Functional analysis of the zebrafish glucocorticoid receptor beta-isoform

In the present study, we have used the zebrafish to investigate the functional role of the glucocorticoid receptor beta-isoform (GRbeta). A transgenic zebrafish line, Tg(hsp70:Gal4, UAS:EGFP-grbeta), was generated, showing heat shock-inducible expression of a GFP-tagged GRbeta. After heat shock treatment at 1 and 2 days post fertilization (dpf), 3 dpf larvae (control and transgenic) were treated with beclomethasone. A microarray study was performed to study transcriptional effects of GRbeta: its effect on transcription in the absence of beclomethasone (intrinsic transcriptional activity) and its effects on beclomethasone-induced transcriptional changes (dominant-negative activity). Although the microarray results revealed minor transcriptional effects of GRbeta expression, these data could not be validated using qPCR. It was therefore concluded that GRbeta did not show any transcriptional activity under these conditions. This microarray study was designed to determine the transcriptional effect of the glucocorticoid receptor beta-isoform. For this purpose, a transgenic zebrafish line, Tg(hsp70:Gal4, UAS:EGFP-grbeta), was generated showing heat shock-inducible expression of a GFP-tagged GRbeta. Control (wild type) and transgenic embryos were heat-shocked (2.5 hours at 37°C) at 1 and 2 dpf. At 3 dpf, larvae were treated with vehicle or beclomethasone (25 microM) for six hours. This resulted in 4 treament groups: con/veh, con/beclo, GRbeta/veh, GRbeta/beclo. RNA was isolated from 20 larvae per treatment group. Four individual experiments were performed, and the resulting quadruplicate samples were analyzed by microarray, using a common reference approach.