Cellular and Molecular Mechanisms of Behavioral Dysfunction in a Zebrafish Model of CHARGE Syndrome

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Project Summary CHARGE syndrome is a rare, multi-system disorder that affects approximately 1 in 10,000 live births. The most common symptoms include ocular coloboma, choanal atresia, heart defects, genital abnormalities, ear malformations, and an array of neuro-behavioral difficulties. Sensory under- or over-load, motor impairments, enhanced pain, sleep disorders, attention deficit hyperactivity disorder (ADHD), obsessive compulsive disorder (OCD), intellectual disability, anxiety, and autism are all frequently observed in CHARGE. Treatments for these behavioral symptoms are limited, highlighting an area of critical need in understanding the mechanisms underlying these defects in order to develop new therapeutic approaches. Two-thirds of CHARGE cases are caused by loss-of-function mutations in chd7, which encodes a DNA-binding, ATP-dependent chromatin remodeling protein. chd7 is highly expressed in the developing vertebrate brain and regulates transcription of several key neurodevelopmental genes, but direct cellular and molecular links between chd7 function and behavioral regulation have not been established. This project leverages the larval zebrafish model, which has rapidly emerged as a powerful system for investigating the development and function of behavioral circuits as well as human genetic disease. Using CRISPR/Cas9 we have established a chd7-null line and have characterized several morphological and behavioral phenotypes reflective of CHARGE. In response to acoustic stimuli, chd7 mutants perform normal short-latency startle responses (SLCs) but have impaired long-latency escape responses (LLCs). Similarly, chd7 mutants respond normally to increases in illumination but are deficient in responding to decreases in illumination. These deficits are independent of morphological defects in the eyes and ears, indicating that chd7 likely regulates specific behavioral circuits in the brain. In Aim 1 we will systematically interrogate the known circuit elements driving these behaviors using in vivo calcium imaging and cell-specific rescue to locate the sites of chd7 action. We will then comprehensively define the brain regions that are dependent on chd7 using whole-brain morphometry and activity analyses. In Aim 2 we will apply state-of- the-art proteomic and transcriptomic approaches to identify molecular pathways that link chd7 with these changes in brain structure and function. By analyzing samples from three developmental timepoints, we will also define the temporal dynamics of these changes. Finally, we will use a systematic CRISPR/Cas9 approach to validate the top proteomics- and transcriptomics-based chd7 targets in vivo by measuring brain development and behavior in mutant larvae. Overall, the results of this work will establish direct links between chd7, its molecular targets, and behavioral circuits. Furthermore, these aims will generate a powerful set of broadly useful resources for interrogating the cellular and molecular bases of chd7-dependent neural development.