Automated Zebrafish Chorion Removal and Single Embryo Placement: Optimizing Throughput of Zebrafish Developmental Toxicity Screens

Abstract

The potential of the developing zebrafish model for toxicology and drug discovery is limited by inefficient approaches to manipulating and chemically exposing zebrafish embryos—namely, manual placement of embryos into 96- or 384-well plates and exposure of embryos while still in the chorion, a barrier of poorly characterized permeability enclosing the developing embryo. We report the automated dechorionation of 1600 embryos at once at 4 h postfertilization (hpf) and placement of the dechorionated embryos into 96-well plates for exposure by 6 hpf. The process removed ≥95% of the embryos from their chorions with 2% embryo mortality by 24 hpf, and 2% of the embryos malformed at 120 hpf. The robotic embryo placement allocated 6-hpf embryos to 94.7% ± 4.2% of the wells in multiple 96-well trials. The rate of embryo mortality was 2.8% (43 of 1536) from robotic handling, the rate of missed wells was 1.2% (18 of 1536), and the frequency of multipicks was <0.1%. Embryo malformations observed at 24 hpf occurred nearly twice as frequently from robotic handling (16 of 864; 1.9%) as from manual pipetting (9 of 864; 1%). There was no statistical difference between the success of performing the embryo placement robotically or manually.

Toxicity of chlorine to zebrafish embryos

“…Conversely, one might consider not treating embryos with chlorine at all for specific toxicology or behavior experiments as we still have not elucidated all the potential long term effects of these chlorine treatments. The SARL laboratory’s rapid through put testing facility indeed does not bleach embryos prior to testing, largely because bleaching interferes with pronase-mediated chorion removal (Mandrell et al 2012). …”

Metabolomic analysis to define and compare the effects of PAHs and oxygenated PAHs in developing zebrafish

“…To maximize chemical exposure and uptake, the fertilized embryos were enzymatically dechorionated using a previously described method (Mandrell et al, 2012). Briefly, these embryos were developmentally staged according to Kimmel et al (1996) and at 4 h post fertilization (hpf), they were incubated in 82 mg L −1 of pronase (Sigma-Aldrich; St Louis, MO, USA) at room temperature, gently agitated for 3 min and then rinsed thoroughly using an automated dechorionating system.…”

15 years of zebrafish chemical screening

“…Here again, is a surmountable challenge, which will eventually be worked out. Several platforms are currently in development, including two originally designed for C. elegans screens and at least two designed directly for zebrafish embryos [40, 41]. At least one automated embryo placement system has been used in a zebrafish high-throughput chemical screen [42].…”