Impaired effect of BHC80 gene knock-down on the cardiac development in zebrafish.
- Author:
Jia-Yun HOU
1
,
2
,
3
;
Dong-Li SONG
;
Da-Qing JIN
;
Jing-Ying HU
;
Xiang-Dong WANG
Author Information
1. Zhongshan Hospital, Fudan University, Shanghai 200032, China
2. School of Life Sciences, Fudan University, Shanghai 200433, China
3. Key Laboratory of Molecular Medicine, Ministry of Education, Fudan University, Shanghai 200032, China. xiangdong.wang@clintransmed.org.
- Publication Type:Journal Article
- MeSH:
Animals;
Embryonic Development;
genetics;
Gene Expression Regulation, Developmental;
Gene Knockdown Techniques;
Heart;
embryology;
Histone Deacetylases;
genetics;
Mice, Knockout;
Oligonucleotides, Antisense;
RNA, Messenger;
Zebrafish;
embryology;
Zebrafish Proteins;
genetics
- From:
Acta Physiologica Sinica
2013;65(5):547-552
- CountryChina
- Language:Chinese
-
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.
