Transcriptomic Analysis Of The Effects Of Brwd3 On The Development Of Nervous System In Zebrafish

Nervous system plays a very important role in involved in the regulation of animal body consciousness and psychological action to the behavior of the individual ability.Given abnormal development of the nervous system,patients will present a variety of neurological problems.Consequently,investigation of the causes of these diseases,especially their genetic variations,in partly,can provide clues of the development of the nervous system.Previous studies found that BRWD3(bromodomain and WD repeat domain containing 3)was significantly correlated with the occurrence of neuropsychiatric diseases,such as intellectual developmental disorder(IDD)and epilepsy.However,the influence and mechanism of BRWD3 genetic variants on occurrence of nervous system development was still not clear.As chromatin-modifying and transcriptional regulatory factor,BRWD3 is composed of several WD40 repeats(WDR)and a bromodomain(Brd)domains,which are widely distributed in various organs of the human body,especially in the brain.BRWD3 mainly mediates the interaction within proteins and regulates the transcription of multiple genes or multiple loci.Through previous studies and literature tracking,combined with the pathogenic characteristics of the disease,we speculate that the BRWD3 gene may affect the development of the nervous system of the body through transcriptional regulation of downstream neural development-related genes.The aim of this study was to investigate whether BRWD3 is involved in the regulation of the expression of genes involved in the development of the nervous system and provides clues to the mechanisms of action between genes and diseases.In this study,the brwd3 gene of zebrafish was mutagenized by Morphholino method,and then the 10 th stage of zebrafish embryo development(segmentation 10 somites,10s)was used as the research material of nervous system development,which was tested by transcriptome sequencing method.In the experimental group and the control group,the differentially expressed genes were affected by brwd3,and on this basis,the regulatory network of brwd3 on the development of the zebrafish nervous system was constructed.Based on the traditional transcriptome analysis,this study carried out a series of optimizations on genome selection,differential expression threshold adjustment and differential gene screening were adjusted.Combined with existing databases and data mining methods,it was speculated that brwd3 transcriptionally regulated target genes,and the regulation of brwd3 in the development of zebrafish nervous system was studied through network construction and regulate the network.Result: 1)in this study,16879 of total genome gene(25118)of zebrafish,were mapped and identified successful.Within these gene,there are 7754 genes were expressed in the brain of zebrafish embryos.compering with wild-type embryos,474 genes showed a differentially expression in brwd3-knock-down zebrafish embryo.Among these DEGs,182 genes were up-regulated,and 292 down-regulated,defined with the criterion |log2FC| ≥ 1 and FDR ≤ 0.05.2)within DEGs genes,a GO functional analysis indicated that,65 DEGs genes were enriched in 3 biological functional items which have rather close relation with to nervous system development,they are "sensory organ development(P = 3.27E-03)","nervous system development(P = 2.65E-06)","neuron differentiation(P = 2.16E-03)".And then,short time-series expression miner(STEM)analysis and KEGG pathway enrichment analysis were used to filter these DEGs gene,further.Finally,23 genes whose expression may be affected by brwd3 were identified.In addition,these genes enriched in Wnt signaling pathway-one of important pathway in neuro-system development.3)Combined with String and other database information,a regulatory network of brwd3 gene was conducted by which brwd3 may be impacted the nervous system development of zebrafish.In this regulatory network,brwd3 mediates the increased expression of fzd8 a to activate the Wnt signaling pathway and regulate the downstream target genes,thereby affecting the regulatory mechanism of nervous system development.Moreover,the BRWD3 action network constructed by us has also been verified in the random detection of q PCR and the detection of genes in the key nodes of the network(R = 0.89,P = 3e-06).Subsequently,RNAi experiments of BRWD3 in two cell lines also shown a consistency with our constructed brwd3 regulatory network.The expression of genes in this network will be changed and consistent with our supposed trend,when the expression of BRWD3 was knock-down by RNAi interference technology in SH-SY5 Y and MCF-7 cell lines.Moreover,the changes in the expression levels of these genes are consistent with the brwd3 regulatory network constructed in this study.This study used the zebrafish model to explore the mechanism by which the brwd3 gene affects the development of the nervous system through expression and regulation,and at the same time constructed a network of roles for the development of the nervous system.However,this brwd3 regulatory network in zebrafish neuro-system development,and the interaction mechanism among genes within this network needs further research and excavation.