Expression And Function Characters Of Zebrafish Brwd3 In Embryonic Development

In recent years,increasing evidence shows that BRWD3 mutation is associated with mental retardation X-linked 93(MRX93)and cancer,but its pathogenic and even normal molecular function remains unclear.We previously analyzed its interacting proteins and downstream genes to understand its molecular function.However,the results in vivo are lacking.This study investigated the characters of expression and function of zebrafish brwd3 in embryonic development to lay the foundation for understanding brwd3 roles in zebrafish embryonic development.In this study,we cloned the zebrafish brwd3 gene,based upon transcriptome sequencing and genome information using polymerase chain reaction combined with sequencing.We explored its conservative and evolutionary characteristics among different species and analyzed the physical and chemical properties of BRWD3 protein.According to the obtained sequence information,we designed primers and used real-time quantitative PCR to study the transcriptional expression level,and used the whole embryo in situ hybridization to observe the distribution of brwd3 in different embryonic developmental stages of zebrafish.To understand its function,we used morpholino to construct brwd3 mutant in zebrafish,simulating the characteristics of BRWD3 truncated mutations in human beings.We then screened the related genes affected by brwd3 truncating mutations,using transcriptome sequencing.We functionally annotated and clustered the downstream candidate genes to construct the brwd3 molecular interaction networks related to brain development.The results showed:(1)We obtained brwd3 containing the full-length coding sequence,which was 6894 bp in size,and encoded 1742 amino acids.It was highly conservative in humans and many typical models such as the mouse.The amino acid similarity between the zebrafish brwd3 and human BRWD3 is 59.98%.The zebrafish brwd3 was predicated as 195.505 k Da of molecular weight,a hydrophilic and not a secreted protein.(2)The expression pattern was revealed that brwd3 presented as a maternal expression pattern and widely distributed.When it developed in the gastrula period,the expression of brwd3 gradually decreased.When it developed in the segmentation period,the expression of brwd3 gradually rised.It reached a peak at 24 hours post fertilization,and then the expression decreased.When it developed in the pharyngula period,it was found to be specifically expressed in the brain.(3)Among the downstream genes with transcriptome sequencing,we selected 49 differentially expressed genes related to brain development.These genes affected by brwd3 mutation were enriched in Wnt signaling pathway,cell social connection and MAPK signaling pathway.The transcriptional level of zebrafish mef2 ca,the homologous gene of human MEF2 C previously identified with Ch IP-Sequence,was decreased in the brwd3 mutant zebrafish.We thus detected the temporal and spatial expression pattern of mef2 ca in wild-type zebrafish and confirmed that the co-expression of brwd3 and mef2 ca,which were involved in zebrafish embryonic brain development.Summarily,we investigated the expression and function characters of zebrafish brwd3 in embryonic development.The results support that brwd3 regulates zebrafish brain development interacting with mef2 ca,which provides a clue for the understanding of the pathogenesis of BRWD3 mutation leading to MRX93.