Functional Analysis of SHANK3 In Zebrafish

Background: Mutations in SHANK3 can lead to autism spectrum disorders. Functionally analysis of SHANK3 has been restricted to cultured cells and to mouse models. We are developing in vivo systems for detailed, rapid functional analyses of autism gene, including SHANK3. Zebrafish, which represent a long-standing model for embryology and development, has attractive aspects as a model system for neurodevelopmental disorders. Its external, transparent embryonic development, high fecundity, and rapid life cycle make it organism particularly well-suited to the molecular genetic analysis of vertebrate neurodevelopment and facilitate studying gene function in vivo.

Objectives: To make use of the zebrafish as a model system to examine functional aspects of SHANK3 and to understand the pathological mechanisms underlying SHANK3 deficiency syndromes.

Methods: Gene knockdown was achieved by microinjection splice-blocking morpholinos into 1-2 cell stage zebrafish embryos. Early embryo development defects were scored and escape responses assessed. Changes in neurogenesis were examined using early neurogenesis markers including neurogenin1 transgenic fish (NGN1:EGFP) and anti-acetylated tubulin antibody.

Results: Double knockdown models of zebrafish Shank3 genes (3a and 3b) were generated. By using RT-PCR and sequencing, we validated the loss of native mRNA and the existence of aberrantly spliced mRNA products with premature stop site in the gene knockdown morphants. Morphological defects and delayed escape responses were observed in the Shank3 morphants. We are now introducing alternate human SHANK3 isoforms in rescue experiments as a means of understanding the function of SHANK3 isoforms.

Conclusions: SHANK3 plays important roles in early neurogenesis. Changes in neuronal function were observed in Shank3 morphants as assessed by escape responses. Zebrafish represent a useful model system to functionally assess autism genes.