- and 2000-fold excess of unlabeled wildtype and a 2000-fold excess of mutated oligonucleotides, corresponding to the Biniou binding sites Bin1, Bin2, Bin3 and Bin4, were performed to reveal specificity. (B) Shown is the D. melanogaster visceral mesoderm enhancer region. Boxes label the four Biniou core binding site motifs, respectively. Arrows indicate the beginning and end of the deletion in the tested construct hand-3rd-intronΔ1074–1374::GFP (see result section). Same region was used for EMSA. (C) Sequence alignment of known Biniou binding sites, including the HFH-8 core consensus binding site. The colored nucleotides match the HFH-8 consensus sequence. β3Tub Biniou binding sites are from , Dpp Biniou binding sites are from . (vm) visceral mesoderm; (lg) lymph glands. (D) Transgenic embryos carrying the whole 3rd intron of the hand gene but with mutated Biniou binding sites as indicated (see methods section for details). Only the combination of mutated Bin2 and Bin4 sites lead to a total loss of reporter gene expression in the visceral mesoderm while expression in the heart is unaffected. (E) Model for the regulation of hand in visceral mesoderm. The activation of hand in the visceral mesoderm depends on Jeb/Alk signaling [10,11,29] and is likely mediated by the direct binding of Biniou to the identified visceral enhancer. Our results furthermore indicate that hand activity might be repressed in the visceral fusion compentent cells by the Gli-like transcription factor Lame duck (fc = founder cells, fcm = fusion competent myoblasts).