CRISPR/Cas9-induced shank3b mutant zebrafish display autism-like behaviors

Abstract

BackgroundHuman genetic and genomic studies have supported a strong causal role of SHANK3 deficiency in autism spectrum disorder (ASD). However, the molecular mechanism underlying SHANK3 deficiency resulting in ASD is not fully understood. Recently, the zebrafish has become an attractive organism to model ASD because of its high efficiency of genetic manipulation and robust behavioral phenotypes. The orthologous gene to human SHANK3 is duplicated in the zebrafish genome and has two homologs, shank3a and shank3b. Previous studies have reported shank3 morphants in zebrafish using the morpholino method. Here, we report the generation and characterization of shank3b mutant zebrafish in larval and adult stages using the CRISPR/Cas9 genome editing technique.MethodsCRISPR/Cas9 was applied to generate a shank3b loss-of-function mutation (shank3b−/−) in zebrafish. A series of morphological measurements, behavioral tests, and molecular analyses were performed to systematically characterize the behavioral and molecular changes in shank3b mutant zebrafish.Resultsshank3b−/− zebrafish exhibited abnormal morphology in early development. They showed reduced locomotor activity both as larvae and adults, reduced social interaction and time spent near conspecifics, and significant repetitive swimming behaviors. Additionally, the levels of both postsynaptic homer1 and presynaptic synaptophysin were significantly reduced in the adult brain of shank3b-deficient zebrafish.ConclusionsWe generated the first inheritable shank3b mutant zebrafish model using CRISPR/Cas9 gene editing approach. shank3b−/− zebrafish displayed robust autism-like behaviors and altered levels of the synaptic proteins homer1 and synaptophysin. The versatility of zebrafish as a model for studying neurodevelopment and conducting drug screening will likely have a significant contribution to future studies of human SHANK3 function and ASD.Electronic supplementary materialThe online version of this article (10.1186/s13229-018-0204-x) contains supplementary material, which is available to authorized users.

Predictive neural processing in adult zebrafish depends on shank3b

“…Shank3b mutants (shank3b -/-) showed no obvious developmental or behavioral deficits upon superficial inspection as adults, consistent with previous observations (Liu et al, 2018). To confirm that shank3b mutants nevertheless showed altered responses to sensory input we first analyzed the behavior of freely swimming adult zebrafish towards a movie of conspecifics ( Figure 5A).…”

“…These fish showed an enhanced attraction to movies of conspecifics that was phenocopied by the overexpression of the dominant-negative shank3b isoform shank3b ΔC , indicating that specific effects on behavior were caused by the disruption of shank3b function. Consistent with this conclusion, a previous study reported that a mutation in shank3b resulted in reduced social interactions and enhanced repetitive behaviors in adult zebrafish (Liu et al, 2018).…”

“…Here we focused on the adult zebrafish with a mutation in shank3b. A disruption of this gene has been reported to result in repetitive behavior and reduced social interactions in adult zebrafish (Liu et al, 2018), consistent with phenotypes of shank3 mutations in other species. All comparisons were made between homozygous mutant and wild-type (wt) siblings from the same crossings.…”

Abnormal repetitive behaviors in zebrafish and their relevance to human brain disorders

“…Like in rodents, disruption of some ASD-related genes provokes ARB-like phenotypes in zebrafish. For example, SHANK3 knockout zebrafish display aberrant circling, thigmotaxis, corner-tocorner swimming and 'looped' figure-8 swimming [105]. With high homology of SHANK3 between rodents and zebrafish (Table 2), such fish ARBs resemble stereotypies in mouse mutants of this gene [106][107][108].…”

“…Albeit potentially reflecting increased anxietylike behavior in some contexts (e.g., anxiogenic center avoidance), this response may also represent a pathological repetitive behavior (e.g., evoked by psychostimulants, such as nicotine) relevant to stereotypic peripheral hyperlocomotion, commonly seen in rodents (e.g., following psychostimulant drugs) Selected examples of genetic models of aberrant repetitive behaviors in zebrafish.SHANK3 (left panel)is an autism-related gene that encodes postsynaptic density protein (PSD, binding to glutamatergic NMDA receptors) whose ablation in mice impairs synaptic transmission. Knockdown of SHANK in zebrafish up-regulates NMDA receptor and evokes ARB-like repetitive circling, corner-to-corner and figure-8 swimming (top view), according to[105]. A synaptic ras GTPase-activating protein SYNGAP1 (right panel) is another key protein involved in synaptic transmission, whose hypofunction in mice induces precocious maturation of synapses and increases synaptic transmission.NMDA receptor interact with postsynaptic density-95 (PSD-95) protein, which binds to SYNGAP.…”