Advancements in zebrafish applications for 21st century toxicology

Abstract

The zebrafish model is the only available high-throughput vertebrate assessment system, and it is uniquely suited for studies of in vivo cell biology. A sequenced and annotated genome has revealed a large degree of evolutionary conservation in comparison to the human genome. Due to our shared evolutionary history, the anatomical and physiological features of fish are highly homologous to humans, which facilitates studies relevant to human health. In addition, zebrafish provide a very unique vertebrate data stream that allows researchers to anchor hypotheses at the biochemical, genetic, and cellular levels to observations at the structural, functional, and behavioral level in a high-throughput format. In this review, we will draw heavily from toxicological studies to highlight advances in zebrafish high-throughput systems. Breakthroughs in transgenic/reporter lines and methods for genetic manipulation, such as the CRISPR-Cas9 system, will be comprised of reports across diverse disciplines.

Keywords: CRISPR; High-throughput; Toxicology; Transgenic/Reporter; Zebrafish.

Figure 1

Conceptual image of the zebrafish data stream

Figure 2

Example of embryonic zebrafish high-throughput screening (HTS) platform. Embryos synchronized at a specific developmental stage are selected, screened for viability, and placed into well plates. Embryos are generally exposed to chemicals between 6–120 hours post fertilization (hpf). Morphological evaluations and behavioral assays are frequently conducted during (1) the early pharyngula stage at 24 hpf when the heart is first clearly visible in a distinct pericardial sac and body/tail flexions initiate with development of the sensory-motor system; and (2) free-swimming larvae represented by inflation of the swim bladder, largely completed developmental morphogenesis, and rapid growth (Haffter et al., 1996; Kimmel et al. 1995; Noyes et al. 2015; Truong et al. 2014).

Figure 3

Representative images from embryonic transgenic zebrafish. (A and B) The Tg(cyp1a:nls-egfp) line can be used as a surrogate for AHR activity to identify the target tissues of chemical exposure. Embryos were continuously exposed to a chemical starting at 6 hpf and imaged at 48 hpf (A) and 120 hpf (B), with noticeable cyp1a expression in the liver at 120 hpf (white arrow). (C and D) The Tg(fli:gfp) line, which expresses GFP in endothelial cells of the entire vasculature, were injected with glioblastoma cells (red) into the brain of 4 dpf larvae (C) and reimaged at 7 dpf (D) in order to capture the invasion and migration behavior of the brain cancer cells. (E–G) Immunohistochemistry was used to determine the expression pattern of various genes in the hair cells of the lateral line neuromast of 4 dpf larvae. (E) 2D composite image stained with antibodies targeting otoferlin (blue), acetylated tubulin (green), and maguk (red). (F) 2D composite image stained with antibodies targeting otoferlin (green) and vglut3, a synaptic vesicle marker (red). (G) 3D composite image stained with DAPI and the synaptic protein ribeye (red clusters). While images (E–G) are not from a transgenic line, the images were included to highlight the ability to capture high quality in situ expression patterns of genes across development, which is the function of transgenic reporter lines.