Effects Of Atrnl1 Mutation On The Nervous System Development And Behavior Of Zebrafish

Background: With development of genetic and genomic detection techniques,deeper understanding has been gained about rare genetic etiology of neurodevelopmental disorders.Autism Spectrum Disorder(ASD)is a class of neurodevelopmental disorders characterized by extensive and persistent social disturbances and restrictive,repetitive interests or behaviors since early development.At present,it is not clear yet about the etiology and pathogenesis of ASD,but more and more studies have confirmed that genetic factors play a very important role in the pathogenesis.Though four patients with ATRNL1 gene mutation have similar clinical features of neurodevelopmental disorders,the functions of ATRNL1 gene and the pathogenesis of its mutation are not yet understood well,which need further investigations.CRISPR/Cas9 technology is a widely used gene editing method to construct gene mutant of cell lines or animal models.Zebrafish,as a genetic model with unique advantages,has a high similarity with humans in structure and functions of nervous system and behavior patterns,which is becoming increasingly popular in research of neurodevelopmental diseases.In zebrafish,ATRNL1 gene has two homologous genes of atrnl1 a and atrnl1 b with a high conservative property among species.This study chose zebrafish as the experimental animal model and used the CRISPR/Cas9 system to build up a zebrafish strain with single or double mutations of atrnl1 a and atrnl1 b genes.The early developmental morphology,behavioral performance and transcriptome of the mutants were detected to figure out the influence of atrnl1 gene mutations on zebrafish nervous development,behavior and m RNA expression,laying the foundation for further research on the functions of ATRNL1 gene and its role in the development of nervous system,as well as the possible pathogenic mechanism.Methods: Through in situ hybridization and RT-q PCR experiments,the expression patterns of atrnl1 a and atrnl1 b genes in the early developed zebrafish was constructed.The CRISPR/Cas9 system was designed to edit the atrnl1 a an atrnl1 b genes of zebrafish and strains of single or double genes mutants were set up.The early development morphology of mutants was observed,and the behavior experiments of light/dark stimulation,novel tank test,social test,mirror test and T-maze test were performed to detect abnormal phenotypes in mutant larvae and adult zebrafish.Finally,RNA of mutant and wild-type zebrafish was extracted by Trizol for transcriptome sequencing.Results: Via in situ hybridization and RT-q PCR experiment,atrnl1 a and atrnl1 b genes had highly specific expression in the zebrafish brain tissue,and atrnl1 b gene acted as a maternal effect gene with higher expression level than atrnl1 a.By using the CRISPR/Cas9 technology,zebrafish strains with atrnl1 gene mutations were successfully established and validated by Sanger sequencing and RT-q PCR,which proved a decrease on m RNA level after atrnl1 genes knockout.Measurement of body length and eye distance of larvae revealed a phenotype of microcephaly in mutants.Behavioral experiments confirmed reactive decline and impaired motor function in mutant larvae,and showed alleviative anxiety,impaired social interaction,less aggression,learning and memory disability in mutant adult zebrafish,suggesting that mutants probably had neurodevelopmental disorders.RNA-seq detection screened out 588 differentially expressed genes in mutants,including 349 up-regulated genes and 239 down-regulated genes.Among these,15 differentially expressed genes were found to be risk genes of ASD in the SFARI database.Functional enrichment analysis of these differentially expressed genes indicated that ATRNL1 was highly likely to be involved in the regulation of biological processes related to cell cycle.Conclusions: This study constructed a mutant zebrafish model with atrnl1 genes knockout and found a phenotype of microcephaly and a series of abnormal behaviors in mutants related to neurodevelopmental disorders.Analysis of the transcriptome sequencing results speculated that atrnl1 genes probably function in the regulation of cell cycle.Further researches on the functions of ATRNL1 gene and its role in the nervous system development are still worthy and necessary.