Table_1_Assessment of Autism Zebrafish Mutant Models Using a High-Throughput Larval Phenotyping Platform.XLSX

In recent years, zebrafish have become commonly used as a model for studying human traits and disorders. Their small size, high fecundity, and rapid development allow for more high-throughput experiments compared to other vertebrate models. Given that zebrafish share >70% gene homologs with humans and their genomes can be readily edited using highly efficient CRISPR methods, we are now able to rapidly generate mutations impacting practically any gene of interest. Unfortunately, our ability to phenotype mutant larvae has not kept pace. To address this challenge, we have developed a protocol that obtains multiple phenotypic measurements from individual zebrafish larvae in an automated and parallel fashion, including morphological features (i.e., body length, eye area, and head size) and movement/behavior. By assaying wild-type zebrafish in a variety of conditions, we determined optimal parameters that avoid significant developmental defects or physical damage; these include morphological imaging of larvae at two time points [3 days post fertilization (dpf) and 5 dpf] coupled with motion tracking of behavior at 5 dpf. As a proof-of-principle, we tested our approach on two novel CRISPR-generated mutant zebrafish lines carrying predicted null-alleles of syngap1b and slc7a5, orthologs to two human genes implicated in autism-spectrum disorder, intellectual disability, and epilepsy. Using our optimized high-throughput phenotyping protocol, we recapitulated previously published results from mouse and zebrafish models of these candidate genes. In summary, we describe a rapid parallel pipeline to characterize morphological and behavioral features of individual larvae in a robust and consistent fashion, thereby improving our ability to better identify genes important in human traits and disorders.

近年来，斑马鱼已成为研究人类性状与疾病的常用模式生物。其体型小巧、繁殖力强且发育迅速，相较于其他脊椎动物模型可开展更多高通量实验。鉴于斑马鱼与人类共享超过70%的基因同源物，且其基因组可通过高效CRISPR（成簇规律间隔短回文重复序列）技术进行便捷编辑，如今我们可快速构建靶向几乎任意目的基因的突变体。遗憾的是，我们对突变幼虫进行表型分析的能力未能跟上这一发展步伐。
为应对这一挑战，我们开发了一套可自动化、并行化获取单条斑马鱼幼虫多项表型数据的实验方案，涵盖形态学特征（如体长、眼面积及头部尺寸）与运动/行为学指标。通过在多种条件下对野生型斑马鱼开展检测，我们确定了可避免显著发育缺陷或物理损伤的最优实验参数：包括在两个时间点[受精后3天（3 dpf）与受精后5天（5 dpf）]对幼虫进行形态学成像，以及在5 dpf时开展行为运动追踪。
作为概念验证，我们针对两株新构建的CRISPR编辑斑马鱼突变品系开展了测试：这两株品系携带syngap1b与slc7a5的预测功能缺失等位基因，二者分别是与人类自闭症谱系障碍、智力障碍及癫痫相关的两个基因的同源基因。借助优化后的高通量表型分析方案，我们成功复现了此前针对这些候选基因的小鼠与斑马鱼模型所发表的研究结果。
综上，我们构建了一套可稳定、一致地表征单条幼虫形态学与行为学特征的快速并行流程，从而提升了我们筛选与人类性状及疾病相关的关键基因的能力。