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

Lack of the FMR1 gene product causes fragile X syndrome, the commonest inherited cause of mental impairment. We know little of the roles that fragile X related (FXR) gene family members (FMR1, FXR2 and FXR1) play during embryonic development. Although all are expressed in the brain and testis, FXR1 is the principal member found in striated and cardiac muscle. The Fxr1 knockout mice display a striated muscle phenotype but it is not known why they die shortly after birth; however, a cardiac cause is possible. The zebrafish is an ideal model to investigate the role of fxr1 during development of the heart. We have carried out morpholino knockdown of fxr1 and have demonstrated abnormalities of striated muscle development and abnormal development of the zebrafish heart, including failure of looping and snapping of the atrium from its venous pole. In addition, we have measured cardiac function using high-speed video microscopy and demonstrated a significant reduction in cardiac function. This cardiac phenotype has not been previously described and suggests that fxr1 is essential for normal cardiac form and function.

Lessons from BWS twins: complex maternal and paternal hypomethylation and a common source of haematopoietic stem cells.

The Beckwith-Wiedemann syndrome (BWS) is a growth disorder for which an increased frequency of monozygotic (MZ) twinning has been reported. With few exceptions, these twins are discordant for BWS and for females. Here, we describe the molecular and phenotypic analysis of 12 BWS twins and a triplet; seven twins are MZ, monochorionic and diamniotic, three twins are MZ, dichorionic and diamniotic and three twins are dizygotic. Twelve twins are female. In the majority of the twin pairs (11 of 13), the defect on chromosome 11p15 was hypomethylation of the paternal allele of DMR2. In 5 of 10 twins, there was additional hypomethylation of imprinted loci; in most cases, the loci affected were maternally methylated, but in two cases, hypomethylation of the paternally methylated DLK1 and H19 DMRs was detected, a novel finding in BWS. In buccal swabs of the MZ twins who share a placenta, the defect was present only in the affected twin; comparable hypomethylation in lymphocytes was detected in both the twins. The level of hypomethylation reached levels below 25%. The exchange of blood cells through vascular connections cannot fully explain the degree of hypomethylation found in the blood cell of the non-affected twin. We propose an additional mechanism through which sharing of aberrant methylation patterns in discordant twins, limited to blood cells, might occur. In a BWS-discordant MZ triplet, an intermediate level of demethylation was found in one of the non-affected sibs; this child showed mild signs of BWS. This finding supports the theory that a methylation error proceeds and possibly triggers the twinning process.

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

The Fxr gene family is composed of three members, FMR1, FXR1 and FXR2. The FMR1 gene is involved in the fragile X syndrome, whereas for the other two members, no human disorder has been identified yet. An appropriate animal model to study in vivo gene function is essential to unravel the cellular function of the gene products FMRP, FXR1P and FXR2P, respectively. In Xenopus tropicalis both Fmr1 and Fxr1 were identified; however, unexpectedly Fxr2 was not. Here we describe the characterization of both Fmrp and Fxr1p in Xenopus tropicalis. Fmrp is expressed ubiquitously throughout the embryo during embryonic development, whereas Fxr1p shows a more tissue-specific expression particularly during late embryonic development. In adult frogs both proteins are highly expressed in most neurons of the central nervous system and in all spermatogenic cells in the testis. In addition, Fxr1p is also highly expressed in striated muscle tissue. Western blotting experiments revealed only one prominent isoform for both proteins using different tissue homogenates from adult frogs. Thus, for in vivo gene function studies, this relative simple animal model may serve as a highly advantageous and complementary model.

Characterisation of Fmrp in zebrafish: evolutionary dynamics of the fmr1 gene.

Fragile X syndrome is the most common inherited form of mental retardation. It is caused by the lack of the Fragile X Mental Retardation Protein (FMRP), which is encoded by the FMR1 gene. Although Fmr1 knockout mice display some characteristics also found in fragile X patients, it is a complex animal model to study brain abnormalities, especially during early embryonic development. Interestingly, the ortholog of the FMR1 gene has been identified not only in mouse, but also in zebrafish (Danio rerio). In this study, an amino acid sequence comparison of FMRP orthologs was performed to determine the similar regions of FMRP between several species, including human, mouse, frog, fruitfly and zebrafish. Further characterisation of Fmrp has been performed in both adults and embryos of zebrafish using immunohistochemistry and western blotting with specific antibodies raised against zebrafish Fmrp. We have demonstrated a strong Fmrp expression in neurons of the brain and only a very weak expression in the testis. In brain tissue, a different distribution of the isoforms of Fmrp, compared to human and mouse brain tissue, was shown using western blot analysis. Due to the high similarity between zebrafish Fmrp and human FMRP and their similar expression pattern, the zebrafish has great potential as a complementary animal model to study the pathogenesis of the fragile X syndrome, especially during embryonic development.

Transport kinetics of FMRP containing the I304N mutation of severe fragile X syndrome in neurites of living rat PC12 cells.

Lack of fragile X mental retardation protein (FMRP) causes the fragile X syndrome, a common form of inherited mental retardation. The syndrome usually results from the expansion of a CGG repeat in the FMR1 gene with consequent transcriptional silencing of FMR1. However, one missense mutation (Ile304Asn) was reported in the second KH domain of the protein involved in RNA binding. The protein containing this mutation showed an impaired function, leading to an extremely severe phenotype. In the present report, we have studied the role of FMRP I304N in living PC12 cells to better understand the (dys) function of this mutant FMRP. We have generated an FMR1 I304N-EGFP stably transfected PC12 cell line with an inducible expression system (Tet-On) for regulated expression of the FMRP I304N-EGFP fusion protein. After Dox-induction, FMRP I304N-EGFP was localized in the neurites of PC12 cells; however, no granules were formed as has been recently demonstrated for the normal FMRP. Time-lapse microscopy in combination with bleaching technology illustrated that although FMRP I304N-EGFP does not form visible granules, the transport into the neurites is microtubule dependent. Immunoprecipitation with antibodies against GFP demonstrates that FMRP I304N-EGFP coprecipitate with both the 60S ribosomal protein P0 and FXR1P, suggesting that the mutant FMRP is still able to form complexes, however, with different characteristics compared to normal FMRP.

Characterization of Fxr1 in Danio rerio; a simple vertebrate model to study costamere development.

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.