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.糖皮质激素受体（glucocorticoid receptor, GR）可在被糖皮质激素（glucocorticoid, GC）激素激活后调控基因表达。在斑马鱼中存在两种GR剪接变体：经典GRα亚型（GRα）以及GRβ亚型（GRβ），目前GRβ的确切功能仍不明确。本研究以斑马鱼胚胎为模型，探究了GRα与GRβ的转录调控功能：通过向胚胎注射两种剪接阻断型吗啉代寡核苷酸（morpholino），其中一种可同时敲低GRα和GRβ两种亚型，另一种靶向GR前体mRNA（pre-mRNA）的可变剪接以偏向产生GRβ亚型，同时还注射了GRβ mRNA以实现特异性的GRβ过表达。随后对提取自受精后30小时（hours post fertilization, hpf）的胚胎总RNA开展转录组分析（transcriptome profiling）。本研究结果显示，GRβ并未作为GRα的显性负向抑制剂发挥功能，且GRα可调控两个主要参与胚胎代谢过程的不同基因簇。本项基因芯片（microarray）研究旨在明确GRα与GRβ的敲低及过表达所产生的生物学效应。为此，我们将处于1-2细胞期的野生型（AB/TL品系）斑马鱼胚胎分别注射标准对照吗啉代寡核苷酸（SC-MO）、可同时敲低GRα与GRβ两种亚型的剪接阻断型吗啉代寡核苷酸（MO1）、靶向GR前体mRNA可变剪接以偏向生成GRβ亚型的剪接阻断型吗啉代寡核苷酸（MO2），或GRβ mRNA。在受精后24小时（24 hpf）时，每组胚胎均接受地塞米松（dexamethasone，原文笔误为dexamathasone）或溶媒（vehicle，缩写为veh）处理6小时，最终得到8个处理组：SC-MO/溶媒、SC-MO/地塞米松、MO1/溶媒、MO1/地塞米松、MO2/溶媒、MO2/地塞米松、GRβ mRNA/溶媒、GRβ mRNA/地塞米松。孵育结束后收集胚胎，从每个处理组的20枚胚胎中提取总RNA。本研究共开展3次独立实验，采用通用参考样本法（common reference approach）对获得的三重重复样本进行基因芯片分析。