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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.
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