Zebrafish as a Model to study Human Disease: Functional Studies of the FXR Proteins

The FMR1 protein, FMRP, has been extensively studied due to its involvement in the fragile X syndrome, which is mainly characterised by mental retardation, macroorchidism and facial dysmorphologies. The FMR1 gene is transcriptionally inactivated by the methylation of its promoter region due to the expansion of a CGG repeat. Thus the fragile X syndrome is caused by the absence of the protein FMRP. FMRP is part of a small RNA binding protein family, which also include FXR1P and FXR2P. Due to the high homology of important functional domains and similar expression patterns in the brain, FXR1P and FXR2P are thought to have a similar function to FMRP. All three proteins are ubiquitously expressed with high expression in brain and testis. In addition, FXR1P is highly expressed in striated muscle tissue. The cellular function of the FXR proteins has been studied by the generation of knockout mouse models for all three genes. The Fmr1 KO mice display similar features as fragile X patients, including learning and memory abnormalities and macroorchidism. Further characterisation of the Fmr1 KO mice showed a reduced pruning and/or maturation of spines, which normally occurs during late embryonic and postnatal development. Fxr1 KO mice showed a severe striated muscle phenotype and died shortly after birth. This early lethality indicates that cellular Fxr1p function is critical during embryonic development. Fxr2 KO mice showed a discrete behavioural phenotype, but needs further characterisation to determine whether Fxr2p has a role during embryonic development. Processes during embryonic development are difficult to study in the mouse model due to in utero development of the embryos. For this reason, we have chosen the zebrafish (Danio rerio) as a model to study the FXR protein function especially during embryonic development. The zebrafish is a well-established simple vertebrate animal model. Their ability to be kept in large numbers and the ease of breeding make them easy to maintain. Due to its transparent embryos that develop externally the zebrafish represents an ideal model to study processes during embryonic development. In addition, the embryos develop quickly from a single cell to something that is recognizable as a fish after 24 hours of development. The favourite method to study gene function in zebrafish is the generation of a morpholino-mediated knockdown zebrafish. This morpholino technology is based on the injection of synthetic antisense oligonucleotides into a 1-8 cell stage embryo, which subsequently binds to the mRNA molecule and thereby preventing translation. The introduction of this thesis (chapter 1) describes the use of zebrafish as a vertebrate animal model with special emphasis to human disease (part 1) and introduces the FXR protein family with its three individual members (part 2). The aim of this thesis is to gain insight in the function of the FXR proteins, and their role during embryonic development particularly using the zebrafish as a model system.