Human 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. CRISPR/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. shank3b^−/− 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. We 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.

Background: Behavioral changes in animals reflect functional changes in their central nervous system. Neuroactive drugs that act on different neural pathways can induce specific behavioral responses; therefore, it is possible to infer the activities of neuroactive drugs by studying the behavioral changes induced by drugs of interest in animals. Methods: In this study, AB strain zebrafish larvae at 7 days post fertilization (dpf) were treated with different concentrations of drugs that act on different neural pathways. Changes in the swimming distances of zebrafish larvae under different illumination conditions and the differences in locomotor activities between light and dark conditions (lighting motor index) were analyzed. Results: Among the drugs studied, different concentrations of sulpiride had no effect on larval locomotor activity either under light or dark conditions. Progressively decreased spontaneous movements were observed in zebrafish larvae treated with increasing doses of MK-801 and valproic acid. With increasing concentrations of pentylenetetrazole and yohimbine, the spontaneous movement of larval zebrafish presented a bell-shaped response. When the illumination changed from light to dark, zebrafish larvae not treated with drugs demonstrated increased locomotor activities. However, high levels of yohimbine, pentylenetetrazole decreased the degree of change in the lighting motor index. Conclusions: In conclusion, drugs that affect different neural pathways exert different influences on the locomotor activities of zebrafish larvae. This study presents an initial effort to establish a framework that correlates the drug activities and the behavioral responses of zebrafish larvae under drug treatments, which may provide a potential identification of the pathways of novel drugs with neurological activities through their behavioral influences.

Objectives: microRNA‐29 (miR‐29) family have shown different expression patterns in cardiovascular diseases. Our study aims to explore the effect and mechanism of miR‐29 family on cardiac development.Materials and methods: A total of 13 patients with congenital heart disease (CHD) and 7 controls were included in our study. Tissues were obtained from the right ventricular outflow tract (RVOT) after surgical resection or autopsy. The next‐generation sequencing was applied to screen the microRNA expression profiles of CHD. Quantitative RT‐PCR and Western blot were employed to measure genes expression. Tg Cmlc2: GFP reporter zebrafish embryos were injected with microRNA (miRNA) to explore its role in cardiac development in vivo. Dual‐luciferase reporter assay was designed to validate the target gene of miRNAs. CCK‐8 and EdU incorporation assays were performed to evaluate cardiomyocyte proliferation.Results: Our study showed miR‐29b‐3p expression was significantly increased in the RVOT of the CHD patients. Injection of miR‐29b‐3p into zebrafish embryos induced higher mortality and malformation rates, developmental delay, cardiac malformation and dysfunction. miR‐29b‐3p inhibited cardiomyocyte proliferation, and its inhibitor promoted cardiomyocyte proliferation in vitro and in vivo. Furthermore, we identified that miR‐29b‐3p influenced cardiomyocyte proliferation by targeting NOTCH2, which was down‐regulated in the RVOT of the CHD patients.Conclusion: This study reveals that miR‐29b‐3p functions as a novel regulator of cardiac development and inhibits cardiomyocyte proliferation via NOTCH2, which provides novel insights into the aetiology and potential treatment of CHD.