Role Of Autism Risk Gene CHD8 In Neurodevelopment And Behavior Of Zebrafish

Aims:Autism Spectrum Disorders（ASD）are complex,lifelong and early-onset neurodevelopmental disorders with complex etiology and clinical symptoms,often associated with various comorbidities,for which no significant and effective treatments have been identified.Therefore,research on the pathogenesis and treatment of ASD is still a challenge.Genetic factors are dominant in the etiology of ASD,and CHD8 is one of the most frequently mutated genes with the highest penetrance among ASD risk genes.Studies on the association between CHD8 mutations and ASD have been reported,but the biological mechanism of CHD8 mutations causing ASD is still controversial and without clear direction.Further study on the molecular mechanism of CHD8 affecting neurological development and CHD8 mutation causing autism is essential for future precision medicine,therefore,this study will use zebrafish as a model organism to investigate the effect of chd8 on neurological development and its molecular mechanism.Methods:In this study,we used whole embryo in situ hybridization and real-time quantitative fluorescence PCR to study the expression of chd8,and CRISPR/Cas9 to construct a mutation model for preliminary observation and analysis of mutant developmental morphology;after obtaining stable and heritable mutants,we then analyzed the effect of chd8 knockout on zebrafish development through open field experiment,novel tank experiment,bright and dark box experiment,three tank experiment,mirror experiment and cluster experiment.The effect of chd8 knockdown on zebrafish behavior was investigated by in situ hybridization,real-time fluorescence quantitative PCR,and immunofluorescence assays to study the effect of chd8 knockdown on neurological development,and transcriptome sequencing was performed on juvenile and adult brain tissue.Finally,single-cell transcriptome sequencing was performed using juvenile brain tissues to investigate the effects of chd8 gene on different cells of the nervous system and to further explore the potential mechanisms of its effects on the development of the nervous system.Results:chd8 was widely expressed in early embryonic development and concentrated in the head after 24 hpf.The chd8 knockout homozygotic mutants were successfully constructed by CRISPR/Cas9 technology.chd8^del25/del25 larvae showed impaired locomotion activity,increased anxiety and increased sleep time.chd8^del25/del25adult zebrafish displayed impaired locomotion activity,increased anxiety,decreased social ability and repetitive stereotypic behaviors.chd8^del25/del25 larvae showed increased proliferative cells in the nervous system,increased expression of neural progenitor marker genes and decreased expression of glutamatergic neuronal marker genes.Transcriptome analysis revealed that chd8 may regulate the expression of genes related to cell cycle,mitosis,ion transport,synaptic signaling,visual system and other biological pathways.A total of 17 clusters of cell types were analyzed,in which the chd8^del25/del25 group had an increased proportion of neural progenitor cells and a decreased proportion of glutamatergic neurons.The expression of transcription factors egr1,jun and tefa was reduced in the chd8^del25/del25 group,while egr1 was mainly expressed in glutamatergic neurons and its expression was highly correlated with chd8.Conclusions:A stable and heritable mutational model of chd8 knockout in zebrafish was successfully constructed.chd8^del25/del25 zebrafish exhibit a behavioral phenotype similar to the symptoms of ASD patients.chd8 knockout caused macrocephaly,abnormal neuron differentiation,and increased cell proliferation in the nervous system in larvae.chd8 may affect the neurological expression of zebrafish by regulating the expression of genes related to several key biological pathways.Knockout of chd8 leads to reduced or abnormal differentiation of glutamatergic neurons,and this abnormality may be associated with altered expression of transcription factors such as egr1.