[Impaired effect of BHC80 gene knock-down on the cardiac development in zebrafish]

The effect of BHC80 ( BRAF35-HDAC complex protein BHC80 ) on development was not well studied, because BHC80 gene knock-out mice died in one day after birth. Interestingly, zebrafish embryos could live, even if their important organs like cardiac system had severe dysfunction, as 25%-40% O2 were supplied through their skin. Therefore, we established a model of BHC80 gene knock-down zebrafish embryos, and explored the effect of BHC80 on the early embryonic development. We designed morpholino antisense oligonucleotides ( MO ) and injected them into zebrafish embryos to interrupt the correct translation of BHC80 mRNA at one or two cells stage which was proved by RT-PCR analysis. We set up 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. The embryonic heart phenotype and cardiac function were monitored by fluorescence microscope and were analyzed in vmhc:gfp transgenic zebrafish which express green fluorescent protein in ventricle and compared between standard control-MO and BHC80-MO2 groups．The results showed that BHC80-MO2 microinjection effectively knocked down the BHC80 gene expression，beacause 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 is 8 ng per embryo, as minority embryos had abnormal phenotype in 4 ng and 6 ng per embryo groups and most embryos died in10 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. Our results showed that the abnormal cardiac phenotype and cardiac dysfunction of BHC80-MO2 embryos indicated the probable reason for the BHC80 gene knock-out mice death, which would provide a good research model to clarify the mechanism of cardiac development．