Abstract
ABSTRACTZebrafish provide a unique opportunity for drug screening in living animals, with the fast-developing, transparent embryos allowing for relatively high-throughput, microscopy-based screens. However, the limited availability of rapid, flexible imaging and analysis platforms has limited the use of zebrafish in drug screens. We have developed an easy-to-use, customisable automated screening procedure suitable for high-throughput phenotype-based screens of live zebrafish. We utilised the WiScan® Hermes High Content Imaging System to rapidly acquire brightfield and fluorescent images of embryos, and the WiSoft® Athena Zebrafish Application for analysis, which harnesses an Artificial Intelligence-driven algorithm to automatically detect fish in brightfield images, identify anatomical structures, partition the animal into regions and exclusively select the desired side-oriented fish. Our initial validation combined structural analysis with fluorescence images to enumerate GFP-tagged haematopoietic stem and progenitor cells in the tails of embryos, which correlated with manual counts. We further validated this system to assess the effects of genetic mutations and X-ray irradiation in high content using a wide range of assays. Further, we performed simultaneous analysis of multiple cell types using dual fluorophores in high throughput. In summary, we demonstrate a broadly applicable and rapidly customisable platform for high-content screening in zebrafish. This article has an associated First Person interview with the first author of the paper.
Highlights
Zebrafish provide an excellent model for human disease and offer a unique opportunity for in vivo small-molecule phenotypic drug screening
This is exemplified by dmPGE2, which entered clinical trials as a therapy for patients undergoing umbilical blood cord transplantation, having been found to enhance haematopoietic stem cells in a zebrafish screen using in situ hybridisation (North et al, 2007; Hagedorn et al, 2014)
Automatic detection of zebrafish embryos and internal anatomy in multiplexed fluorescence imaging with the co-developed WiSoft® Athena image analysis platform Effective high-content screening (HCS) necessitates simple and fast image acquisition to allow for high throughput
Summary
Zebrafish provide an excellent model for human disease and offer a unique opportunity for in vivo small-molecule phenotypic drug screening. Unlike most in vivo screening platforms, thousands of animals can be imaged within days, allowing for a relatively high-throughput screen, with the advantages of screening in intact living animals. The utility of such in vivo screens has been demonstrated by the rapid repurposing of identified compounds into clinical trials. This is exemplified by dmPGE2, which entered clinical trials as a therapy for patients undergoing umbilical blood cord transplantation, having been found to enhance haematopoietic stem cells in a zebrafish screen using in situ hybridisation (North et al, 2007; Hagedorn et al, 2014). ORC-13661, identified in zebrafish screens of hair cells in zebrafish embryos (Owens et al, 2008; Chowdhury et al, 2018; Kitcher et al, 2019) is currently in clinical trials as an agent to prevent hearing loss from aminoglycoside antibiotic-induced hair loss
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