Assessment of Autism Zebrafish Mutant Models Using a High-Throughput Larval Phenotyping Platform

Abstract

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.

De novo mutations within metabolism networks of amino acid/protein/energy in Chinese autistic children with intellectual disability

“…Amino acids, especially branched chain amino acids (BCAAs) which comprise as much as 30% of proteins in the cell, are also related to long-term modifications of synapses. By studying the mutation of BCKDK (a metabolizing enzyme of BCAAs) [ 77 , 78 ] and SLC7A5 (a neutral amino acid transporter) [ 79 ], people know that these amino acids are also used as neurotransmitters and as metabolic intermediates in the etiology of ASD, ID, and other mental diseases [ 80 ]. Moreover, oxygen consumption, a major index for energy metabolism in the brain, accounts for about one-fifth of the total consumption of the human body.…”

Deleterious Variation in BR Serine/Threonine Kinase 2 Classified a Subtype of Autism

“…Then the body length was measured as the distance between the farthest most left and right of the larvae (Figure 3B). Measurements of optic tectum (OT) were also performed as the brain width directly behind the eyes in the dorsal view following previously study (Colón-Rodríguez et al, 2020; Figure 3B). All the measurements were conducted using ImageJ software.…”

Diisopropylfluorophosphate (DFP) volatizes and cross-contaminates wells in a common 96-well plate format used in zebrafish larvae toxicology studies

“…Zebrafish are increasingly used for medium-to-high-throughput toxicological assays (Cassar et al, 2020;Tal, Yaghoobi, & Lein, 2020). Static waterborne exposures of ZF larvae in 96-well plates is used extensively, particularly when screening large chemical libraries or phenotyping mutant models (Colón-Rodríguez et al, 2020;Dach et al, 2019;Tal et al, 2020;Truong et al, 2020). Microinjection is a possible alternative to aqueous exposure, but it is more time-consuming and not as amenable to medium-tohigh throughput screening (Schubert, Keddig, Hanel, & Kammann, 2014).…”