title: [Impaired effect of BHC80 gene knock-down on the cardiac development in zebrafish].
abstract: The effect of BHC80 (a component of BRAF-HDAC complex) on development was not well studied, because BHC80 gene knock-out mice died in one day after birth. Interestingly, zebrafish embryos can live, even if their important organs like cardiac system has severe dysfunction, as 25%-40% O2 are supplied through their skin. Therefore, a model of BHC80 gene knock-down zebrafish embryos was established to explore the effect of BHC80 on the early embryonic development. BHC80-morpholino antisense oligonucleotides 2 (BHC80-MO2) was designed and injected into zebrafish embryos to interrupt the correct translation of BHC80 mRNA at one or two cells stage, which was proved by RT-PCR analysis. Two control groups, including non-injection group and control-MO (con-MO) injection group, and four different doses of BHC80-MO2 injection groups, including 4 ng, 6 ng, 8 ng and 10 ng per embryo were set up. The embryonic heart phenotype and cardiac function were monitored, analyzed and compared between con-MO and BHC80-MO2 groups by fluorescence microscope in vmhc:gfp transgenic zebrafish which express green fluorescent protein in ventricle. The results showed that BHC80-MO2 microinjection effectively knocked down the BHC80 gene expression, because the BHC80-MO2 group emerged a new 249 bp band which reduced 51 bp compared to 300 bp band of con-MO group in RT-PCR analysis, and the 51 bp was the extron 10. The abnormal embryo rate rose with the increase of BHC80-MO2 dosage. The proper BHC80-MO2 injection dosage was 8 ng per embryo, as minor embryos had abnormal phenotype in 4 ng and 6 ng per embryo groups and most embryos died in 10 ng per embryo group. BHC80-MO2 embryos exhibited abnormal cardiac phenotype, including imbalance of the proportion of heart ventricle to atrium, incomplete D-loop, even tubular heart, slow heart rates and cardiac dysfunction. The results from a model of BHC80 gene knock-down zebrafish embryos show that the abnormal cardiac phenotype and cardiac dysfunction of BHC80-MO2 embryos may be one of the probable reasons for the BHC80 gene knock-out mice death, which would provide a good research model to clarify the mechanism of cardiac development.
authors.affiliation: Zhongshan Hospital, Fudan University, Shanghai 200032, China; School of Life Sciences, Fudan University, Shanghai 200433, China; Key Laboratory of Molecular Medicine, Ministry of Education, Fudan University, Shanghai 200032, China. xiangdong.wang@clintransmed.org.
authors.forename: Jia-Yun
authors.initials: JY
authors.lastname: Hou
authors.forename: Dong-Li
authors.initials: DL
authors.lastname: Song
authors.forename: Da-Qing
authors.initials: DQ
authors.lastname: Jin
authors.forename: Jing-Ying
authors.initials: JY
authors.lastname: Hu
authors.forename: Xiang-Dong
authors.initials: XD
authors.lastname: Wang
mesh_terms: D000818:Animals; D047108:Embryonic Development / Q000235:genetics*; D018507:Gene Expression Regulation, Developmental; D055785:Gene Knockdown Techniques; D006321:Heart / Q000196:embryology*; D006655:Histone Deacetylases / Q000235:genetics*; D018345:Mice, Knockout; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D015027:Zebrafish / Q000196:embryology*; D029961:Zebrafish Proteins / Q000235:genetics
chemical_list: D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D029961:Zebrafish Proteins; D006655:Histone Deacetylases