Effects Of TANC2 On Early Neurodevelopment And Behavior In Zebrafish

BackgroundNeurodevelopmental disorders are a class of disorders that start during childhood,widely affecting brain development and function.Both genetic and environmental factors are associated with neurodevelopmental disorders,among which de novo mutation is proposed to be one of the important pathogenic mechanisms.Functional genomic studies have shown that synaptic dysfunction,chromatin conformational changes and gene expression are associated with neurodevelopmental disorders.Whole exon sequencing and gene chip analysis demonstrate that TANC2 is a potential risk gene for neurodevelopmental disorders.Of the 28 previously reported cases of neurodevelopmental disorders with TANC2mutations,22 were de novo mutations.The clinical manifestations of these patients include autistic symptoms,intellectual disorders,language disorders and motor disorders,as well as other neurological and behavioral symptoms.TANC2 is defined as a postsynaptic scaffolding protein and co-locates with PSD95 in the postsynaptic membrane,regulating the formation of dendritic spines.Loss of TANC2 alleles causes embryonic lethality in mice and the heterozygous mice show impaired learning and memory.Although TANC2 has been identified as a neurodevelopmental disorder risk gene,its impacts on early development of nervous system and behaviors remain unclear.MethodBefore constructing the tanc2 knockout model,the alignments of amino acid sequence and exon sequence between human TANC2 and zebrafish tanc2 were performed respectively to determine the genetic conservation.Meanwhile,in situ hybridization and RT-q PCR of tanc2 in 3 to 72 hpf wild type zebrafish embryos were performed to determine its spatio-temporal expression profile.Finally,tanc2 mutant lines of zebrafish were established by CRISPR-Cas9 technique.After obtaining tanc2 mutant zebrafish,larval morphological observations were firstly performed by measuring body length and head area of 3 dpf zebrafish larvae.To observe alterations in neurotransmitter systems,in situ hybridization and RT-q PCR of neurotransmitter-related marker genes were performed in 48 hpf zebrafish embryos.To observe changes in brain architecture,dissected brains of 48 to120 hpf embryos/larvae were stained with acetylated Tubulin and SV2 antibody.Finally,48 hpf embryos were stained for p H3 and 28 hpf embryos were stained for TUNEL.Finally,behavioral analysis was performed to determine the behavioral alterations.For zebrafish larvae,acousto-optic stimulation experiments were performed firstly in 5 dpf zebrafish,followed by sleep rhythm-activity analysis of 4 to 6 dpf zebrafish larvae.For adult zebrafish,the anxiety-like behavior,social behavior,aggressive behaviors a well as learning and memory ability were detected in 6-months-old male zebrafish.ResultsWe found that there were two copies of tanc2 gene in zebrafish and both the amino acid sequence and exons of the main structural domain between human and zebrafish were very conserved.The maternal-effect gene tanc2 was found to be mainly expressed in the nervous system during development,and its expression was accompanied by the development of the nervous system.Finally,two tanc2 knockout zebrafish lines,tanc2a^Δ5b^Δ31 and tanc2a^Δ14b^Δ29,were successfully constructed using CRISPR-Cas9 gene editing.We found that the body length and the lateral brain area of mutant was increased compared with wild-type zebrafish,demonstrating that the growth and development of mutant was accelerated.At the molecular level,the mutant zebrafish larvae showed increased cell proliferation and decreased cell apoptosis,but the overall brain structure of mutant zebrafish had no significant changes.At the neurotransmitter level,the expression levels of neurotransmitter marker genes in mutant zebrafish were transiently altered during development.We found that mutant zebrafish larvae had no significant changes in reaction to acousto-optic stimuli but showed sleep disturbances and altered rhythms.In addition,adult mutant zebrafish showed reduced anxiety,mild impairment of social preference,reduced aggressive behavior,but no significant changes in learning and memory ability.Conclusiontanc2 affects the early development of nervous system in zebrafish.tanc2 knockout promotes cell proliferation,inhibits apoptosis and therefore accelerates growth and development.Loss of tanc2 affects sleep and social interaction in zebrafish.These results suggest that TANC2 is a pathogenic gene for neurodevelopmental disorders.