Analysis of the in vivo function of the zebrafish fragile X gene family

Abstract

Fragile X Syndrome is the leading cause of inherited mental retardation in humans. This X-linked disorder results from underexpression of the Fragile X mental retardation related protein(FMR1P) due to expansion and hypermethylation of a CGG trinucleotide repeat in the 5V UTR region of the Fragile X gene(fmr1). Fmr1 has two autosomal homologs, fxr1 and fxr2, which together comprise a family of highly conserved RNAbinding proteins thought to be involved with mRNA transport and translation, although their precise function(s) in development have not been determined. Expression analyses of these three zebrafish genes have been conducted by this laboratory and others. We have begun an analysis of the in vivo role of the Fragile X gene family in early zebrafish development through the use of morpholino(mo) oligonucleotide knockdowns. We are injecting 1–8 cell stage zebrafish embryos with mo-derived anti-sense reagents designed against the translation start sites of the three zebrafish Fragile Xrelated genes. Injected embryos are assayed for gross morphologic defects, as well as changes in organization of the nervous system through detection of axon tracts with anti-acetylated tubulin antibody. The well-described development of the early zebrafish nervous system, along with the ability to study dose-dependent inactivation of these target sequences individually and in combination should be powerful tools to help discern the in vivo function(s) of the Fragile X gene family in development.