Whole-organism chemical screening can circumvent bottlenecks that impede drug discovery. However, in vivo screens have not attained throughput capacities possible with in vitro assays. We therefore developed a method enabling in vivo high-throughput screening (HTS) in zebrafish, termed automated reporter quantification in vivo (ARQiv). In this study, ARQiv was combined with robotics to fully actualize whole-organism HTS (ARQiv-HTS). In a primary screen, this platform quantified cell-specific fluorescent reporters in >500,000 transgenic zebrafish larvae to identify FDA-approved (Federal Drug Administration) drugs that increased the number of insulin-producing β cells in the pancreas. 24 drugs were confirmed as inducers of endocrine differentiation and/or stimulators of β-cell proliferation. Further, we discovered novel roles for NF-κB signaling in regulating endocrine differentiation and for serotonergic signaling in selectively stimulating β-cell proliferation. These studies demonstrate the power of ARQiv-HTS for drug discovery and provide unique insights into signaling pathways controlling β-cell mass, potential therapeutic targets for treating diabetes.DOI: http://dx.doi.org/10.7554/eLife.08261.001

The zebrafish has emerged as an important model for whole-organism small-molecule screening. However, most zebrafish-based chemical screens have achieved only mid-throughput rates. Here we describe a versatile whole-organism drug discovery platform that can achieve true high-throughput screening (HTS) capacities. This system combines our automated reporter quantification in vivo (ARQiv) system with customized robotics, and is termed ‘ARQiv-HTS’. We detail the process of establishing and implementing ARQiv-HTS: (i) assay design and optimization, (ii) calculation of sample size and hit criteria, (iii) large-scale egg production, (iv) automated compound titration, (v) dispensing of embryos into microtiter plates, and (vi) reporter quantification. We also outline what we see as best practice strategies for leveraging the power of ARQiv-HTS for zebrafish-based drug discovery, and address technical challenges of applying zebrafish to large-scale chemical screens. Finally, we provide a detailed protocol for a recently completed inaugural ARQiv-HTS effort, which involved the identification of compounds that elevate insulin reporter activity. Compounds that increased the number of insulin-producing pancreatic beta cells represent potential new therapeutics for diabetic patients. For this effort, individual screening sessions took 1 week to conclude, and sessions were performed iteratively approximately every other day to increase throughput. At the conclusion of the screen, more than a half million drug-treated larvae had been evaluated. Beyond this initial example, however, the ARQiv-HTS platform is adaptable to almost any reporter-based assay designed to evaluate the effects of chemical compounds in living small-animal models. ARQiv-HTS thus enables large-scale whole-organism drug discovery for a variety of model species and from numerous disease-oriented perspectives.

SUMMARYKnockdown of protein function by antisense oligonucleotides has been used to understand the protein function not only in development but also in human diseases. Recently, Vivo-Morpholinos, chemically modified morpholinos which penetrate the cells, have been used in adult experimental animal models to alter the splicing and thereby change the protein expression. Until now, there have been no such studies using Vivo-Morpholinos, to evaluate hemostatic function in adult animals. We injected αIIb Vivo-Morpholinos intravenously into adult zebrafish. Thrombocyte function was assayed by time to aggregation assay of the citrated blood, annexin V binding to thrombocytes, and gill bleeding. The thrombocyte functional inhibition occurred in 24 hrs after αIIb Vivo-Morpholinos injection and reached a maximum in 48 hrs. However, in 72 hrs, the inhibition was no longer observed. Reduction of annexin V binding to thrombocytes and increased gill bleeding were observed 48 hrs after αIIb Vivo-Morpholino injections. The action of the αIIb Vivo-Morpholino was demonstrated by the presence of an alternatively spliced αIIb mRNA and the reduction of αIIb in thrombocytes of fish treated with αIIb Vivo-Morpholino. These results provide the first proof of principle that thrombocyte function can be inhibited by thrombocyte-specific Vivo-Morpholinos in adult zebrafish and presents an approach to knockdown thrombocyte-specific genes to conduct biochemical studies in thrombocytes. This study also provides the first antisense antithrombotic approach to inhibit thrombocyte function in adult zebrafish.

Abstractvon Willebrand factor (vWF) is a large protein involved in primary hemostasis. A dysfunction in this protein or an insufficient production of the protein leads to improper platelet adhesion/ aggregation, resulting in a bleeding phenotype known as von Willebrand disease (vWD). To gain a better understanding of vWF interactions in vivo, the use of zebrafish as a model is ideal because of the transparency of the embryos and larvae. In this article, we examined the presence and function of vWF in hemostasis of zebrafish utilizing a variety of molecular methods. Using RT-PCR and antibody staining, we have shown that vWF mRNA is present in thrombocytes. Through antibody staining, we demonstrated vWF is synthesized in blood vessels. The role of zebrafish vWF in hemostasis was established through knockdown methods using vWF morpholino (vWF MO) antisense oligonucleotides. Embryos injected with vWF MO at the one to four cell stages resulted in a bleeding phenotype. Injection of embryos with vWF MO also caused an increase in time to occlusion within arteries in larvae upon laser induced injury. We then used vWF-specific Vivo-Morpholinos (VMO) to induce vWF knockdown in adult zebrafish by targeting the exon homologous to the human exon 28 of the vWF gene. The reduced ristocetin-mediated agglutination of thrombocytes in a plate tilting assay, using blood from adult zebrafish injected with VMO, provided evidence that vWF is involved in the hemostatic process. We also administered desmopressin acetate to larvae and adults which resulted in enhanced aggregation/ agglutination of thrombocytes. Zebrafish genome database analysis revealed the presence of GPIbβ gene. It also revealed the exon of zebrafish vWF gene corresponding to exon 28 of human vWF gen is highly similar to the exon 28 of human vWF gene, except that it has an insertion that leads to a translated peptide sequence that separates the two A domains coded by this exon. This exon is also conserved in other fishes. In summary, we established that zebrafish vWF has a role similar to that of vWF found in humans, thus, making zebrafish a useful model for studying the cell biology of vWF in vivo.