Transcriptional effects of glucocorticoid receptor alpha and beta signaling in zebrafish

The glucocorticoid receptor (GR) regulates gene expression upon activation by glucocorticoid (GC) hormones. In zebrafish, two GR splice variants exist: the canonical GR α-isoform (GRα), and the GR β-isoform (GRβ). The exact function of GRb remains elusive. We have investigated the transcriptional role of GRa and GRb in the zebrafish embryo model by injecting mebryos with two splice-blocking morpholinos (one leading to knockdown of both GR α- and β-isoforms, and another targeting the alternative splicing of the GR pre-mRNA in favor of the GR β-isoform) and with GRβ mRNA (resulting in specific GRβ overexpression). Transcriptome profiling was performed on total RNA isolated from 30 hpf embryos. Our results show that GRb does not act as a dominant-negative inhibitor of GRa, and that GRa regulates two distinct gene clusters, which are mainly involved in the metabolism of the embryo. This microarray study was designed to determine the effect of knockdown and overexpression of GRa and GRb. For this purpose, wild type (AB/TL) zebrafish embryos were injected at the 1-2 cell stage with a standard control morpholino (SC-MO), a splice-blocking morpholino leading to knockdown of both GR α- and β-isoforms (MO1), a splice-blocking morpholino targeting the alternative splicing of the GR pre-mRNA in favor of the GR β-isoform (MO2), or GRb mRNA. At 24 hpf, each group was treated with dexamathasone (or vehicle) for 6 hr. This resulted in 8 treament groups: SC-MO/veh, SC-MO/dex, MO1/veh, MO1/dex, MO2/veh, MO2/dex, GRb mRNA/veh, GRb mRNA/dex. After the incubation period, embryos were collected and RNA was isolated from 20 embryos per treatment group (30 hpf). Three individual experiments were performed, and the resulting triplicate samples were analyzed by microarray, using a common reference approach.