Relationship between the expression pattern of BVES and TOF. (A) The phenotype of RVOT stenosis in TOF diagnosed by echocardiography. RVOT, right ventricular outflow tract; AO, aorta; PA, pulmonary artery; RPA, right pulmonary artery; LPA, left pulmonary artery. CT, Control, the normal phenotype; TOF, tetralogy of fallot, the mal-phenotype of TOF patients. (B) qRT-PCR detected the expression of BVES. (C) Western blot detected the expression of BVES in downregulated samples. (D) Quantification of BVES by greyscale analysis. CT, control, RVOT tissue of normal controls; TOF, tetralogy of fallot, hypertrophic RVOT tissue of patients; n: number of samples. *p < 0.05; **p < 0.01; ***p < 0.001. The error bar shows the mean and SD.

Detection of SHF gene expression. CT, control, RVOT tissue of normal controls; TOF, tetralogy of fallot, hypertrophic RVOT tissue of patients; n, Number of samples. *p < 0.05; **p < 0.01. The error bar shows the mean and SD.

bves knockdown led to abnormal cardiac looping in zebrafish. (A) Survival ratio of wild type and morphants with 1.25, 2.0 and 5.0 ng of bves Mo at 72 hpf. WT, wild type; bves-1.25 ng, bves morphants with injection of 1.25 ng of bves Mo; bves-2.0 ng, bves morphants with injection of 2.0 ng of bves Mo; bves-5.0 ng, bves morphants with injection of 5.0 ng of bves Mo. (B) bves morphants showed cardiac defects at 48 hpf for the cmlc2:dsRed transgenic fish (a: atrium; v: ventricle), which were divided into four types based on the degree of cardiac dysplasia. H-0, the normal phenotypes; H-I, the moderate phenotypes; H-II, the strong phenotypes; H-III, the severe phenotypes. The position of the heart is marked by compass lines. ca, caudal; cr, cranial; do, dorsal; ve, ventral. Scale bar: 50 μm. (C) Data statistics of the different cardiac phenotypes in (B). (D) The morphants at 72 hpf with a left-looping heart in the ventral view. ca, caudal; cr, cranial; r, right; l, left. Scale bar: 50 μm. WT: wild type; bves Mo: bves morphants; bves Mo + bves mRNA, coinject bves morpholino and bves mRNA.

bves knockdown led to the phenotype of outflow tract stenosis in zebrafish with flia:eGFP. (A) qRT-PCR detected the expression of elnb in zebrafish at 72 hpf. WT, wild type; bves Mo, bves morphants. *p < 0.05. (B) qRT-PCR detected the expression of ELN in human tissue exapmles. CT, control, RVOT tissue of normal controls; TOF, tetralogy of fallot, hypertrophic RVOT tissue of patients. *p < 0.05. (C) bves morphants showed outflow tract defects at 72 hpf in Tg (flia:eGFP) zebrafish. The left column shows the outflow tract at the cardiac end-systolic stage, and the right column shows the end-diastolic stage. The words that marked by red color is the width of outflow tract. ca, caudal; cr, cranial; r, right; l, left. Scale bar = 15 μm. (D) Statistics about the width of outflow at the end-systolic stage in (C). Compared with the WT, the width of the outflow tract in bves knockdown mutants decreased by approximately 50% (WT, 9.5 μm; bves Mo, 5.0 μm, p < 0.01 compared with WT; bves Mo + bves mRNA, 7.5 μm, p < 0.01 compared with WT; p < 0.01 compared with bves Mo). OT, outflow tract; *Significance analysis with WT; ^#Significance analysis with bves Mo. **p < 0.01; ^##p < 0.01. n, number of samples. The error bar shows the mean and SD. bves Mo: bves morphants; WT: wild type; bves Mo + bves mRNA, coinject bves morpholino and bves mRNA.

bves regulates the development of the outflow tract via SHF. (A) SHF gene expression was detected in bves morphants by qRT-PCR at 48 hpf. (B) Overexpression of BVES led to upregulation of transcriptional activity at the promoters of GATA4, NKX2.5 and HAND2, downregulation of transcriptional activity at the promoters of TBX1, and no change in transcriptional activity at the promoters of TBX20 and MEF2C. pCMV-Vector, empty vector of pCMV-Myc; pCMV-BVES, the vector overexpressing BVES; pGL3-Vector, empty vector of pGL3-Bias; pGL3-Promotor, vector containing the promotor of gene. Red *: significance analysis with pGL3-Vector group; Black *: significance analysis with pGL3-Promotor + pCMV-Vector group. *p < 0.05; **p < 0.01; ***p < 0.001. (C) Outflow tract phenotype of the wild type, bves Mo injected, bves Mo and nkx2.5 mRNA coinjected in end-systolic at 72 hpf in Tg (flia:eGFP). The words that marked by red color is the width of outflow tract. ca, caudal; cr, cranial; r, right; l, left. Scale bar = 15 μm. (D) The width of the outflow tract at the end-systolic stage in (C). Compared with the WT and bves Mo, the width of the outflow tract was partially rescued by nkx2.5 mRNA (WT, 9.5 μm, bves Mo, 5.0 μm, p < 0.01 compared with WT; bves Mo + nkx2.5 mRNA, 6.3 μm, p < 0.01 compared with WT, p < 0.05 compared with bves Mo). n, number of samples. OT, outflow tract. *Significance analysis with WT; ^#Significance analysis with bves Mo. ^#p < 0.01; **p < 0.01. The error bar shows the mean and SD. WT: wild type; bves Mo: bves morphants; bves Mo + nkx2.5 mRNA, coinject bves morpholino and nkx2.5 mRNA.