Zebrafishology, study design guidelines for rigorous and reproducible data using zebrafish

Abstract

The zebrafish (Danio rerio) is one of the most widely used research model organisms funded by the United States' National Institutes of Health, second only to the mouse. Here, we discuss the advantages and unique qualities of this model organism. Additionally, we discuss key aspects of experimental design and statistical approaches that apply to studies using the zebrafish model organism. Finally, we list critical details that should be considered in the design of zebrafish experiments to enhance rigor and data reproducibility. These guidelines are designed to aid new researchers, journal editors, and manuscript reviewers in supporting the publication of the highest-quality zebrafish research.

Fig. 1. Examples of pigmentation patterns in zebrafish larvae due to genetic or chemical perturbations.

Zebrafish are, by convention, oriented with their head to the left and tail to the right. The two common orientations are a lateral/side view (a, c, e) or a top/dorsal view (b, d, f). Labeled in the WT 5 pdf larvae (a) is the eye and swim bladder. a A side view of WT pigmentation pattern of the TLF line. b From the top view, the WT pigment obscures the zebrafish brain and upper digestive tract. c–d The zebrafish casper mutant line prevents most pigmentation from forming except for the eyes and around the swim bladder. E-F) WT embryos were exposed to PTU after gastrulation (at the start of somitogenesis) but before pigment started forming at 24 hpf. PTU can be added after the start of pigment formation, but this will cause an incomplete loss of pigment. Adding PTU prior to somitogenesis will inhibit gastrulation, and the embryos will not develop correctly. When comparing the WT and the WT + PTU zebrafish, despite these larvae coming from the same clutch, the PTU has slowed down development (compare swim bladder size and yolk). This is one example of when careful timing is important for direct comparison work.

Fig. 2. Important milestones in zebrafish development.

Shown are critical milestones in zebrafish development (note images are not shown to scale). However, the embryos and larvae are mm in size, with the single cell at fertilization being 1 mm, whereas the juveniles and adults are cm in size. The mid-blastula transition (MBT) is when the time between cell cycles increases, and cell synchrony is lost. Zygotic genome activation quickly follows, and maternal transcripts begin to be depleted. Gastrulation begins by just over 5 hpf, and somitogenesis begins 5 h later. The neural tube is formed by ~17 h, and primary neurogenesis begins at around 2 dpf, with secondary neurogenesis beginning at 3 dpf. The zebrafish have a mature nervous system by 4 dpf. The heart starts beating by 24 h, and the larval kidney (the pronephros) begins to function at around 2 dpf. The yolk sac is depleted by ~5–6 dpf. The mature digestive system is fully functional by 7 dpf, but the larvae can begin free feeding as early as 5 dpf^,. The gonads begin to differentiate in the juvenile fish by around 20–25 dpf. At ~3 months, the fish is fully mature and capable of mating. All times are developmental stages when the fish is developing at the standard 28.5 °C.

Fig. 3. The zebrafish as a developing organism.

a Whole mount in situ hybridization mRNA stain of Short stature homeobox 1 (shox1) throughout the first 5 days of development exemplifies how a gene can change intensity (black arrows point to the brain) and location of expression (red arrows point to the fin bud, which is only expressed in 1.5–3 dpf; black arrowhead points to the pharyngeal arches which begin expression at 2 dpf) in a relatively short period of time. b The VEGF receptor (kdr-like) florescent line (Tg(kdrl:Hsa.HRAS-mCherry)s916) demonstrates how quickly an organ system, the vasculature, changes and develops in the first 4 days of zebrafish development. ISV—intersegmental vessels (a—arterial, v—venous); DA—dorsal aorta; PCV—posterior cardinal vein.

Fig. 4. Defining replicates in the zebrafish model.

a Using a single mating pair’s progeny, technical replicates can be conducted (the Eppendorf tube stands in for any data output with a–c representing three technical replicates). This data will only give information on the variation due to the experimental method. b Biological replicates are also necessary to sample the population appropriately. Using the progeny from different mating pairs ensures genetic diversity, which is the basis of biological variability. c A single experiment typically uses many different mating pairs to create biological diversity within the pool of progeny tested. The experiment shown here has a single technical replicate with multiple conditions (control, con; treatment 1, Tx 1; treatment 2, Tx 2). However, technical replicates are achieved using these progenies, but these are not adequate biological replicates. d Multiple experiments using different tanks of outcrossed adult fish allow for both technical and biological replicates.