A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Abstract

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.

Prostaglandin signaling regulates nephron segment patterning of renal progenitors during zebrafish kidney development

“…The effect of each compound was annotated as to whether the experimental dosage was associated with WT development, an expansion in segment(s) (P+, PCT+, DE+) or a restriction in segment(s) (P-, PCT-, DE-) (Figure 1C, Figure 1—source data 1). The compounds that led to alterations in nephrogenesis included numerous RA pathway agonists and antagonists, such as 4-hydroxyphenylretinamide (4-HPR), a synthetic analog of all-trans RA (Figure 1D) (Poureetezadi et al, 2014). Compared to WTs, exposure to 1 mM 4-HPR led to an expansion of the PCT, caudal shift of the DE, and a dramatic expansion of the interval between these segments where the PST normally emerges, suggestive of an expanded PST segment (Figure 1D) (Poureetezadi et al, 2014).…”

“…The compounds that led to alterations in nephrogenesis included numerous RA pathway agonists and antagonists, such as 4-hydroxyphenylretinamide (4-HPR), a synthetic analog of all-trans RA (Figure 1D) (Poureetezadi et al, 2014). Compared to WTs, exposure to 1 mM 4-HPR led to an expansion of the PCT, caudal shift of the DE, and a dramatic expansion of the interval between these segments where the PST normally emerges, suggestive of an expanded PST segment (Figure 1D) (Poureetezadi et al, 2014). The observation that molecules which impact the RA pathway were flagged as hits in the screen provided an important positive control for our experimental system, given the well-established effects of RA levels on renal progenitors (Wingert et al, 2007; Wingert and Davidson, 2011; Li et al, 2014; Cheng and Wingert, 2015; Marra and Wingert, 2016; Drummond et al, 2017).…”

“…Zebrafish wild-type (WT) embryos were arrayed and treated with small molecules using the ICCB Known Bioactives Library as described (Poureetezadi et al, 2014). Zebrafish embryos were staged at 2 hpf, then at least 30 fertilized embryos were arrayed into the chambers of 24-well plates and incubated at 28°С in E3 media until just prior to 4 hpf.…”

Insights into kidney stem cell development and regeneration using zebrafish

“…This is an important advance for pursuing research about nephrogenesis in this segment during development and regeneration. Additional tools and transgenic lines such as this are needed to advance our ability to visualize renal regeneration, as they could be combined with chemical genetics to identify signals that modulate regeneration and the development of renal progenitors during embryogenesis [99] . Processes that are happening in regeneration that we cannot "see" must also be evaluated, such as renal cell signaling, gene activation, and cell fate decisions.…”

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

“…Tools for molecular analysis, such as gene expression 35–37 , have been utilized with high resolution to determine the features of nephrogenesis 38–40 . Experimental manipulations for testing of small molecules through chemical genetics are well established in zebrafish and applicable to the kidney 41–43 . Indeed, in recent years, zebrafish have been applied to model nephrotoxicity of agents ranging from acetominophen to mycotoxins and aristolochic acid 44–48 .…”