Fig. 2. SMAD consensus sites bind MH1 domain of SMAD4 and are required for BMP response of CAR3. (A) Oligonucleotides used for SMAD protein gel shifts are shown as colored horizontal lines over a sequence of 200 bp BMPRE, representing chick Nkx2.5 genomic sequences from +2150 to +2350. Nucleotide positions in BMPRE are renumbered from 1-200 for convenience. Consensus SMAD-binding sites or SBEs are shown boxed in green; numerical designations are shown in green circles. Nucleotide substitutions made for mutant oligos and reporters are displayed in red under their cognate sites, as are correspondingly altered sequences within the BMPRE (SBE1, nucleotides 35-65; SBE2, nucleotides 80-120; SBE3, nucleotides 120-160). (B) Gel shifts performed with purified GST, GST-SMAD1 MH1 or GST-SMAD4 MH1 domain proteins on labeled wild-type (lanes 1-3, 7-9 and 13-15, respectively) or mutant (mut, lanes 4-6, 10-12 and 16-18, respectively) SBE oligomers. (C) P19 cell BMP response assays for wild-type Nkx2.5-lux-CAR3 (left) or Nkx2.5-lux-BMPRE (right) reporters, or cognate reporters bearing specific SBE mutations shown in A. Nkx2.5-lux constructs are shown schematically over bar graph. (D-O) Representative X-gal stained F0 transient transgenic mice embryos injected with wild-type or SBE mutant (mut) Nkx2.5-lacZ-CAR3 reporters. Embryonic stages are in the bottom left-hand corners. Whole-mount embryo at stages E7.5 (D,G,J,M) and E10.5 (E,H,K,N) and E10.5 hearts (F,I,L,O) are shown as designated for Nkx2.5-lacZ-CAR3 WT (D-F), mut SBE1 (G-I), mut SBE2 (J-L) and mut SBE3 (M-O) as shown in A. Cardiac staining shown was representative of the following numbers of transient transgenic embryos bearing Nkx2.5-lacZ constructs driven by CAR3 reporters: wild type, 3/5 E7.5 and 4/4 E10.5 embryos displayed expression in the cardiac crescent and forming heart; CAR3mut SBE1, 2/4 E7.5 embryos displayed robust transgene expression in the cardiac crescent and 9/9 E10.5 embryos either failed to express the transgene or displayed very weak transgene expression in the outflow tract; CAR3mut SBE2, 7/7 E7.5 embryos displayed robust transgene expression in the cardiac crescent and 2/2 E10.5 embryos failed to express the transgene; CAR3mut SBE3, 7/7 E7.5 embryos and 3/3 E10.5 embryos either failed to express the transgene or displayed residual transgene expression in the outflow tract. Abbreviations are as in previous figures. RA, right atrium; LA, left atrium; LV, left ventricle.