Study On The Role Of BRD4 Gene In The Regulation Of Myeloid Hematopoiesis By Using Zebrafish Model

Objective:The temporal and spatial expression of brd4 gene in wild-type zebrafish was initially analyzed.The stable brd4 knock-out zebrafish mutant established by CRISPR/Cas9 gene editing technology was used to explore the role of brd4 gene in the zebrafish myeloid hematopoietic differentiation.At the same time,we used JQ1,the specific small molecule inhibitor of BRD4 protein,to treat wild-type zebrafish embryos in order to further verify the function of brd4 gene and deeply understand the theoretical basis of the regulation of the hematopoietic development.Method:（1）The antisense RNA probe was designed and constructed based on the zebrafish brd4 mRNA CDS coding region.The spatiotemporal expression of brd4in wild-type zebrafish was analyzed by whole embryo in situ hybridization（WISH）and RT-PCR.（2）Extraction of zebrafish genomic DNA and RNA,PCR amplification,gene sequencing and RT-PCR were carried out to identify zebrafish mutants.（3）The wild type zebrafish embryo was treated with the small molecule inhibitor JQ1 to further explore the function of brd4 in myeloid differentiation.（4）The number and distribution of mature neutrophils were detected by Sudan black B staining.（5）The number and distribution of macrophages were detected by neutral red in vivo staining experiments.（6）The temporal and spatial expression levels of myeloid hematopoietic specific marker genes mpx,lyz,l-plastin and mfap4 were detected by whole-mount in situ hybridization（WISH）assay.Result:（1）The brd4 mRNA molecular probe was successfully constructed.And brd4 gene was inherited maternally and expressed widely and non-specifically in early-staged WT zebrafish.（2）Compared with wild-type or heterozygous mutant zebrafish,the survival rate of brd4^-/-zebrafish decreased and usually died at around13dpf,which indicating that brd4 gene plays a pivotal role in the growth and development of zebrafish.（3）The antisense RNA probes of zebrafish neutrophil and mononuclear macrophage differentiation marker genes were successfully prepared by in vitro transcription of T7 transcriptase,and they had good specificity.（4）Sudan Black B staining（SBB）detected a significant reduction in the number of mature neutrophils in brd4 knockout homozygous mutants;while neutral red in vivo staining assay showed that the number of macrophages in brd4^-/-was not significantly different from that of wild-type zebrafish.（5）Whole-mount in situ hybridization（WISH）assay showed that the expression levels of granulocyte-associated genes mpx and lyz in brd4^-/-zebrafish were significantly lower than those in wild-type zebrafish,but there were no significant differences in the expression levels of mononuclear related genes l-plastin and mfap4.（6）The phenotypic analysis results of the WT zebrafish treated with JQ1 small molecule inhibitor were consistent with the above experimental results.Conclusion:Knocking out brd4 gene and continuous treatment of zebrafish with JQ1 drugs can lead to the neutrophil differentiation and development retardation and neutropenia without affecting the differentiation and development of mononuclear macrophages,suggesting that the brd4 gene is indispensable for normal granulopoiesis.And the specific molecular regulation mechanism and corresponding function need to be further studied.