Abstract
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.
Highlights
Diabetes is associated with reductions in pancreatic β-cell mass, curing diabetic patients will require β-cell replacement therapy. β cells can be replaced by transplantation of pancreatic islets (Vardanyan et al, 2010)
We reasoned that the β/δ-reporter line would allow us to detect compounds affecting endocrine differentiation and/or proliferation of β cells or their progenitors since both would cause an increase in YFP reporter signal (Figure 1C)
We leveraged the high-throughput capacity afforded by automated reporter quantification in vivo (ARQiv) to reduce false-call rates using qHTS principles—that is, titration-based primary screening (Inglese et al, 2006)
Summary
Diabetes is associated with reductions in pancreatic β-cell mass, curing diabetic patients will require β-cell replacement therapy. β cells can be replaced by transplantation of pancreatic islets (Vardanyan et al, 2010). Diabetes is associated with reductions in pancreatic β-cell mass, curing diabetic patients will require β-cell replacement therapy. Β cells can be replaced by transplantation of pancreatic islets (Vardanyan et al, 2010). Placing animal models at the start, rather than the end, of the drug discovery process has the potential to circumvent high attrition rates that have plagued in vitro high-throughput screening (HTS) over the past two decades. The zebrafish is an ideal vertebrate model system for whole-organism-based drug discovery (Zon and Peterson, 2005). A chemical derivative of prostaglandin E2 (16,16 dimethyl prostaglandin E2), originally identified for the capacity to induce increased hematopoietic stem cell (HSC) numbers in zebrafish embryos, recently completed Phase I and entered Phase II clinical trials as a means of enhancing engraftment of cord blood transplants in leukemic patients (Cutler et al, 2013)
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