High-throughput functional analysis of autism risk genes

这项研究的中心目标是阐明与癌症密切相关的基因的功能。 自闭症谱系障碍（ASD）在脊椎动物大脑发育的基本过程中。而 全外显子组测序导致越来越多的“高置信度”ASD（hcASD）风险基因， 我们对hcASD基因表达的机制的理解， 破坏改变了特定细胞类型和神经回路的发育，导致行为功能障碍， 仍然有限。这项研究的长期目标是阐明基本的生物学机制， ASD，这将为发现靶向药物治疗提供急需的途径。 在这里，我们将利用斑马鱼作为体内生物相关系统的独特优势， 多个hcASD基因的并行快速功能分析，包括：（i）直接可视化基础的能力， 通过透明胚胎在整个脊椎动物大脑中的神经发育过程和神经活动;（ii） 大后代，其对于进行高通量体内筛选以鉴定小分子是理想的 行为表型的抑制因子;（iii）易于遗传操纵，因此我们已经 在10个顶级hcASD基因中产生了斑马鱼突变体，我们将在本研究中进行分析。我们的中央 一种假设是，hcASD基因的斑马鱼突变体将表现出可量化的形态学、简单的行为学 和电路水平的表型，将汇聚在共同的途径，提供新的见解的作用， 这些基因在发育中的脊椎动物大脑中。这一假设是基于我们的研究证据， ASD风险基因CNTNAP 2的斑马鱼突变体，揭示了GABA能和 一种新的ASD基因相关突变体的药理学抑制剂 行为表型为了验证这一假设，我们将追求以下目标：1）识别可量化的大脑 使用光片成像和CNS的自动化深度表型分析对10种hcASD突变体进行表型分析。 特异性标记物; 2）使用新的， 自动视觉惊吓试验，并进行小分子筛选，以确定突变惊吓的抑制因子 表型;和3）表征神经回路缺陷的变化的感觉处理行为， hcASD突变体，并确定使用全脑的药理学抑制剂的回路水平机制， 活体双光子成像这种方法具有很强的创新性，是第一个分析多个 hcASD基因在结构、行为和电路水平上并行，使我们能够从风险中快速进步。 基因发现，以阐明与ASD相关的会聚途径。因此，我们预计， 这项研究将导致我们对ASD基础生物学的理解取得重大进展，并提供一条途径， 在发现基于机制的药物治疗方面取得了进展。

The central objective of this research is to elucidate the function of genes that are strongly associated with autism spectrum disorders (ASD) in fundamental processes of vertebrate brain development. While whole-exome sequencing has led to a growing list of “high confidence” ASD (hcASD) risk genes, which are beginning to converge on common pathways, our understanding of the mechanisms by which hcASD gene disruption alters the development of specific cell types and neural circuits, resulting in behavioral dysfunction, remains limited. The long-term goal of this research is to illuminate the basic biological mechanisms underlying ASD, which will provide a much-needed avenue for the discovery of targeted pharmacological treatments. Here, we will capitalize on the unique advantages of zebrafish as an in vivo, biologically relevant system for the rapid functional analysis of multiple hcASD genes in parallel, including: (i) the ability to directly visualize basic neurodevelopmental processes and neural activity in a whole vertebrate brain through transparent embryos; (ii) large progenies, which are ideal for conducting high-throughput in vivo screens to identify small molecule suppressors of behavioral phenotypes; and (iii) ease of genetic manipulation, such that we have already generated zebrafish mutants in 10 top hcASD genes, which we will analyze in the present study. Our central hypothesis is that zebrafish mutants of hcASD genes will display quantifiable morphological, simple behavioral, and circuit-level phenotypes that will converge on common pathways, providing new insights into the roles of these genes in the developing vertebrate brain. This hypothesis is based on evidence from our study of zebrafish mutants of the ASD risk gene, CNTNAP2, which revealed dysregulation of GABAergic and glutamatergic signaling and identified a novel pharmacological suppressor of an ASD gene-associated mutant behavioral phenotype. To test this hypothesis, we will pursue the following aims: 1) Identify quantifiable brain phenotypes across 10 hcASD mutants using light-sheet imaging and automated deep phenotyping of CNS- specific markers; 2) Conduct high-throughput pharmaco-behavioral profiling of hcASD mutants using a novel, automated visual-startle assay and perform small molecule screens to identify suppressors of mutant startle phenotypes; and 3) Characterize neural circuit deficits underlying altered sensory processing behaviors in hcASD mutants and identify the circuit-level mechanisms of pharmacological suppressors using whole-brain in vivo two-photon imaging. This approach is highly innovative and is the first to analyze the function of multiple hcASD genes in parallel at the structural, behavioral, and circuit levels, allowing us to progress rapidly from risk gene discovery to the elucidation of convergent pathways with relevance to ASD. Therefore, we expect that this research will lead to critical advances in our understanding of the basic biology of ASD and provide a path forward in the discovery of mechanism-based pharmacological treatments.