Characterization ofFxr1inDanio rerio; a simple vertebrate model to study costamere development

Abstract

SUMMARY The X-linked FMR1 gene, which is involved in the fragile X syndrome, forms a small gene family with its two autosomal homologs, FXR1 and FXR2. Mouse models for the FXR genes have been generated and proved to be valuable in elucidating the function of these genes, particularly in adult mice. Unfortunately, Fxr1 knockout mice die shortly after birth, necessitating an animal model that allows the study of the role of Fxr1p, the gene product of Fxr1, in early embryonic development. For gene function studies during early embryonic development the use of zebrafish as a model organism is highly advantageous. In this paper the suitability of the zebrafish as a model organism to study Fxr1p function during early development is explored. As a first step, we present here the initial characterization of Fxr1p in zebrafish. Fxr1p is present in all the cells from zebrafish embryos from the 2/4-cell stage onward; however, during late development a more tissue-specific distribution is found, with the highest expression in developing muscle. In adult zebrafish, Fxr1p is localized at the myoseptum and in costamere-like granules in skeletal muscle. In the testis, Fxr1p is localized in immature spermatogenic cells and in brain tissue Fxr1p displays a predominantly nuclear staining in neurons throughout the brain. Finally, the different tissue-specific isoforms of Fxr1p are characterized. Since the functional domains and the expression pattern of Fxr1p in zebrafish are comparable to those in higher vertebrates such as mouse and human, we conclude that the zebrafish is a highly suitable model for functional studies of Fxr1p.

Two members of the Fxr gene family, Fmr1 and Fxr1, are differentially expressed in Xenopus tropicalis

“…4G). This Fxr1p expression pattern during embryonic development and adult stages showed high similarities with the expression patterns described for man, mice and fish (Dube et al, 2000, Engels et al, 2004, Khandjian et al, 1998, Tamanini et al, 1997. Both Fmrp and Fxr1p share the nuclear labelling during very early development which suggests a specific nuclear function during this short period of development.…”

“…Unlike the slow-growing, tetraploid Xenopus laevis, however, Xenopus tropicalis is diploid and has a relatively short (<5 months) life cycle. A high level of sequence conservation of the FXR genes throughout various vertebrates (mouse, rat, zebrafish) has been demonstrated (Engels et al, 2004). This was used to screen the Sanger Institute X. tropicalis EST database with sequences from the human FXR family members to find orthologues of the X. tropicalis Fxr genes.…”

“…The high expression of Fxr1p in skeletal muscle tissue at late embryonic stages and in adult frogs suggests a role for Fxr1p in myogenesis. Similar to striated muscle tissue from mouse and zebrafish, the characteristic granular labelling of Fxr1p present in skeletal muscle from frogs suggests that Fxr1p is located in specific structures of the muscle tissue, called costameres (Dube et al, 2000, Engels et al, 2004, Mientjes et al, 2004. In man and mice, different isoforms of Fxr1p have been described for the different tissues, due to alternative splicing (Bakker et al, 2000, Huot et al, 2001, Khandjian et al, 1998, Tamanini et al, 1997.…”

Reduction in fragile X related 1 protein causes cardiomyopathy and muscular dystrophy in zebrafish

“…(hours postfertilisation) as previously described (van't Padje et al, 2005). Briefly, following blocking of gels, an antibody against zebrafish Fxr1p (Affi5, 1:500) was applied (Engels et al, 2004). Following washing, a peroxidase conjugated secondary antibody (goat antirabbit Igs; 1:5000) was used to detect specific antibody binding and reported by chemiluminescence (ECL Kit, Amersham, UK).…”

“…The FXR1 gene shows evolutionary conservation, and morpholino (MO) knockdown in Xenopus has suggested abnormal somite formation (Huot et al, 2005). We have previously shown that the functional domains of FXR genes are conserved in zebrafish and that the expression pattern of Fxr1 in zebrafish is similar to human and mouse (Engels et al, 2004). Furthermore, the mRNA sequence around the initiator codon of fxr1 is known and it has therefore proved possible to investigate the effects of translational silencing using morpholino techniques.…”