Construction Of A Zebrafish Mutant Model Of Gigyf1 Gene And Its Effects And Functions On The Nervous System Development

BackgroundAutism spectrum disorder（ASD）is a collective term for a wide range of neurodevelopmental disorders.The main characteristics were different degrees of language development disorders,interpersonal communication dysfunction,repetitive stereotypes,and narrow interest areas that appear in infants and young children.Studies have shown that ASD often has abnormalities in the development of the nervous system and brain structures such as the corpus callosum and cortex.The etiology of ASD is complex and there are many interactions among heredity,neurodevelopment and environmental factors.Among them,genetic factors play a major role.GIGYF1 gene was found to be one of the high-risk pathogenic genes of ASD through the collection of a large number of clinical sample information in the early stage,but the specific molecular mechanism of GIGYF1 gene mutation induced to ASD is still unclear,and whether its deletion will cause abnormal development of the nervous system is also absent.Recently,based on efficient and convenient genetic manipulation technology and the strong behavioral phenotype,zebrafish has gradually become a more popular model animal in the field of ASD research.Zebrafish genome has undergone the entire duplication during the evolution process.The orthologous gene of the human GIGYF1gene has two homologous genes in zebrafish,namely gigyf1a and gigyf1b.Therefore,we used CRISPR/Cas9 gene editing technology to construct a single homozygous mutant model of gigyf1a and gigyf1b in zebrafish.In order to prevent the functional redundancy of gigyf1a or gigyf1b genes,we also constructed a double homozygous mutant of gigyf1ab model,and made preliminary morphological observation of the above mutant model.Through behavioral experiments on larvae and adults,it was further determined whether mutations in the gigyf1 gene would cause abnormal behaviors in zebrafish.At the same time,we carried out transcriptomics and proteomics tests on double mutant larvae in order to discover the molecular pathways that gigyf1gene may participate in early neurodevelopment,and provide directions for further research on specific molecular mechanisms.This study mainly includes the following three parts:Part I:Spatiotemporal expression profile of gigyf1 gene and construction of mutant modelObjective:To explore the spatiotemporal expression profiles of gigyf1a and gigyf1b,and the single and double mutant models of gigyf1a and gigyf1b genes in zebrafish were constructed,which lay a good foundation for the follow-up study of mechanism of neural development.Methods:Digoxin-labeled in situ hybridization and RT-qPCR experiments were used to detect the spatiotemporal expression of gigyf1a and gigyf1b genes.Then effective g RNA and Cas9 m RNA were designed for single-cell injection of zebrafish.After three generations of screening,we obtain the single mutant line of gigyf1a and gigyf1b,and above-mentioned single mutant line were hybridized with each other to obtain double mutant line.Results:Both gigyf1a and gigyf1b are maternally expressed genes with similar spatiotemporal expression profiles.We successfully obtained two single mutant lines of gigyf1a and gigyf1b genes,and a double mutant line of gigyf1ab by CRISPR/Cas9 gene editing technology.The single mutant lines of gigyf1a and gigyf1b showed no obvious abnormalities in morphology,while the double mutant line showed shortened body length and reduced interorbital distance.Conclusion:Both single and double mutant lines of gigyf1a and gigyf1b were successfully obtained,and the morphological development of the double mutant line was abnormal.PartⅡ:gigyf1 homozygous mutation causes zebrafish autism-like behaviorObjective:To determine whether the loss function of gigyf1 gene could cause behavioural abnormalities in zebrafish.Methods:The larvae and adult zebrafish lines(gigyf1a^Δ16/Δ16 to gigyf1a^+/+,gigyf1b^Δ4/Δ4 to gigyf1b^+/+,gigyf1b^Δ23/Δ23 to gigyf1b^+/+,gigyf1a^Δ16/Δ16;gigyf1b^Δ4/Δ4to gigyf1a^+/+;gigyf1b^+/+)in the same period were used for the behavioral tests,which including light and dark stimulation experiments,new tank experiments,shoaling experiments,three-cylinder social experiments and mirror experiments.Results:Double mutant larvae of gigyf1ab showed reduced motor function in light and dark stimulation experiments.In the new tank experiment,adult double mutant zebrafish always swim at the bottom of the tank all the time,showing significantly higher anxiety and fear behavior compared with wild type.Simultaneously,the double mutants had significant social dysfunction and decreased aggressive behavior in the shoaling experiment,the three-cylinder social experiment and the mirror experiment.But the single mutant line of gigyf1a and gigyf1b did not show significantly abnormal behavioral phenotype compared with wild type in the above-mentioned behavioral experiments.Conclusion:The double mutant line of gigyf1ab showed significantly behavioral abnormalities related to the autism phenotype compared with the wild type.Part III:Transcriptomics and proteomics analysisObjective:To investigate the changes of transcriptome and proteomics in zebrafish brain after the gigyf1 gene was knockout.Methods:Three tubes of 4.3 dpf gigyf1ab double mutant and wild type larvae head tissues were obtained respectively.Total RNA or total protein of each tube was extracted for RNA sequencing（RNA-seq）and proteomics（TMT）detection.Results:Transcriptome sequencing results showed that differentially expressed genes（DEGs）are enriched in various nervous systems and biological processes,the development of the eye and visual system,and there contained several ASD risk genes in this DEGs group.Proteomics sequencing revealed that differential expression proteins（DEPs）induced by gigyf1 mutations may be involved in a variety of biological function,such as the regulation of the immune system,the regulation of steroid hormone responses,and the transport of lipids.Conclusion:The gigyf1 gene may participate in a variety of biological functions and thus play an abnormal neurobiological role at the molecular level.This may provided experimental directions for the next study of the molecular function of gigyf1.