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

项目摘要 CHARGE综合征是一种罕见的多系统疾病，影响约1/10，000的活产婴儿。最 常见症状包括眼缺损、后鼻孔闭锁、心脏缺陷、生殖器异常、耳畸形、 畸形和一系列神经行为障碍感觉负荷不足或过重，运动障碍， 增强疼痛、睡眠障碍、注意缺陷多动障碍（ADHD）、强迫症 (OCD)智力障碍、焦虑和自闭症都是CHARGE中常见的。治疗这些 行为症状是有限的，突出了一个领域的关键需要了解的机制 这些缺陷的基础，以开发新的治疗方法。三分之二的案件是 由chd 7中的功能缺失突变引起，chd 7编码DNA结合、ATP依赖性染色质 重塑蛋白chd 7在发育中的脊椎动物脑中高度表达， 几个关键的神经发育基因，但直接的细胞和分子之间的联系chd 7的功能， 行为规范尚未建立。该项目利用了斑马鱼幼虫模型， 作为一个强大的系统，它迅速出现，用于研究行为回路的发展和功能， 以及人类遗传疾病。使用CRISPR/Cas9，我们已经建立了一个chd 7-空系，并具有 表征了反映CHARGE的几种形态学和行为表型。为了响应声学 chd 7突变体表现出正常的短潜伏期惊吓反应（SLC），但长潜伏期惊吓反应受损。 逃避反应（LLC）。类似地，chd 7突变体对光照的增加反应正常，但缺乏 对光照减少的反应。这些缺陷是独立的形态缺陷的眼睛 这表明chd 7可能调节大脑中特定的行为回路。在目标1中， 使用体内钙成像系统地询问驱动这些行为的已知电路元件， 细胞特异性拯救以定位CHD 7作用的位点。然后，我们将全面定义大脑区域， 依赖于使用全脑形态测量和活性分析的CHD 7。在目标2中，我们将应用状态- 现有技术的蛋白质组学和转录组学方法来鉴定将CHD 7与这些 大脑结构和功能的变化。通过分析三个发育时间点的样本，我们还将 定义这些变化的时间动态。最后，我们将使用系统的CRISPR/Cas9方法， 通过测量大脑发育，验证基于蛋白质组学和转录组学的体内chd 7靶点 和行为之间的关系总的来说，这项工作的结果将在chd 7、其 分子靶点和行为电路此外，这些目标将产生一套强大的、广泛有用的 询问chd 7依赖性神经发育的细胞和分子基础的资源。

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.