A Chemical Screening Procedure for Glucocorticoid Signaling with a Zebrafish Larva Luciferase Reporter System

Abstract

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids can lead to severe side effects. Test systems are needed to search for novel compounds influencing glucocorticoid signaling in vivo or to determine unwanted effects of compounds on the glucocorticoid signaling pathway. We have established a transgenic zebrafish assay which allows the measurement of glucocorticoid signaling activity in vivo and in real-time, the GRIZLY assay (Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY). The luciferase-based assay detects effects on glucocorticoid signaling with high sensitivity and specificity, including effects by compounds that require metabolization or affect endogenous glucocorticoid production. We present here a detailed protocol for conducting chemical screens with this assay. We describe data acquisition, normalization, and analysis, placing a focus on quality control and data visualization. The assay provides a simple, time-resolved, and quantitative readout. It can be operated as a stand-alone platform, but is also easily integrated into high-throughput screening workflows. It furthermore allows for many applications beyond chemical screening, such as environmental monitoring of endocrine disruptors or stress research.

Glucocorticoid deficiency causes transcriptional and post-transcriptional reprogramming of glutamine metabolism

“…A fully functional stress axis leading to the release of cortisol by the interrenal gland, the fish counterpart to the mammalian adrenal gland [ 53 ], is present at four to five days of development [ 32 , 38 , 76 ]. Several zebrafish mutants [ 19 , 23 , 33 , 60 ] and transgenic reporter lines [ 6 , 8 , 24 , [28] , [29] , [30] , 37 , 44 , 63 , 75 , 76 ] of steroid hormone synthesis and action have been established. They allow for comprehensive analysis of in vivo processes and for high-throughput compound screenings to identify novel drug targets.…”

Zebrafish biosensor for toxicant induced muscle hyperactivity

“…Molecular endpoints derived from toxicogenomic responses provided other endpoints that allow us to demonstrate the presence of compounds with a specific, potentially tissue-specific mode of action 3 4 5 6 . This observation has led to the development of transgenic biosensor lines that utilize genetic regulatory elements to drive the expression of fluorescent proteins and thereby report the presence of target classes of substances in the environment, such as estrogenic compounds 7 8 9 10 , dioxins 11 , heavy metals 12 13 14 , goitrogens 15 and glucocorticoids 16 17 18 . Interestingly, many of these biosensor genes exhibit tissues preferably responsive to particular classes of pollutants 6 .…”

MondoA regulates gene expression in cholesterol biosynthesis-associated pathways required for zebrafish epiboly

“…For recording of in vivo bioluminescence from a ChoRE reporter, a mixture composed of the 2xChoRE luciferase reporter construct (100 ng/µl; see above, ‘cell culture’), phenol red (0.1%) and either 150 mM 2-DG or water was injected into zygotes. The injected embryos were transferred into 96-well plates containing E3 medium with 0.5 mM luciferin (E3L) at high/oblong stage for bioluminescence measurement as described ( Weger et al, 2013a ; Weger et al, 2012 ; Weger et al, 2013b ). Bioluminescence was measured at sphere stage and subtracted against background luminescence from wells without embryos.…”