The zebrafish as a model system for glucocorticoid receptor research

Abstract

Glucocorticoids regulate a plethora of physiological processes, and are widely used clinically as anti-inflammatory drugs. Their effects are mediated by the glucocorticoid receptor (GR), a ligand-activated transcription factor. Currently, zebrafish embryos are being developed into a model system for GR research, since they are easy to manipulate genetically and their phenotype can easily be visualized because of their transparent bodies. In addition, the zebrafish GR gene shows a relatively high level of similarity with its human equivalent. First, both the zebrafish and the human genome contain only a single gene encoding the GR. In all other fish species studied thus far, two GR genes have been found. Second, the zebrafish contains a C-terminal GR splice variant with high similarity to the human GRβ, which has been shown to be a dominant-negative inhibitor of the canonical GRα and may be involved in glucocorticoid resistance. Thus, zebrafish embryos are potentially a useful model system for glucocorticoid receptor research, but currently only a limited number of tools is available. In this review, we discuss which tools are available and which need to be developed, in order to exploit the full potential of the zebrafish as a model system for GR research.

Introduction

In the present review we will discuss how research on the glucocorticoid receptor (GR) may benefit from using the zebrafish (Danio rerio) as an animal model system. The zebrafish could be a valuable tool, both in fundamental studies on the molecular mechanisms of GR action and in applied research like screening of glucocorticoid drugs. We will present the advantages of this model system for GR research. However, since the zebrafish has mostly been used as an animal model in the field of developmental biology, several specific tools required for research on the GR in zebrafish are lacking. We will give an overview of which tools are already available and which tools need to be developed in order to exploit the full potential of the zebrafish as a model system for GR research.

Glucocorticoids are steroid hormones that are secreted by the adrenal gland after stress and in a circadian rhythm. In humans and fish, the main endogenous glucocorticoid is cortisol, whereas corticosterone is the main glucocorticoid in rodents. These hormones regulate a wide range of processes, like the immune response (Barnes, 2006), neural activity and behavior (de Kloet et al., 2005), metabolism (Wang, 2005) and bone formation (Migliaccio et al., 2007). They are well known for their anti-inflammatory effects, and are widely used clinically to treat immune-related diseases like asthma and rheumatoid arthritis. Synthetic analogs of glucocorticoids are among the most prescribed drugs in the world.

The effects of glucocorticoids are mediated by an intracellular receptor, the glucocorticoid receptor (GR). This receptor is a member of the family of steroid receptors, which in turn belong to the superfamily of nuclear receptors (Zhang et al., 2004). Like all nuclear receptors, the GR acts as a ligand-activated transcription factor, and it is well conserved among vertebrate animal species (Bridgham et al., 2006). It consists of a large N-terminal domain, involved in transcriptional activation, a small DNA binding domain which contains two zinc-fingers and a C-terminal ligand-binding domain (Giguere et al., 1986). In the absence of hormone, the GR resides in the cytoplasm where it forms a complex with heat shock proteins and immunophilins (Pratt and Toft, 2003). Upon ligand binding, the receptor dissociates from the complex and translocates to the nucleus. There the activated GR can bind to glucocorticoid response elements (GREs) in the promoter region of target genes and interact with transcriptional cofactors (Beato and Klug, 2000). In this way, gene transcription of the downstream gene is activated and this process is called transactivation. Alternatively, the GR can inhibit gene expression induced by other transcription factors like nuclear factor(NF)-κB and activator protein(AP)-1 (De Bosscher et al., 2008). This process is called transrepression and forms the basis of the anti-inflammatory action of glucocorticoids, since these transcription factors are involved in the transcription of many pro-inflammatory genes. The exact mechanism of transrepression has not been elucidated yet, but physical interaction between GR and the other transcription factor and recruitment of specific transcriptional cofactors appears to be involved.

The zebrafish has many advantages over other vertebrate animal model systems (Trede et al., 2004, Lieschke and Currie, 2007, Hsu et al., 2007, Levraud et al., 2008). It is small, easily maintained and breeds well under laboratory conditions. Each female can produce hundreds of eggs per day, that are fertilized externally. Upon fertilization, the embryos develop rapidly and most organ systems have been formed 5 days later. The ex utero development makes the zebrafish embryos easily accessible for transient genetic manipulation by microinjection of DNA, mRNA or morpholinos, which are antisense DNA oligonucleotides that can alter protein synthesis in the developing embryo by blocking a specific translation start site or a splice donor or acceptor site. The embryos are transparent, which allows for microscopic imaging at the subcellular level, especially when performed in combination with fluorescent labeling of specific cells or proteins. Furthermore, an increasing number of transgenic and mutant zebrafish lines are available, as well as several zebrafish cell lines derived from embryos and adult tissues, that can be used as a complementary tool allowing more refined biochemical characterizations (Driever and Rangini, 1993, Chen et al., 2002). The zebrafish genome, as available in the zv7 assembly on the Ensembl website (http://www.ensembl.org/index.html), is virtually complete. Seventy percent of the genome has been sequenced with > 99.999% accuracy. For the rest of the genome, a so-called whole genome shotgun approach has been used, which has a coverage of 5.5 times. The sequence database has been compared to the data obtained from a double haploid zebrafish line.