High-throughput functional analysis of autism risk genes

这项研究的中心目标是阐明密切相关的基因的功能。 自闭症谱系障碍(ASD)是脊椎动物大脑发育的基本过程。而当 全外显子组测序导致了越来越多的高置信度自闭症(HcASD)风险基因，这些基因是 开始在共同的途径上收敛，我们对hcASD基因机制的理解 干扰会改变特定细胞类型和神经回路的发育，导致行为障碍， 仍然是有限的。这项研究的长期目标是阐明潜在的基本生物学机制 ASD，这将为发现靶向药物治疗提供一个急需的途径。 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基因并行的快速功能分析，包括：(I)直接可视化基本的能力 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) 大的子代，这是进行高通量体内筛选以识别小分子的理想选择 行为表型的抑制者；以及(Iii)基因操作的简便性，以至于我们已经 在10个顶级hcASD基因中产生了斑马鱼突变体，我们将在本研究中对其进行分析。我们的中央 假设hcasd基因的斑马鱼突变体将表现出可量化的形态，简单的行为， 和电路级别的表型，这些表型将汇聚在共同的路径上，为了解 这些基因存在于发育中的脊椎动物大脑中。这一假设是基于我们对 斑马鱼ASD风险基因CNTNAP2的突变，揭示了GABA能和 谷氨酸能信号转导并鉴定了ASD基因相关突变体的一种新的药理抑制因子 行为表型。为了验证这一假设，我们将追求以下目标：1)确定可量化的大脑 10个hcASD突变体的表型，使用光片成像和自动化的CNS- 特定标记；2)使用一种新型的， 自动视觉惊吓检测和进行小分子筛选以识别突变惊吓的抑制子 表型；以及3)表征导致感觉处理行为改变的神经回路缺陷 HcASD突变体，并利用全脑功能鉴定药物抑制因子的电路水平机制 活体双光子成像。这种方法具有很高的创新性，是第一次分析多个 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 基因发现有助于阐明与ASD相关的汇聚途径。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，并为我们提供一条途径 在发现基于机制的药理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.