Fig. 2. Identification of the chick Cer left-side enhancer. (A) Deletion analysis of chick Cer (cCer) cis-regulatory sequences. The genomic organization of cCer is depicted at the top. cCer 5' sequences (black boxes) were fused to the reporter EGFP gene (green boxes) to determine the activity of each DNA fragment. The FoxH1 elements (red; F1 and F2) and the SMAD element (orange; S) are depicted in the reporter constructs. The presence (+) or absence (-) of EGFP expression in the anterior mesendoderm and its derivatives (AM) and in the left-side mesoderm (LSM) from electroporated chick embryos is listed on the right. Each result is representative of at least 12 embryos. LSM expression was disrupted in embryos electroporated with Cer0.34 or shorter constructs. Cer0.12-EGFP expression was very weak and ubiquitous (low). (B) Nucleotide sequence of the 5'-flanking region of cCer. Binding sites for the transcription factors FoxH1 (F1 and F2; orange), SMAD (S; yellow), GATA (green) and Nkx-2.5 (light blue), and a putative TATA box (purple), are outlined. Two transcription initiation sites were identified by RLM-RACE at positions -26 and -29 upstream of the ATG (arrowheads). Point mutations were introduced into the F1, S and F2 sites, as indicated. The morpholino antisense oligo sequence (MO) and its control oligo with five mismatches (CoMO) are outlined in pink. (C) Site-directed mutagenesis analysis of FoxH1- and SMAD-binding elements. LSM expression was specifically abolished in embryos transfected with constructs carrying deletions or mutations (^*) in the FoxH1 (F1del, F1mut, F2del and F2mut) or SMAD (Sdel and Smut) elements. (D) Enhancer analysis of potential regulatory sequences of cCer. Fragments of the cCer 5' region (PCR1-5) and sequences of the FoxH1 and SMAD elements (FSF, FF and FS) were subcloned into an enhancer-less vector carrying the human beta-globin minimal promoter (blue boxes) upstream of the EGFP coding sequence. LSM expression was detected in embryos electroporated with the PCR3, PCR5 and FSF constructs (which contained all of the F1, F2 and S elements), but not in those electroporated with the PCR1, PCR2, PCR4, FF and FS constructs (which lacked at least one of those sites). EGFP fluorescence was observed in the AM of embryos electroporated with each of the EGFP reporter constructs tested, with the exception of PCR4. FS-EGFP expression was not tested in the AM cells (nd). +/-, presence/absence of EGFP expression; AM, anterior mesendoderm and its derivatives; CoMo, control morpholino oligo sequence; EGFP, enhanced green fluorescence protein; F1/F2, FoxH1-binding sites; LSM, left-side mesoderm; MO, morpholino antisense oligo sequence; nd, not tested. S, SMAD-binding site.