Automated Reporter Quantification In Vivo: High-Throughput Screening Method for Reporter-Based Assays in Zebrafish

Abstract

Reporter-based assays underlie many high-throughput screening (HTS) platforms, but most are limited to in vitro applications. Here, we report a simple whole-organism HTS method for quantifying changes in reporter intensity in individual zebrafish over time termed, Automated Reporter Quantification in vivo (ARQiv). ARQiv differs from current “high-content” (e.g., confocal imaging-based) whole-organism screening technologies by providing a purely quantitative data acquisition approach that affords marked improvements in throughput. ARQiv uses a fluorescence microplate reader with specific detection functionalities necessary for robust quantification of reporter signals in vivo. This approach is: 1) Rapid; achieving true HTS capacities (i.e., >50,000 units per day), 2) Reproducible; attaining HTS-compatible assay quality (i.e., Z'-factors of ≥0.5), and 3) Flexible; amenable to nearly any reporter-based assay in zebrafish embryos, larvae, or juveniles. ARQiv is used here to quantify changes in: 1) Cell number; loss and regeneration of two different fluorescently tagged cell types (pancreatic beta cells and rod photoreceptors), 2) Cell signaling; relative activity of a transgenic Notch-signaling reporter, and 3) Cell metabolism; accumulation of reactive oxygen species. In summary, ARQiv is a versatile and readily accessible approach facilitating evaluation of genetic and/or chemical manipulations in living zebrafish that complements current “high-content” whole-organism screening methods by providing a first-tier in vivo HTS drug discovery platform.

Immunomodulation-accelerated neuronal regeneration following selective rod photoreceptor cell ablation in the zebrafish retina

“…Retinas in which only rod cells were ablated demonstrated effective loss and regeneration of YFP rod cells (Fig. 4 C-C′′′), consistent with previous results (20). Coablated retinas exhibited effective loss of YFP cells and a reduction in rod cell regeneration kinetics through day 4 of recovery ( Fig.…”

“…The majority of the procedures applied and all transgenic lines used, Tg(rho: YFP-Eco.NfsB)gmc500, Tg(mpx:GFP)uwm1, Tg(mpeg1.1:LOX2272-LOXP-dTomato-LOX2272-Cerulean-LOXP-EYFP)w201, Tg(mpeg1.1:NTR-EYFP)w202, and Tg(gfap: GFP)m2001, have been previously published (8,20,22,39,41,47). Brief descriptions of the experimental design are outlined here; additional details can be found in SI Materials and Methods and references provided.…”

“…Following Mtz removal, microglia underwent a second wave of proliferation within the INL during the recovery phase. Interestingly, this second wave was coincident with the initiation of rod cell regeneration (20).…”

“…Importantly, the NTR/Mtz-based ablation system elicits apoptotic cell death (18), a hallmark of neurodegenerative disease (19). To model retinitis pigmentosa, we created a transgenic line expressing a YFP-nitroreductase fusion protein specifically in rod photoreceptor cells-Tg(rho:YFP-Eco.NfsB)gmc500 (hereafter, rho: YFP-NTR)-enabling Mtz-induced ablation of rod cells (20). To investigate neuroimmune interactions occurring during retinal degeneration, intravital multicolor time-lapse imaging (14,21,22) was used to monitor and quantify the behavioral responses of transgenically labeled immune cell types to rod cell death.…”

“…The kinetics of rod cell regeneration was assessed using fluorescent plate reader-based quantification (i.e., automated reporter quantification in vivo, ARQiv) of YFP signal levels in individual larvae (20,51,52). Using this method, we had previously shown full regeneration of rod cells as early as day 5-6 of recovery (20). For comparison with Dextreated larvae, the level of YFP signal observed in Mtz-treated control larvae at day 4 of recovery was normalized to 100% regeneration.…”