Functional Analysis of Genes Strongly Associated with Autism Spectrum Disorders in a Zebrafish Model System

Background: Whole-exome sequencing has rapidly expanded the list of ASD-associated genes, while co-expression network analysis is beginning to reveal points of spatio-temporal convergence among these genes. Moreover, these new ASD risk genes are elucidating novel biological mechanisms, such as chromatin modification (CHD8) and ion channels (SCN2A), complementing earlier genetic findings, which implicated cell adhesion molecules and synaptic proteins (e.g. CNTNAP2) in the biology of ASD. We expect these biological mechanisms to disrupt common neural pathways leading to an ASD phenotype, yet delineating these neural pathways remains a challenge. Therefore, a critical next step in elucidating ASD neuropathology is the development of in vivo models that allow for the visualization and quantitative assessment of neural pathways in the developing CNS.

Objectives: To develop zebrafish as a model system for the functional analysis of ASD risk genes. These studies will capitalize on two key advantages of zebrafish: 1) transparent embryos that enable visualization of neural circuits in real time during brain development; and 2) large progenies, which allow for the conduct of quantitative profiling assays and small-molecule chemical screens.

Methods: We utilized zinc finger nuclease and transcription activator-like effector nuclease (ZFN, TALEN) technology to target the zebrafish orthologs of the ASD risk genes CHD8, SCN2A, and CNTNAP2. We analyzed the development of axon tracts, along with excitatory and inhibitory neurons during early embryonic stages to correlate these findings with the readout from behavioral assays. Moreover, we conducted large-scale quantitative behavioral profiling in zebrafish knockouts 4-7 days post fertilization (dpf) to characterize differences in the neural circuitry underlying rest-wake activity.

Results: Multiple lines of zebrafish knockouts were generated carrying deleterious germline mutations in CHD8, SCN2A, and CNTNAP2. CNTNAP2 knockouts show subtle abnormalities in axon organization at early developmental stages and disruption of forebrain commissure formation. Furthermore, these CNTNAP2 knockouts demonstrate increased susceptibility to chemically-induced seizures. SCN2A knockouts display motor abnormalities beginning at 4-5 dpf and do not survive past 11 dpf. Preliminary analysis of quantitative behavioral profiling of all knockouts reveals individual behavioral “fingerprints” indicative of significant differences in rest-wake architecture. The identification of such behavioral phenotypes offers a path forward in illuminating the specific neural circuitry that is disrupted in these knockouts.

Conclusions: These experiments provide evidence for the strength and feasibility of zebrafish as a tractable model system for the functional analysis of ASD risk genes and for testing specific hypotheses generated from genomic analyses in humans. While ongoing studies will assess excitatory-inhibitory neuron development in knockouts, preliminary evidence suggests these pathways may be disrupted given alterations in the behavioral profiles of knockouts, and in particular, the increased susceptibility of CNTNAP2 knockouts to drug-induced seizures. We anticipate that our approach of utilizing large-scale quantitative profiling of zebrafish knockouts of ASD genes will allow the testing and discovery of convergent biological mechanisms underlying the pathophysiology of ASD and aid in the identification of novel therapeutic targets.